Dr. Ricardo Becker Médico Especialista em cirurgias Ortopédicas Oncológicas e Sarcomas.
Rua Tobias da Silva, 126 - Porto Alegre - RS.
Dr. Ricardo Becker Médico Especialista em cirurgias Ortopédicas Oncológicas e Sarcomas.
Rua Tobias da Silva, 126 - Porto Alegre - RS.
Patricia Jacqueline Schneider BSc, Nathan Evaniew MD, PhD, Paula McKay BSc, Michelle Ghert MD
Background Several challenges presently impede the conduct of prospective clinical studies in orthopaedic oncology, including limited financial resources to support their associated costs and inadequate patient volume at most single institutions. This study was conducted to prioritize research questions within the field so that the Musculoskeletal Tumor Society (MSTS), and other relevant professional societies, can direct the limited human and fiscal resources available to address the priorities that he stakeholders involved believe will have the most meaningful impact on orthopaedic oncology patient care. Questions/purposes The purpose of this study was to use a formal consensus-based approach involving clinicianscientists and other stakeholders to identify the top priority research questions for future international prospective clinical studies in orthopaedic oncology. Methods A three-step modified Delphi process involving multiple stakeholder groups (including orthopaedic oncologists, research personnel, funding agency representation, and patient representation) was conducted. First, we sent an electronic questionnaire to all participants to solicit clinically relevant research questions (61 participants; 54% of the original 114 individuals invited to participate returned the questionnaires). Then, participants rated the candidate research questions using a 5-point Likert scale for five criteria (60 participants; 53% of the original group participated in this portion of the process). Research questions that met a priori consensus thresholds progressed for consideration to an in-person consensus meeting, which was attended by 44 participants (39% of the original group; 12 countries were represented at this meeting). After the consensus panel’s discussion, members individually assigned scores to each question using a 9-point Likert scale. Research questions that met preset criteria advanced to final ranking, and panel members individually ranked their top three priority research questions, resulting in a final overall ranking of research priorities. Results A total of 73 candidate research questions advanced to the consensus meeting. In the end, the consensus panel identified four research priorities: (1) Does less intensive surveillance of patients with sarcoma affect survival? (2) What are the survival outcomes over time for orthopaedic oncology implants? (3) Does resection versus stabilization improve oncologic and functional outcomes in oligometastatic bone disease? (4) What is the natural history of untreated fibromatosis?
Conclusions The results of this study will assist in developing a long-term research strategy for the MSTS and, possibly, the orthopaedic oncology field as a whole. Furthermore, the results of this study can assist researchers in guiding their research efforts and in providing a justified rationale to funding agencies when requesting the resources necessary to support future collaborative research studies that address the identified orthopaedic oncology priorities.
Sarcomas are a rare and heterogeneous group of cancers that represent\1% of all malignancies . However, as a result of their clinical behavior, multidisciplinary management, and complex multimodal treatment, the impact to patients is significant and the cost of care is substantial [7, 13, 27]. Moreover, the skeleton is the third most common organ affected by metastatic cancer . In the advent of improved medical treatment of many cancers, bone metastases are becoming increasingly prevalent because patients with cancer are living longer with their disease [1, 2]. Many critical questions surrounding the surgical management of patients with bone and soft tissue tumors and metastatic bone disease remain unanswered, but funding to support prospective clinical research is meager in comparison to basic science research . Furthermore, a recent qualitative study determined that orthopaedic oncology collaborators are enthusiastic to conduct further research, provided that the research questions are feasible and address clinically relevant endpoints . Therefore, it is important to identify research priorities through a systematic and thoughtful process.
An effective research system should address health issues, interventions, and outcomes of concern within a given field that are considered important by both clinicians
and patients. The Delphi method is one approach that can be utilized to amass the expertise and opinions from various stakeholders in an area and use it to determine a suitable set of research priorities through consensus. This method has been previously used in the development of research agendas in other specialties. For example, the
Acute Kidney Injury Network (AKIN) utilized a Delphi process to construct a research agenda to guide future clinical and translational acute kidney injury (AKI) research [17, 21]. This collaborative network has since conducted multiple studies that address many of the identified priorities and has generated evidence to improve the are of patients with AKI [11, 16, 25]. Their work also exemplifies the impact a formal research agenda can have on clinical care in a given field. However, research priorities have yet to be identified in the field of orthopaedic oncology.
The purpose of this study was to use a consensus-based approach to prioritize research questions within orthopaedic oncology so that efforts and available resources can
then be streamlined toward addressing these priorities.
Materials and Methods
The protocol for this initiative was previously reported elsewhere in further detail . Briefly, a three-step modified Delphi process was conducted between April and November 2016, consisting of a qualitative assessment (Phase I), a rating evaluation (Phase II), and a consensus panel meeting (Phase III) (Fig. 1). During the qualitative assessment, participants were asked to identify a maximum of three research questions that they believed most urgently needed answering to guide patient care. In the rating evaluation, participants were asked to individually rate each candidate research question based on the definitions provided of five criteria that are considered crucial to the development of a realistic yet compelling research question: scientific merit, clinical significance, innovation, relevance, and feasibility (Table 1). Research questions from the rating evaluation that met the a priori consensus thresholds outlined in detail subsequently were brought forward to the consensus panel meeting for facilitated discussion. Thereafter, each consensus panel member anonymously scored each candidate research question; those that met the predetermined criteria were then brought forward for final ranking. The consensus panel members were then asked to rank their top three research questions, which were then distributed to all participants for validation.
Before conducting this consensus-based process, ethics approval was received from the Hamilton Integrated Research Ethics Board (Project No. 1765). For the qualitative assessment and rating evaluation, written informed consent was not obtained from participants because the completion and submission of questionnaires were considered implied consent. For the in-person consensus meeting participants, written informed consent was obtained before the commencement of the meeting; however, for remote participants, written informed consent was not obtained because access to the remote response system and submission of responses were again considered implied consent. All suggested research questions, scores, and rankings were kept strictly confidential and all identifiers were removed before any analysis. Data with direct identifiers were available to only one member of the planning committee (PJS).
Fig. 1 A flow diagram shows the Moving Forward Through Consensus Orthopaedic Oncology Research Program planning initiative.
An initial invitation to gauge interest in participating in this research program planning initiative was distributed to 351 individuals by email on May 24, 2016. The invitation was sent to all clinicians on the Prophylactic Antibiotic Regimens in Tumor Surgery (PARITY) network distribution list, which includes both individuals who have eitherexpressed an interest or are actively participating in the PARITY trial (NCT No. 01479283)  as well as members of the Musculoskeletal Tumor Society (MSTS). Of the 351 individuals, 117 responded to the email (33%). Almost all of these 117 respondents (114 individuals) indicated that they would be interested in participating in all phases of this initiative with only two individuals indicating that they did not wish to participate and one individual wanting to participate only in the consensus meeting
Phase I: Qualitative Assessment–Soliciting Research Questions of Interest
To be eligible for the qualitative assessment, participants had to be a clinician who was either: (1) interested or actively participating in the PARITY trial; or (2) an active or candidate MSTS member. An Orthopaedic Research and Education Foundation (OREF) representative and a clinical research manager of an orthopaedic oncology unit at a major clinical site (with extensive practical experience in orthopaedic oncology clinical research) were also included. A total of 114 individuals were invited to participate. Sixty-one individuals (54% response rate) provided complete responses to the Phase I questionnaire (Fig. 1). Of the participants, all but one were orthopaedic surgeons (98%), and almost all (90%) were men. Of the orthopaedic surgeons who participated, 88% had completed a fellowship in orthopaedic oncology. Participants represented orthopaedic oncology practices from 16 countries (Table 2).
*In instances where the OREF representative participated, the total will equal more than the total number of participants because the OREF representative was also an orthopaedic oncologist (and, therefore, was counted twice); OREF = Orthopaedic Research and Education Foundation.
The invitation to complete a web-based, open-ended questionnaire (with unrestricted answers) was distributed by email on June 14, 2016. The questionnaire was not accompanied by any additional facilitators or literature reviews. Rather, participants were asked to review the literature and consult with colleagues as they saw fit before proposing their ideas. The questionnaire also requested some demographic data and any potential financial or intellectual conflicts of interest. The questionnaire remained online for 4 weeks, and reminder emails were sent approximately every 7 days after the initial invitation to those who had not yet completed the questionnaire.
Phase II: Rating Evaluation–Rating of Research
The same eligibility criteria for qualitative assessment were maintained for the rating evaluation. In total, 113 individuals (one individual withdrew) were invited to participate in this part of the study. Sixty individuals (59 complete, one incomplete; 53% response rate) responded to the rating evaluation questionnaire (Fig. 1). Of the participants, all but one were orthopaedic surgeons (97%); however, one of these orthopaedic surgeons specialized in veterinary medicine. Over two-thirds of the participants (85%) were men. Of the orthopaedic surgeons who participated, 88% had completed a fellowship in orthopaedic oncology. Participants represented orthopaedic oncology practices from 16 countries (Table 2).
The second phase of the modified Delphi process used a web-based questionnaire that asked participants to rate each candidate research question individually on a 5-point Likert scale for five criteria: scientific merit, clinical significance, innovation, relevance, and feasibility (Table 3). Participants were also provided with the opportunity to further clarify or add to research questions, if necessary, and recommend additional research questions. Participants were invited to complete this questionnaire by email on August 10, 2016. The questionnaire remained online for 4 weeks, and reminder emails were sent approximately 7 days after the initial invitation to individuals who had not yet completed the questionnaire.
Phase III: Consensus Meeting–Vetting and Ranking of Research Priorities
Rather than utilize a random sample that is representative of the target population, the Delphi method uses a consensus panel of those invested in the process and its outcomes. There is little agreement on the definition of an expert . Therefore, for the purposes of the current study, all participants from the qualitative assessment and rating evaluation were invited to participate as well as representatives from patient advocacy groups, the MSTS, and OREF. One hundred one individuals were invited to participate. In response to numerous requests, mostly from international clinicians, we opted to also provide a web-based response system and conferencing capabilities, which allowed us to ensure that the consensus panel membership was geographically diverse. The consensus panel included a total of 44 individuals from 12 countries (Fig. 1). The panel was comprised of one patient representative, one clinical research manager, and 42 were orthopaedic surgeons. The OREF selected a representative to attend the meeting, who is also an orthopaedic oncologist. Of the orthopaedic surgeon members, half (50%) were between the ages of 30 and 40 years, and almost two-thirds (57%) had been in practice for no more than 10 years (Table 2).
The consensus meeting was held on October 7, 2016. The meeting was facilitated by an experienced and independent meeting facilitator with extensive prior involvement in strategic planning initiatives. At this meeting, the members of the consensus panel were given the opportunity to discuss the eligible candidate research questions as a group. A semistructured agenda that had flexible time parameters to allow for discussion and questions was followed, as previously published . The consensus meeting was recorded, and all consensus panel members were informed that it was being recorded before the start of the meeting.
Absolute Scoring Stage
Onsite participants were provided with an audience response system device to anonymously score research questions throughout the meeting. Remote participants were provided with access to anonymously score research questions through the Internet in real time throughout the meeting. All candidate research questions wereindividually discussed by members of the consensus panel, thereby providing an opportunity for members to reconsider their Phase II ratings in light of other members’ views. However, priority was given to the discussion of higher ranking tiers. For each question, the facilitator first asked the consensus panel for any remarks in support of the research question; then the facilitator asked for any comments against the research question before asking for any additional comments. When differences in the ratings from Phase II appeared to have resulted from ambiguity in the wording of a research question, the members also used this time to agree on revised wording. After each question’s discussion, the members anonymously assigned a score using the response system.
Final Ranking Stage
Utilizing a web-based questionnaire, the candidate research questions that met the predetermined criteria as outlined below were subsequently distributed to the consensus panel members who were asked to rank their top three research questions . All individuals (100%) involved in the consensus meeting provided their final rankings.
Phase I: Qualitative Assessment–Soliciting Research Questions of Interest
The questionnaire responses were compiled for review. Responses from the qualitative assessment were initially reviewed by an orthopaedic oncologist (MG) on the planning committee. Similar ideas were clustered together into emerging research questions, and duplicate responses were removed. This review was independently repeated by a health research methodology expert (NE) on the planning committee. The two reviewers then met to discuss any differences produced from these independent reviews and reach a consensus on the list of candidate research questions that would progress to the next phase.
Phase II: Rating Evaluation–Rating of Research Questions
The planning committee compiled the ratings for each research question as well as any additional research questions. The results of the questionnaire were reviewed to determine whether each candidate research question met the predetermined consensus thresholds (Table 4). Research questions that met either the inclusion or nonconsensus thresholds progressed to Phase III for review by the consensus panel. Candidate research questions that met the exclusion consensus threshold were not brought forward for review.
Phase III: Consensus Meeting–Vetting and Ranking of Research Priorities
Absolute Scoring Stage Consensus panel members anonymously assigned a score on a 9-point Likert scale for each candidate research question (Table 5). A larger Likert scale was used during this stage because it provided a better opportunity for respondents to adequately report their opinions on the relative importance of each candidate research question. This was especially important during this stage considering the large number of candidate research questions that progressed for in-person vetting.
Once the scores were compiled, questions meeting one of the following predetermined criteria were brought forward for final ranking: 100% of respondents scored the
candidate research question as a 7, 8, or 9 or at least 10% of respondents scored the research question as a 9. If none of the research questions met these criteria, it was also decided a priori that the top 10 scoring candidate research questions would be brought forward for final ranking .
Final Ranking Stage Consensus panel members then ranked their top three research questions. The corresponding point system was used to determine the highest ranking candidate research questions (Table 6). We decided a posteriori that a multiple regression analysis of the consensus meeting data, as stated inthe study’s protocol , was not appropriate given the relatively small final sample size and the large number of priority research questions brought forward by the participants.
Phase I: Qualitative Assessment–Soliciting Research
Questions of Interest
In total, 175 candidate research questions were proposed by the respondents. After the qualitative assessment review, during which 106 duplicates were removed, it was determined that 69 candidate research questions would progress to the rating evaluation phase (Appendix 1 [Supplemental materials are available with the online version of CORR1.]).
Phase II: Rating Evaluation–Rating of Research
In total, 60 individuals (59 complete, one incomplete) participated in the rating evaluation, rating the 69 candidate research questions on five criteria. Four additional research questions were proposed (Note: after the consensus meeting, none of these four questions were ranked highly.). As per the consensus thresholds (Table 4), only one candidate research question met the inclusion threshold. Sixty-eight research questions met the nonconsensus threshold, and no research questions met the exclusion threshold. Therefore, 73 candidate research questions (69 candidate research questions plus four new suggestions) progressed to the final phase for consideration and assessment at the consensus meeting (Appendix 2 [Supplemental materials are available with the online version of CORR1.]).
As a result of the large number of candidate research questions that met the conditions for advancement, we divided them into the following seven tiers in preparation for the consensus meeting: Tier 1–met inclusion threshold; Tier 2–met nonconsensus threshold (average consensus score C 70%); Tier 3–met nonconsensus threshold (average consensus score = 65%–69%); Tier 4–met nonconsensus threshold (average consensus score = 60%–64%); Tier 5– met nonconsensus threshold (average consensus score = 50%–59%); Tier 6–met nonconsensus threshold (average consensus score\50%); and Tier 7–new suggestions. This was done to minimize time constraints and prioritize discussion of candidate research questions that had higher degrees of consensus in the rating evaluation phase
Phase III: Consensus Meeting–Vetting and Ranking of Research Priorities
Final Research Priorities
The final stage identified four research priorities with proposed study methodologies (Table 7): (1) Does less intensive surveillance of patients with sarcoma affect survival? (2) What are the survival outcomes over time for orthopaedic oncology implants? (3) Does resection versus stabilization improve oncologic and functional outcomes in oligometastatic bone disease? (4) What is the natural history of untreated fibromatosis?
Absolute Scoring Stage
The final top research priorities were identified through the scoring of each research question. Two candidate research questions met the criteria for progression to the final ranking stage. This was considered a priori an insufficient number of candidate research questions to conduct the final ranking, so the mean score was calculated for each research question and the top 10 were brought forward as per protocol . Because one research question that met the criteria for progression did not score in the
top 10 by mean score, we decided that 11 candidate research questions would advance to the final stage of this phase (Table 8).
Final Ranking Stage
After each consensus meeting participant provided their final rankings, the top priority research questions were identified as outlined previously (Table 7). The research question with the fourth highest score was also included in this list because the total number of panel members that ranked this question in their top three was higher than that of both the second and third ranking questions with a final score only marginally lower than that of the third ranking question.
Previous work has demonstrated a lack of high-quality evidence to guide clinical decisions in orthopaedic oncology . As a result of the rarity of bone and soft tissue
tumors, multicenter prospective collaboration is imperative for broadly meaningful research and evidence-based advances in patient care . However, although agreement exists pertaining to the importance of collaborative research and the need for higher quality research in orthopaedic oncology, research priorities remain unclarified. Consensus methods are being increasingly used to develop research agendas in various medical and surgical specialties [17, 20, 23]. Research agendas can assist professional groups in allocating finite research resourcesto clinical investigations likely to provide the greatest value. They can also provide individual researchers with guidance to help prioritize their own endeavors. To establish a research agenda specific to orthopaedic oncology, we brought together international stakeholders and
conducted a modified Delphi process, which identified the following four priorities: (1) Does less intensive surveillance of patients with sarcoma affect survival? (2) What are the survival outcomes over time for orthopaedic oncology implants? (3) Does resection versus stabilization improve oncologic and functional outcomes in oligometastatic bone disease? (4) What is the natural history of untreated fibromatosis?
Our study has some limitations that should be recognized. Although diverse, our sample’s composition was still dominated by individuals from North America, so our
results may reflect a North American perspective that is not applicable to all regions as a result of differences in access to health care, referral patterns, availability of medical interventions, and cultural acceptability of treatment. However, these imbalances in perspective may have been resolved through discussions with international colleagues during the consensus meeting. Furthermore, underrepresentation of distinct groups may have resulted from our sampling method. We invited all interested or active PARITY investigators and MSTS members to participate in this initiative. However, a substantial number of orthopaedic oncologists are not affiliated with either the PARITY trial or MSTS; therefore, they may not be adequately represented in our study. Although we made numerous attempts to contact orthopaedic oncology patient advocacy groups for their input, we were only able to connect with one patient advocate, who was contacted directly and who was interested in supporting orthopaedic oncology research. It was, therefore, fruitful to directly contact patients rather than patient advocacy groups. Nevertheless, future opportunities exist to include a greater number of patient advocates and underrepresented stakeholders such as during the development process of specific research protocols to address the identified priority research questions. Finally, our response rates (33% [117 of 351] for the initial invitation and 54% [61 of 114] and 53% [60 of 113] for the qualitative assessment and rating evaluation questionnaires, respectively) were somewhat lower than those achieved in similar studies in other medical specialties [20, 23]. However, our initial invitation allowed us to specifically select a population that is interested in collaborative research in the field. Considering that our objective was to determine the priority research questions in orthopaedic oncology to drive future collaborative prospective research, this bias may have been favorable and resulted in inherently collaborative participants providing many insightful ideas. Individuals who did not respond to the initial invitation may not have been interested in participating in any collaborative research, may not have expected this initiative to be fruitful, or did not prioritize this initiative above other academic interests.
However, response bias was minimized by allowing individuals to participate in any phase of the study. Future studies that are aimed at more rigorously evaluating
potential sampling biases should include European and Asian orthopaedic oncology societies.
Despite these limitations, the design of the current initiative was structured, thorough, transparent, and aimed to include all invited individuals who expressed an interest. Furthermore, the use of a modified Delphi process for this initiative maximized the benefits of two common consensus approaches—the classic Delphi method and the Nominal Group Technique. Throughout the initiative, anonymity in scoring was maintained, even while the moderated discussion took place; this provided the opportunity for the causes of disagreement to be explored without particularly vocal participants dominating the discussion and overpowering the opinions of others [8, 15]. Therefore, it is unlikely that consensus was forced on participants nor that the priority research questions identified reflect the perspective of any one participant. Finally, the composition of the participant group is also thought to influence the outcome of the consensus process. Other studies have previously demonstrated that homogenous samples select different options than heterogeneous ones considering the same choices . To maximize the feasibility and generalizability of the identified research priorities, we assembled a group of participants from a broad range of healthcare systems, geographic regions, career focus and stage, and prior research experience.
The natural succession of the identification of research priorities is the development of an action plan to answer the priority research questions and to identify viable funding mechanisms to support these research efforts. Surveillance after sarcoma treatment is a subject that spans all disciplines in cancer care. The balance between the cost of intense surveillance with respect to resources and patient quality of life and the potential benefit to identify relapsed disease in a curable stage must take into account many important factors [7, 10, 19, 22, 35]. This is, therefore, a complex question that will require coordinated protocol development among a long list of stakeholders, the most important being the patients themselves. Puri et al.  have published the only relevant randomized controlled trial (RCT) to date in the sarcoma field. This single-center study concluded that overall 3-year survival and diseasefree survival were no different between patients with sarcoma who had more intensive surveillance (CT scans) and those with less (chest radiographs) . However, as a result of the sample size, this trial could not conclusively demonstrate noninferiority in overall survival for a 6- monthly followup visit interval against a 3-monthly interval . In addition, because this was a single-center study, generalizability of the results to other centers and countries is limited. A large international collaborative network will be required to implement a RCT protocol that addresses both the implications for healthcare systems and the preferences of patients with sarcoma. Government-level funding and large-scale grants from cancer research funding agencies would presumably be required to ensure the success of this effort.
Patients with sarcoma are often teenagers and young adults . Therefore, those who survive after treatment may live for many decades. The implants used to reconstruct the extremities after tumor excision are prone to failure for a variety of reasons including infection, fracture, and aseptic loosening [14, 30]. Identifying emerging trends in the characteristics of those requiring revision surgery specific to the available implants may help identify risk factors so that the appropriate resources can be allocated to mitigate those risks. The success of the National Joint Registry (NJR) for England, Wales, Northern Ireland, and the Isle of Man in identifying implant designs that were failing at a proportionately higher rate illustrates the benefits of long-term observational data collection in other orthopaedic specialties . An industry- or governmentfunded prospective implant registry specific to orthopaedic oncology would likely be the ideal approach to address this research priority.
With newer targeted systemic therapy, many patients with metastatic bone disease from primary carcinomas such as breast and renal cancers are living months and years longer with their disease [4, 32]. Therefore, a more aggressive surgical approach to resect entire bone metastases en bloc, as opposed to stabilization without tumor excision in its entirety, may be warranted to improve survival and possibly quality of life. This research priority could be answered with a multicenter RCT with a focus not only on survival rates, but also patient-reported outcomes specific to quality of life. However, such an endeavor would likely require generous government-level and nonprofit support. Similarly, there has been a paradigm shift in the treatment of fibromatosis (desmoid tumors), a benign diagnosis with aggressive local behavior. Once thought to be a surgical disease, it is now understood through retrospective data that with unacceptably high recurrence rates, ‘‘watchful waiting’’ may be the most appropriate management strategy for most patients [9, 12]. However, patients must be convinced that, if left untreated, the natural history of this disease is benign. A prospective multicenter cohort study that follows patients for symptoms and progression would assist in directing the care of patients in future generations. The Desmoid Tumor Foundation would be an ideal funding mechanism for this important study in orthopaedic oncology.
We have identified research priorities for international prospective research in orthopaedic oncology by conducting a three-step modified Delphi process. Top research priorities in orthopaedic oncology include evaluating different postoperative surveillance regimens in patients with extremity sarcoma, understanding the survival outcomes of orthopaedic oncology implants over time, evaluating whether resection versus stabilization improves outcomes in patients with oligometastatic bone disease, and understanding the natural history of untreated fibromatosis. These priority research questions highlight areas where international stakeholders have agreed by consensus that further knowledge would have a significant impact on the clinical care of orthopaedic oncology patients. Therefore, the MSTS and other professional orthopaedic oncology societies whose missions are to promote the advancement of orthopaedic oncology science and patient care may enable and support research efforts that address these priorities. These societies could do so by helping to facilitate the establishment of Working Groups and coordinating Working Group meetings, preferably by designating specific meeting times at annual conferences, to explore the next steps and develop action plans. The limited research funds of these professional societies could also be preferentially allocated to studies concentrating on one of the identified priorities. This research agenda could also be used by researchers to focus their research efforts and provide a rationale in competitive grant applications when applying for the financial resources to support endeavors
directed at answering these priority research questions in orthopaedic oncology.
Acknowledgments We thank Mr Bruce Withrow of Meeting Facilitators International for his objective advice on executing a strategic planning initiative and his impartial and unbiased facilitation of the consensus meeting. Reaching an agreement on these research priorities in orthopaedic oncology would not have been possible
without his expertise. We would also like to thank the following individuals, who participated in at least one phase of this orthopaedic oncology research program planning initiative: Albert Aboulafia MD, MBA (MedStar Franklin Square, Georgetown University, Baltimore, MD, USA); John Abraham MD (The Rothman Institute, Thomas Jefferson University, Philadelphia, PA, USA); Brock Adams MD (MedStar Franklin Square, Georgetown University, Baltimore, MD, USA); Alexandre Arkader MD (The Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA); Annie Arteau MD (L’Hoˆtel-Dieu de Que´bec, CHU de Que´bec,
Universite´ Laval, Que´bec, Canada); Raffi Avedian MD (Stanford University, Redwood City, CA, USA); Tessa Balach MD (University of Chicago, Chicago, IL, USA); André Mathias Baptista MD, PhD (Hospital de Clínicas de São Paulo, Universidade deSão Paulo, São Paulo, Brazil); Ricardo Gehrke Becker MD, PhD (Hospital de
Clínicas de Porto Alegre, Instituto do Câncer Infantil do Rio Grande do Sul, Porto Alegre, Brazil); Joseph Benevenia MD (University Hospital, Rutgers, The State University of New Jersey, Newark, NJ, USA); Marko Bergovec MD (Medical University Graz, Graz, Austria); Nicholas Bernthal MD (University of California Los Angeles, Los Angeles, CA, USA); B. Hudson Berrey MD (Baptist Medical Center, Jacksonville, FL, USA); Justin Bird MD (University of Texas MD Anderson Cancer Center, Houston, TX, USA); Michele Boffano MD (AOU Citta` della Salute e della Scienza, Torino, Italy); Scot Brown MD (University of Wisconsin, Madison, WI, USA); George Calvert MD (Norton Healthcare, Louisville, KY, USA); Edward Cheng MD (University of Minnesota, Minneapolis, MN, USA); Mark Clayer, MBBS (retired; Royal Adelaide Hospital, Adelaide, Australia); Sheila Conway MD (University of Miami, Miami, FL, USA); Benjamin Deheshi MD, MSc (McMaster University, Juravinski
Hospital and Cancer Centre, Toronto, Ontario, Canada); P. D. Sander Dijkstra MD, PhD (Leids Universitair Medisch Centrum, Leiden, The Netherlands); Gregory Domson MD (Virginia Commonwealth University Health System, Richmond, VA, USA); Yee-Cheen Doung MD (Oregon Health and Science University, Portland, OR,
USA); Nicole Ehrhart VMD, PhD (Colorado State University, Fort Collins, CO, USA); Cynthia Emory MD (Wake Forest University, Winston-Salem, NC, USA); Lisa Ercolano MD (Allegheny Health Network, Drexel University, Pittsburgh, PA, USA); Gary Friedlaender MD (Yale University, New Haven, CT, USA); Marcos Galli Serra MD (Hospital Universitario Austral, Universidad Austral, Pilar, Argentina); Barbara Gilligan, Krista Goulding MD (McGill University, Montreal, Quebec, Canada); David Greenberg MD (St Louis University, St Louis, MO, USA); Anthony Griffin MSc (Mount Sinai Hospital, Toronto, Ontario, Canada); James Hayden MD, PhD (Oregon Health and Science University, Portland, OR, USA); John Healey MD (Memorial Sloan-Kettering Cancer Center, Cornell University, New York, NY, USA); Robert Henshaw MD (MedStar Washington Hospital Center, Georgetown University, Washington, DC, USA); Asle Charles Hesla MD (Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden); Werner Hettwer MD (Rigshospitalet, Københavns Universitet, Copenhagen, Denmark); Ronald Hillock MD (Nevada Orthopedic & Spine Center, Las Vegas, NV, USA); Kelly Homlar MD (Augusta University, Augusta, GA, USA); Shintaro Iwata MD, PhD (National Cancer Center Hospital, Tokyo, Japan); Paul Jutte MD, PhD (Universitair Medisch Centrum Groningen, Groningen, The Netherlands); David King MD (Medical College of Wisconsin, Milwaukee, WI, USA); Daniel Lerman MD (University of Maryland, College Park, MD, USA); Adam Levin MD (Johns Hopkins University, Baltimore, MD, USA); Patrick Lin MD (University of Texas MD Anderson Cancer Center, Houston, TX, USA); Francisco Linares MD (Hospital Universitario San Ignacio, Pontifica Universidad Javeriana, Bogota, Colombia); Adam Lindsay MD (University of Connecticut, Farmington, CT, USA); Dieter M. Lindskog MD (Yale University, New Haven, CT, USA); Kevin MacDonald MD (Virginia Mason Medical Center, Seattle, WA, USA); Leonard Marais MBChB, MMed (Grey’s Hospital, University of KwaZulu-Natal, Durban, South Africa); Joel Mayerson MD (Wexner Medical Center, The Ohio State University, Columbus, OH, USA); Richard McGough MD (University of Pittsburgh, Pittsburgh, PA, USA); Nathan Mesko MD (The Cleveland Clinic, Cleveland, OH, USA); Benjamin Miller MD, MS (Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA); David G. Mohler MD (Stanford University, Redwood City, CA, USA); Michael P. Mott MD (Henry Ford Health System, Detroit, MI, USA); Michael E. Mulligan MD (University of Maryland, Baltimore, MD, USA); Miguel Murcia Herna´ndez MD, PhD (Hospital Pablo Tobo´n Uribe, Universidad Pontificia Bolivariana, Medellı´n, Colombia); Jorge Navia MD (deceased; Centro Medico Imbanaco, Universidad del Valle, Cali, Colombia); Lukas Nystrom MD (The Cleveland Clinic, Cleveland, OH, USA); Eduardo J. Ortiz-Cruz MD, PhD (Hospital Universitario La Paz, Universidad Auto´noma de Madrid, Madrid, Spain); Michael Parry MBChB, MD (Royal Orthopaedic Hospital NHS Foundation Trust, Birmingham, Birmingham, UK); Joshua Patt MD, MPH (Carolinas Medical Center, Charlotte, NC, USA); Terrance D. Peabody MD (Northwestern Memorial Hospital, Northwestern University, Chicago, IL, USA); Daniel E. Prince MD, MPH (Memorial Sloan Kettering Cancer Center, Cornell University, New York, NY, USA); Ajay Puri MS (Tata Memorial Centre, Homi Bhabha National Institute, Mumbai, India); R. Lor Randall MD (Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA); Pietro Ruggieri MD, PhD (Universita` di Padova, Padova, Italy); Chigusa Sawamura MD, MPH (Tokyo Medical and Dental University, Tokyo, Japan); Amir Sternheim MD (Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel); Richard Terek MD (Providence VA Medical Center, Brown University, Providence, RI, USA); Joachim Thorkildsen MD, PhD (Oslo University Hospital – Radiumhospitalet, Oslo, Norway); Steven Thorpe MD (UC Davis Comprehensive Cancer Center, University of California, Davis, Sacramento, CA, USA); Robert Turcotte MD (McGill University Health Centre, McGill University, Montreal, Quebec, Canada); Wakenda Tyler MD, MPH (Columbia University Medical Center, Columbia University, New York, NY, USA); Roberto Ve´lez MD, PhD (Hospital Universitario Vall d’Hebron, Universidad Auto`noma de Barcelona, Barcelona, Spain); Matthew Wallace MD, MBA (MedStar Cancer Institute, Baltimore, MD, USA); Ronald P. Williams MD (US Oncology Network–Texas Oncology, Austin, TX, USA); Rosanna Wustrack MD (University of California, San Francisco, San Francisco, CA, USA); and Juan Pablo Zuma´rraga MD, MSc (Hospital de Clı´nicas de Sa˜o Paulo, Universidade de São Paulo, São Paulo, Brazil).
1. American Academy of Orthopaedic Surgeons. Metastatic Bone Disease. Available at: http://orthoinfo.aaos.org/topic.cfm?topic= A00093. Accessed May 12, 2017.
2. Bagi CM. Targeting of therapeutic agents to bone to treat metastatic cancer. Adv Drug Del Rev. 2005;57:995–1010.
3. Baker J, Lovell K, Harris N. How expert are the experts? An exploration of the concept of ‘expert’ within Delphi panel techniques. Nurse Res. 2006;14:59–70.
4. Coppin C, Kollmannsberger C, Le L, Porzsolt F, Wilt TJ. Targeted therapy for advanced renal cell cancer (RCC): a Cochrane systematic review of published randomised trials. BJU Int. 2011;108:1556–1563.
5. Evaniew N, Nuttall J, Farrokhyar F, Bhandari M, Ghert M. What are the levels of evidence on which we base decisions for surgical management of lower extremity bone tumors? Clin Orthop Relat Res. 2014;471:2017–2027.
6. Ghert M, Deheshi B, Holt G, Randall RL, Ferguson P, Wunder J, Turcotte R, Werier J, Clarkson P, Damron T, Benevenia J, Anderson M, Gebhardt M, Isler M, Mottard S, Healey J, Evaniew N, Racano A, Sprague S, Swinton M, Bryant D, Thabane L, Guyatt G, Bhandari M; PARITY Investigators. Prophylactic antibiotic regimens in tumour surgery (PARITY): protocol for a multicentre randomised controlled study. BMJ Open. 2012;2:e002197.
7. Goel A, Christy ME, Virgo KS, Kraybill WG, Johnson FE. Costs of follow-up after potentially curative treatment for extremity soft-tissue sarcoma. Int J Oncol. 2004;25:429–435.
8. Greatorex J, Dexter T. An accessible analytical approach for investigating what happens between the rounds of a Delphi study. J Adv Nurs. 2000;32:1016–1024.
9. Honeyman JN, Theilen TM, Knowles MA, McGlynn MM, Hameed M, Meyers P, Crago AM, La Quaglia MP. Desmoid ibromatosis in children and adolescents: a conservative approach to management. J Pediatr Surg. 2013;48:62–66.
10. Hopkins RB, Goeree R, Longo CJ. Estimating the national wage loss from cancer in Canada. Curr Oncol. 2010;17:40–49.
11. Hoste AEJ, Bagshaw SM, Bellomo R, Cely CM, Colman R, Cruz DN, Edipidis K, Forni LG, Gomersall CD, Govil D, Honore´ PM, Joannes-Boyau O, Joannidis M, Korhonen AM, Lavrentieva A, Mehta RL, Palevsy P, Roessler E, Ronco C, Uchino S, Vazquez A, Andrade EV, Webb S, Kellum JA. Epidemiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study. Intensive Care Med. 2015;41:1411–1423.
12. Huang K, Wang CM, Chen JG, Du CY, Zhou Y, Shi YQ, Fu H. Prognostic factors influencing event-free survival and treatments in desmoid-type fibromatosis: analysis from a large institution. Am J Surg. 2014;207:847–854.
13. Jansen-Landheer MLEA, Krijnen P, Oostindie¨r MJ, Kloosterman-Boele WM, Noordijk EM, Nooij MA, Steup WH, Taminiau AHM, Vree R, Hogendoorn PCW, Tollenaar RAEM, Gelderblom H. Improved diagnosis and treatment of soft tissue sarcoma patients after implementation of national guidelines: a population-based study. Eur J Surg Oncol. 2009;35:1326–1332.
14. Jeys LM, Kulkarni A, Grimer RJ, Carter SR, Tillman RM, Abudu A. Endoprosthetic reconstruction for the treatment of musculoskeletal tumors of the appendicular skeleton and pelvis. J Bone Joint Surg Am. 2008;90:1265–1271.
15. Jones J, Hunter D. Consensus methods for medical and health services research. BMJ. 1995;311:376–380.
16. Kashani K, Al-Khafaji A, Ardiles T, Artigas A, Bagshaw SM, Bell M, Bihorac A, Birkhahn R, Cely CM, Chawla LS, Davison DL, Feldkamp T, Forni LG, Ng Gong M, Gunnerson KJ, Haase M, Hackett J, Honore PM, Hoste EAJ, Joannes-Boyau O, Joannidis M, Kim P, Koyner JL, Laskowitz DT, Lissauer ME, Marx G, McCullough PA, Mullaney S, Ostermann M, Rimmele´ T, Shapiro NI, Shaw AD, Shi J, Sprague AM, Vincent JL, Vinsonneau C, Wagner L, Walker MG, Wilkerson RG, Zacharowski K, Kellum JA. Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care. 2013;17:R25.
17. Kellum JA, Mehta RL, Levin A, Molitoris BA, Warnock DG, Shah SV, Joannidis M, Ronco C; Acute Kidney Injury Network (AKIN). Development of a clinical research agenda for acute kidney injury using an international, interdisciplinary, three-step modified Delphi process. Clin J Am Soc Nephrol. 2008;3:887– 894.
18. Leopold SS. Editor’s Spotlight/Take Five: What are the levels of evidence on which we base decisions for surgical management of lower extremity bone tumors? Clin Orthop Relat Res. 2014;472:3–7.
19. Longo CJ, Deber R, Fitch M, Williams AP, D’Souza D. An examination of cancer patients’ monthly ‘out-of-pocket’ costs in Ontario, Canada. Eur J Cancer Care (Engl). 2007;16:500–507.
20. McIntyre S, Novak I, Cusick A. Consensus research priorities for cerebral palsy: a Delphi survey of consumers, researchers, and clinicians. Dev Med Child Neurol. 2010;52:270–275.
21. Mehta RL, Kallum JA, Shah SV, Molitoris BA, Ronco C, Warnock DG, Levin A; Acute Kidney Injury Network. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care. 2007;11:R31.
22. Nipp RD, Zullig LL, Samsa G, Peppercorn JM, Schraq D, Taylor DH Jr, Abernethy AP, Zafar SY. Identifying cancer patients who alter care or lifestyle due to treatment-related financial distress. Psychooncology. 2016;25:719–725.
23. Ota S, Cron RQ, Schanberg LE, O’Neil K, Mellins ED, Fuhlbrigge RC, Feldman BM. Research priorities in pediatric rheumatology: the Childhood Arthritis and Rheumatology Research Alliance (CARRA) consensus. Pediatr Rheumatol. 2008;6:5.
24. Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 2009;152:3–13.
25. Prowle JR, Liu YL, Licari E, Bagshaw SM, Egi M, Haase M, Haase-Fielitz A, Kellum JA, Cruz D, Ronco C, Tsutsui K, Uchino. S, Bellomo R. Oliguria as predictive biomarker of acute kidneyinjury in critically ill patients. Crit Care. 2011;15:R172.
26. Puri A, Gulia A, Hawalder R, Ranganathan P, Badwe RA. Does intensity of surveillance affect survival after surgery for sarcomas? Results of a randomized noninferiority trial. Clin Orthop Relat Res. 2014;472:1568–1575.
27. Renard AJ, Veth RP, Schreuder HWB, van Loon CJ, Koops HS, van Horn JR. Function and complications after ablative and limbsalvage therapy in lower extremity sarcoma of bone. J Surg Oncol. 2000;73:198–205.
28. Rendon JS, Swinton M, Bernthal N, Boffano M, Damron T, Evaniew N, Ferguson P, Galli Serra M, Hettwer W, McKay P, Miller B, Nystrom L, Parizzia W, Schneider P, Spiguel A, Ve´lez R, Weiss K, Zuma´rraga J, Ghert M. Barriers and facilitators experienced in collaborative research in orthopaedic oncology. Bone Joint Res. 2017;6:307–314.
29. Schneider P, Evaniew N, Rendon JS, McKay P, Randall RL, Turcotte R, Ve´lez R, Bhandari M, Ghert M. Moving forward through consensus: protocol for a modified Delphi approach to determine the top research priorities in the field of orthopaedic oncology. BMJ Open. 2016;6:e011780.
30. Shehadeh A, Noveau J, Malawer M, Henshaw R. Late complications and survival of endoprosthetic reconstruction after resection of bone tumors. Clin Orthop Relat Res. 2010;468:2885– 2895.
31. Skulmoski GJ, Hartman FT, Krahn J. The Delphi methods for graduate research. J Inf Technol Educ. 2007;6:1–21.
32. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783–792.
33. Smith AJ, Dieppe P, Vernon K, Porter M, Blom AW; on behalf of the National Joint Registry of England and Wales. Failure rates of stemmed metal-on-metal hip replacements: analysis of data from the National Joint Registry of England and Wales. Lancet. 2012;379:1199–1204.
34. Sung NS, Crowley WF, Genel M, Salber P, Sandy L, Sherwood LM, Johnson SB, Catanese V, Tilson H, Getz K, Larson EL, Scheinberg D, Reece EA, Slavkin H, Dobs A, Grebb J, Martinez RA, Korn A, Rimoin D. Central challenges facing the national clinical research enterprise. JAMA. 2003;289:1278–1287.
35. Thompson CA, Charlson ME, Schenkein E, Wells MT, Furman RR, Elstrom R, Ruan J, Martin P, Leonard JP. Surveillance CT scans are a source of anxiety and fear of recurrence in long-term lymphoma survivors. Ann Oncol. 2010;21:2262–2266.
36. Yu HH, Tsai YY, Hoffe SE. Overview of diagnosis and management of metastatic disease to bone. Cancer Control. 2012;19:84–91.
Limb salvage with endoprosthetic reconstruction is the current standard practice for the surgical management of lower extremity bone tumors in skeletally mature patients and typically includes tumor resection followed by the functional limb reconstruction with modular metallic and polyethylene endoprosthetic implants.
However, owing to the complexity and length of these procedures, as well as the immunocompromised nature of patients treated with chemotherapy, the risk of surgical site infection (SSI) is high. The primary research objective of the Prophylactic Antibiotic Regimens In Tumor Surgery (PARITY) trial is to assess whether a 5-day regimen of postoperative antibiotics decreases the risk of SSI at 1 year post-operatively compared to a 1-day regimen. This article describes the statistical analysis plan for the PARITY trial.
The PARITY trial is an ongoing multi-center, blinded parallel two-arm randomized controlled trial (RCT) of 600 participants who have been diagnosed with a primary bone tumor, a soft tissue sarcoma that has invaded the bone or oligometastatic bone disease of the femur or tibia that requires surgical resection and endoprosthetic reconstruction. This article describes the overall analysis principles, including how participants will be included in each analysis, the presentation of results, adjustments for covariates, the primary and secondary outcomes, and their respective analyses. Additionally, we will present the planned sensitivity and sub-group
Our prior work has demonstrated (1) high rates of SSI after the treatment of lower extremity tumors by surgical excision and endoprosthetic reconstruction, (2) highly varied opinion and practice among orthopedic oncologists with respect to prophylactic antibiotic regimens, (3) an absence of applicable RCT evidence, (4) extensive support from international investigators to participate in a RCT, and (5) the feasibility of conducting a definitive RCT to evaluate a 5-day regimen of post-operative antibiotics in comparison with a 1-day regimen.
ClinicalTrials.gov NCT01479283. Registered on 24 November 2011
Orthopedic oncology, Bone sarcoma, Randomized controlled trial, Antibiotics, Statistical analysis plan
Limb salvage surgery is the current standard of care in the management of sarcoma of the long bones [1–3]. Advances in chemotherapeutic regimens and imaging
techniques allow for wide resection and functional reconstruction in 95% of patients. The most common type of long-bone reconstruction involves the use of a tumor
prosthesis or endoprosthesis. Due to the complexity and length of surgical resection and reconstruction, as well as the immunocompromised nature of patients treated
with chemotherapy, the risk of surgical site infection (SSI) remains high, which is a devastating complication that often requires staged revision surgery and longterm intravenous antibiotics [4, 5]. The risk for subsequent infection remains high, as does the risk for ultimate amputation [4, 5]. Moreover, patients’ quality-of-life and function following infection are dramatically impacted, as are healthcare costs [6, 7]. However, the most effective antibiotic regimen in preventing post-operative SSI remains controversial, and the current state of practice varies widely, particularly with respect to antibiotic duration . Strategies to prevent SSIs and optimize quality-of-life while mitigating healthcare costs are needed.
The Prophylactic Antibiotic Regimens In Tumor Surgery (PARITY) trial is an ongoing international, multicenter randomized controlled trial (RCT) using a parallel two-arm design to determine whether a long duration (5 days) of post-operative prophylactic antibiotics decreases the risk of SSI when compared to a short duration (1 day) . The protocol for the PARITY trial has been published elsewhere and provides more detail on the trial rationale, eligibility criteria, interventions, data management, and methods for minimizing bias . Briefly, 600 participants 12 years of age or older undergoing surgical excision and endoprosthetic reconstruction of a lower extremity bone tumor across North America, South America, Europe, Australia, Africa, and Asia are randomized to receive either a short (1 day) or long (5 days) duration of post-operative antibiotics. Allocation is concealed using a centralized and automated 24-h computerized randomization platform that allows Internet-based randomization. Randomization is stratified by tumor location (femur or tibia) and clinical site in randomly permuted blocks of 2 and 4. The primary outcome of the study is the development of a SSI, guided by the Centers for Disease Control and Prevention (CDC) National Healthcare Safety Network reporting criteria . Secondary outcomes include the development of antibiotic-related complications (such as gastrointestinal infections, fungal infections), unplanned re-operations, oncologic outcomes, mortality, and patient functional outcomes and quality-of-life at 1 year. Participants are regularly monitored post-operatively by the treating surgeon at 2 weeks, 6 weeks, 3 months, 6 months, 9 months, and 1 year following surgery. Outcome assessors and data analysts are blinded to treatment allocation. The full study
process is shown in Fig. 1.
In this article, we present our planned statistical analyses for the PARITY trial. The statistical analysis plan was finalized and approved on 11 January 2021 (version 1) for the PARITY trial protocol (31 October 2016, version 6) and in accordance with the trial master file, including the data management plan (11 December 2020, version 2). Ethics approval was granted for the Methods Centre at McMaster University (Hamilton Integrated Research Ethics Board No. 12-009) and at each participating clinical site (as per their local ethics committee). This trial is registered on ClinicalTrials.Gov (NCT01479283).
The primary outcome of the PARITY trial is the development of a SSI within 1 year following the initial surgery to treat a lower extremity bone tumor. The primary
analysis is to assess whether a long duration regimen (5 days) of post-operative antibiotics decreases the risk of SSI at 1 year compared to a short duration regimen (1
day). SSIs are classified according to the criteria established by the CDC, which defines a SSI as an infection occurring within 30 days following the operative procedure or within 1 year if an implant is in place and the infection appears to be related to the procedure . The SSI can involve any part of the body that is opened or manipulated during the operative procedure but excludes the skin incision, fascia, or muscle layers. The participant must also present with at least one of the following:
▪ Purulent drainage from the superficial/deep/organ space incision;
▪ Organisms isolated from an aseptically obtained culture of fluid or tissue from the superficial/deep/ organ space incision;
▪ Superficial/deep/organ space incision that is deliberately opened by a surgeon, attending physician, or other designee and is culture-positive OR not cultured and the participant has at least one of the following signs or symptoms: pain or tenderness, localized swelling, redness, or heat; or
▪ Diagnosis of a superficial/deep/organ space incisional SSI by a surgeon or attending physician.
Secondary outcomes include the following:
1) Antibiotic-related complications including, but not limited to, Clostridioides difficile-associated colitis, opportunistic fungal infections, and indwelling catheter-related sepsis;
2) Unplanned re-operations including, but not limited to, amputation, irrigation and debridement, implant revision, and implant exchange;
3) Oncologic events;
4) All-cause mortality;
5) Physician-derived functional outcome as measured y the Musculoskeletal Tumor Society (MSTS)-87 and MSTS-93 scores; and
6) Self-reported functional outcome as measured by the Toronto Extremity Salvage Score (TESS) survey.
The MSTS-87 score is a standardized scoring system that is completed by an individual on the treatment team (preferably the treating surgeon) and measures physical
function after treatment for a musculoskeletal tumor across seven domains: motion, pain, stability, deformity, muscular strength, functional activity, and emotional acceptance. The MSTS-93 score is a standardized scoring system that is also completed by an individual on the treatment team and measures functional outcome after
treatment for a musculoskeletal tumor across six domains: pain, function, emotional acceptance, support, walking ability, and gait. The TESS survey is a selfadministered evaluation tool that was developed to assess physical function and quality-of-life in patients that have undergone limb salvage surgery for tumors of the extremities. The lower extremity portion of the survey contains 30 questions that are framed to ask about the difficulty experienced by the patient in performing each activity over the previous week. The MSTS-87, MSTS93, and TESS surveys are all commonly accepted functional scoring systems in the orthopedic oncology literature [11–13].
SSIs, antibiotic-related complications, re-operations, and mortality will be reviewed by an independent Adjudication Committee.
At the onset of the trial, we calculated that the definitive trial’s sample size would include 460 participants per group, for a total of 920 participants. This sample size
was based on a between-group comparison for the primary outcome of deep SSI following long duration (5 days) or short duration (1 day) prophylactic antibiotics and was calculated to ensure that the study would have a power of 80% to identify differences among the two groups at an alpha level of 0.05, on the basis of an overall event rate of 10% and a presumed 50% or greater reduction in the risk of deep SSI within 1 year.
Prior to the transition from the pilot phase to the definitive phase of the trial, we met with our Steering Committee to finalize the definitive study protocol and processes. At this time, we decided to expand the trial’s primary outcome from deep SSI to any SSI (superficial/ deep/organ space SSI) in order to increase the expected event rate and study power without compromising clinical importance. This adjustment in the primary outcome resulted in an overall pilot phase event rate of 14%, which exceeds the overall event rate of 10% used to calculate the initial sample size. As a result, the definitive trial’s sample size was reduced to 300 participants per arm, for a total of 600 participants to identify the differences among the two groups at an alpha level of 0.05 and to ensure that the study would have a power of 80% using the updated event rate of 14% while maintaining the presumed 50% or greater reduction in the risk of SSI within 1 year.
The current sample size calculation is the standard method to determine sample size in a binary outcome study and will provide a conservative yet similar estimate to the more complicated and complex calculation for a time-to-event analysis. This decision was made as utilizing the more conservative binary outcome estimate would likely account for any unforeseen losses to followup, dropouts, and crossovers, which were considered negligible in our study population, and therefore, adjustments for their occurrences were not warranted at the time of the definitive sample size calculation. The binary outcome study method is also simpler to present in study documents such as grant applications.
This statistical analysis plan follows the JAMA Guidelines for the Content of Statistical Analysis Plans in Clinical Trials . A summary of all planned analyses is provided in Table 1.
All statistical analyses will first be conducted using blinded treatment groups (i.e., antibiotic duration X and duration Y). To do so, the blinded study statistician will
provide complete blinded results labeled antibiotic duration X and duration Y; the remainder of the study team at the Methods Centre will be left unaware of which
treatment groups antibiotic durations X and Y represent. Interpretations for the effect of the antibiotic durations will be documented during a blinded review of the data based upon blinded antibiotic duration X versus Y (i.e., we will determine how to interpret the results if antibiotic duration X proves to be the long duration regimen
(5 days) of post-operative antibiotics versus how to interpret the results if antibiotic duration Y proves to be the long duration regimen (5 days) of post-operative antibiotics) . We will unblind the results by breaking the randomization code following the approval and documentation of the interpretations by the study team. These agreed-upon interpretations will guide the discussion section of the subsequent definitive trial manuscript.
Presentation of data
The trial results will be presented according to the Consolidated Standards of Reporting Trials (CONSORT) guidelines for RCTs . The number of patients screened, included, and excluded will be presented in a flow diagram (Fig. 2). The baseline emographic characteristics, tumor details, and surgical and peri-operative management characteristics of the participants, as well as details of the prophylactic study antibiotic administrations, will be summarized by group. Continuous data will be presented with means and standard deviations [SD], or medians and first and third quartiles [Q1, Q3] for skewed data, and categorical data will be presented as frequencies and proportions (see Tables 2, 3, 4, and 5).
Primary outcome analysis
Our hypotheses for the primary analysis are as follows: Null hypothesis: There is no difference in the risk of SSI at 1 year between the two treatment groups. Alternate hypothesis: There is a difference in the risk of SSI at 1 year between the two treatment groups. The primary analysis will be a Cox proportional hazards analysis with time from surgery to the SSI as the primary outcome. Post-operative prophylactic antibiotic duration (treatment group [1 day versus 5 days]) will be the independent variable, and the Cox regression will also include tumor location (femur or tibia) and clinical site as stratification variables. All clinical sites with fewer than five participants enrolled will be collapsed into a single clinical site when included in our regression model. Participants who did not experience the primary endpoint will be censored at 12 months or the time of the last visit. The proportional hazards assumption of the Cox model will be assessed by examining Schoenfeld residuals. If the independent variable does not meet the assumption of the proportional hazards, we will modify the model to allow the HR to differ throughout the study period guided by the observed data. Results will be reported as HRs with the corresponding 95% CI and associated p values. Kaplan-Meier curves will be
constructed for the two randomized treatment groups. For each treatment group, we will also report superficial SSI, deep SSI, and organ space SSI. The results of the
primary analysis will be presented in Table 6.
Secondary outcomes analysis
We will estimate the effect of post-operative prophylactic antibiotic duration (1 day versus 5 days) on antibiotic-related complications, re-operations, oncologic events, and all-cause mortality at 1 year (Table 6).
Similar to the primary analysis, we will perform a Cox proportional hazards analysis. We will only perform Cox regressions for individual antibiotic-related complications, unplanned re-operations, and oncologic and mortality events if there are enough events. Should there be an insufficient number of events, we will summarize by treatment group and report using descriptive statistics (frequencies and proportions). We will also separately report 4-week, 3-month, and 1-year mortality figures for
future comparison with other studies.
In addition, we will also estimate the effect of postoperative prophylactic antibiotic duration on patient functional outcomes (MSTS-87 and MSTS-93 surveys) and quality-of-life (TESS surveys) at 1 year (Table 7). To do so, we will use multiple linear regression models that include the following independent variables: randomized treatment group, tumor location (femur versus tibia), clinical site, and baseline score. The results will be reported as the mean differences with 95% CIs. Given that functional and quality-of-life outcomes are the most difficult to collect and, therefore, we expect some missing data, we will use multiple imputation to address the missing data for these outcomes should the amount of missing data be considerable but not too substantial. Convention dictates that if more than five but less than 40% of data is missing, the use of multiple imputation is appropriate and warranted .
Sensitivity analyses will be performed for the primary outcome only . We will conduct a competing risks analysis that accounts for deaths and amputation as
competing risks. We will also perform sensitivity analyses for center-effects where we will redo the primary analysis without including the clinical site in the model.
We will also look for prognostic imbalances between the two treatment groups based on the following key variables known to be risk factors for a SSI: total operative time, tumor location, diabetes status, chemotherapy regimen, and radiation treatment. We will complete adjusted analyses to address any possible baseline imbalance between the groups.
At the onset of the PARITY trial, we identified two important sub-groups (tumor type and tumor location), which will be reported according to the standard guidelines . As we near the end of the trial, prior to unblinding, we have identified a further three important sub-groups (sex, age, and peri-operative chemotherapy). We will add a main effect for the sub-group variable and the treatment by sub-group interaction to our primary model described above to assess whether the magnitude of the treatment effect is significantly different between the sub-groups (Fig. 3). This will be repeated separately for each sub-group variable. We will perform the following sub-group analyses with the primary endpoint as the outcome (Fig. 3):
1) Tumor type—the type of tumor will be classified as follows: bone sarcoma, soft tissue sarcoma, or oligometastatic bone disease. We hypothesize that there will be no difference between the tumor types with regard to the association between prophylactic antibiotic duration and risk of infection.
2) Tumor location—the location of the tumor will be classified as follows: femur or tibia (we will not include the stratification variable of tumor location in this analysis). We hypothesize that a long duration (5 days) of prophylactic antibiotics will be more effective relative to a short duration (1 day) in tibial reconstructions than in femoral
3) Sex—sex will be classified as follows: male or female. We hypothesize that there will be no difference between the sexes with regard to the association between prophylactic antibiotic duration and risk of infection.
4) Age—age will be classified as follows: pediatric and young adults (12–30 years of age) or older adults (≥ 31 years of age). We hypothesize that a long duration (5 days) of prophylactic antibiotics will be more effective relative to a short duration (1 day) in the older adult population than in the pediatric and young adult population.
5) Peri-operative chemotherapy—peri-operative chemotherapy will be classified as follows: no chemotherapy or chemotherapy (neoadjuvant or adjuvant or a combination of the two). We hypothesize that a long duration (5 days) of prophylactic antibiotics will be more effective relative to a short duration (1 day) in patients who received chemotherapy than in those who did not receive chemotherapy.
Rather than pre-specifying a threshold p value for making a sub-group claim, we will use the approach suggested by Sun et al. to consider the plausibility of any possible subgroup effects . If a plausible sub-group effect is found, we will further explore the impact of the sub-group on the secondary outcomes. However, due to inadequate sample size and power to conduct the sub-group analyses, these results will be used solely for the generation of hypotheses for further investigations.
No interim analyses are planned due to our desire to avoid spuriously inflated estimates of treatment effects [21, 22]. The PARITY Data and Safety Monitoring Board (DSMB) regularly meets to monitor the study data for participant safety.
Upon trial completion, the primary manuscript with the 1-year follow-up results, whether positive, negative, or neutral, will be submitted for peer review and publication in a top-tier medical journal. The final dataset will be shared through an open access data repository once all analyses are completed.
The PARITY trial began as a pilot of 60 participants in January 2013 . Upon demonstrating study feasibility and securing definitive funding (July 2014), these participants were rolled into the definitive study (N = 600). Recruitment for the definitive study was completed in October 2019, and the final 1-year follow-up data is expected to be completed and collected in December 2020.
CDC: Centers for Disease Control and Prevention; CI: Confidence interval;
CONSORT: Consolidated Standards of Reporting Trials; DSMB: Data and Safety
Monitoring Board; HR: Hazards ratio; ITT: Intention-to-treat;
MSTS: Musculoskeletal Tumor Society; PARITY: Prophylactic Antibiotic
Regimens In Tumor Surgery; RCT: Randomized controlled trial; TESS: Toronto
Extremity Salvage Score; SD: Standard deviation; SSI: Surgical site infection
Full authorship list for the PARITY Investigators:
Steering Committee: Michelle Ghert (Chair, McMaster University), Mohit Bhandari (Co-Chair, McMaster University), Benjamin Deheshi (McMaster University), Gordon Guyatt (McMaster University), Ginger Holt (Vanderbilt University Medical Center), Timothy O’Shea (McMaster University), R. Lor Randall (University of California at Davis Medical Center), Lehana Thabane (McMaster University), Roberto Vélez (Hospital Vall d’Hebron), and Jay Wunder (Mount Sinai Hospital).
Methods Centre: Michelle Ghert [principal investigator]; Patricia Schneider, Victoria Giglio, and Paula McKay [project management]; Sheila Sprague [research methodologist]; Diane Heels-Ansdell [statistical analysis]; and Lisa Buckingham [data management] (McMaster University).
Data and Safety Monitoring Board: Peter Rose (Chair, The Mayo Clinic), Brian Brigman (Duke University Medical Center), and Eleanor Pullenayegum (The Hospital for Sick Children).
Adjudication Committee: Michelle Ghert (Chair, McMaster University), Timothy O’Shea (McMaster University), R. Lor Randall (University of California at Davis Medical Center), and Robert Turcotte (McGill University Health entre).
Participating Clinical Sites:
Juravinski Hospital and Cancer Centre (Hamilton, ON, Canada)—Michelle Ghert (PI), Benjamin Deheshi, and David Wilson
Mount Sinai Hospital (Toronto, ON, Canada)—Peter Ferguson (PI) and Jay Wunder
McGill University Health Centre (Montreal, QC, Canada)—Robert Turcotte (PI) and Krista Goulding
The Ottawa Hospital (Ottawa, ON, Canada)—Joel Werier (PI) and Hesham Abdelbary Vancouver General Hospital (Vancouver, BC, Canada)—Paul Clarkson Hôpital Maisonneuve-Rosemont (Montreal, QC, Canada)—Marc Isler (PI) and Sophie Mottard
CHUQ – L’Hôtel-Dieu de Québec (Québec City, QC, Canada)—Norbert Dion (PI) and Annie Arteau
Vanderbilt University Medical Center (Nashville, TN, USA)—Ginger Holt (PI), Jennifer Halpern, and Herbert Schwartz
Beth Israel Deaconess Medical Center (Boston, MA, USA)—Megan Anderson (PI) and Mark Gebhardt
Boston Children’s Hospital (Boston, MA, USA)—Megan Anderson (PI) and Mark Gebhardt
Huntsman Cancer Institute (Salt Lake City, UT, USA)—Kevin Jones (PI) and R. Lor Randall
Memorial Sloan-Kettering Cancer Center (New York, NY, USA)—John Healey (PI) Hospital Universitario Austral (Buenos Aires, ARG)—Marcos Galli Serra (PI) Royal Adelaide Hospital (Adelaide, SA, AUS)—Mark Clayer (PI)
University of Connecticut Health Center (Farmington, CT, USA)—Adam Lindsay (PI) and Tessa Balach
The Rothman Institute (Philadelphia, PA, USA)—John Abraham (PI) and Scot Brown
Holden Comprehensive Cancer Center (Iowa City, IA, USA)—Benjamin Miller (PI) University of Minnesota Medical Center (Minneapolis, MN, USA)—Edward Cheng (PI)
Wexner Medical Center (Columbus, OH, USA)—Thomas Scharschmidt (PI) and Joel Mayerson
Emory University Orthopedics and Spine Center (Atlanta, GA, USA) – Nickolas Reimer (PI)
Montefiore Medical Center (New York, NY, USA)—David Geller (PI) and Bang Hoang
Stanford University Hospital and Clinics (Palo Alto, CA, USA)—Raffi Avedian (PI) Foothills Medical Centre (Calgary, AB, Canada)—Shannon Puloski (PI) and Michael Monument
Sinai Hospital of Baltimore (Baltimore, MD, USA)—Albert Aboulafia (PI) SUNY Upstate University Hospital (East Syracuse, NY, USA)—Timothy Damron (PI)
Maimonides Medical Center (New York, NY, USA)—Howard Goodman (PI)University of Pittsburgh Medical Center (Pittsburgh, PA, USA)—Kurt Weiss (PI) and Mark Goodman
Massachusetts General Hospital (Boston, MA, USA)—Joseph Schwab (PI)
Franklin Square Medical Center (Baltimore, MD, USA)—Albert Aboulafia (PI)
Albany Medical Center (Albany, NY, USA)—Matthew DiCaprio (PI) and Bradford Palmer
Oregon Health and Science University Hospital (Portland, OR, USA)—Yee-Cheen Duong (PI), Kenneth Gundle, and James Hayden
Johns Hopkins Hospital (Baltimore, MD, USA)—Carol Morris (PI) and Adam Levin
Grey’s Hospital (Pietermaritzburg, ZAF) – Reitze Rodseth (PI) and Leonard Marais Instituto de Ortopedia e Traumatologia da Universidade de São Paulo (São Paulo, BRA)—André Mathias Baptista (PI) and Juan Pablo Zummaraga
Hospital Vall d’Hebron (Barcelona, ESP)—Roberto Vélez (PI) University of California at San Francisco Medical Center (San Francisco, CA, USA)—Rosanna Wustrack (PI), and Richard O’Donnell
Cincinnati Children’s Hospital (Cincinnati, OH, USA)—Joel Sorger (PI) University of Maryland Medical Center (Baltimore, MD, USA)—Daniel Lerman (PI)
University of Florida Health Shands Hospital (Gainesville, FL, USA)—André Spiguel (PI), C. Parker Gibbs, and Mark Scarborough
Leiden University Medical Center (Leiden, NLD)—P.D. Sander Dijkstra (PI) and Michiel van de Sande
All India Institute of Medical Sciences (New Delhi, IND)—Shah Alam Khan (PI) and Venkatesan Sampath Kumar
Medical College of Wisconsin (Milwaukee, WI, USA)—John Neilson (PI) Long Island Jewish Medical Center [Northwell Health] (New Hyde Park, NY, USA)—Howard Goodman (PI)
Dartmouth-Hitchcock Medical Center (Hanover, NH, USA)—Eric Henderson (PI)
Saint Louis University Hospital (St. Louis, MO, USA)—David Greenberg (PI)
University Medical Center Groningen (Groningen, NLD)—Paul Jutte (PI)
The Cleveland Clinic (Cleveland, OH, USA)—Nathan Mesko (PI) and Lukas Nystrom
Children’s Cancer Hospital Egypt (Cairo, EGY)—Ahmed Elghoneimy (PI) Hartford Hospital (Hartford, CT, USA)—Adam Lindsay (PI)
Hospital de Clínicas de Porto Alegre (Porto Alegre, BRA)—Ricardo Becker (PI)
University of Arkansas for Medical Sciences (Little Rock, AR, USA)—Richard Nicholas (PI)
University of California at Los Angeles Medical Center (Los Angeles, CA, USA)—Nicholas Bernthal (PI), Jeffrey Eckhardt, and Francis Hornicek
Medical University Graz (Graz, AUT)—Andreas Leithner (PI) and Marko Bergovec
Singapore General Hospital (Singapore, SNG)—Mann Hong Tan (PI) and Suraya Zainul Abidin
University of California at Davis Medical Center (Sacramento, CA, USA)—Steven Thorpe (PI) and R. Lor Randall
Together, PS and DHA drafted the initial statistical analysis plan. PS drafted the manuscript and incorporated all author edits. DHA critically revised the manuscript for important intellectual content. LT provided important intellectual content to the initial statistical analysis plan and critically revised the manuscript for important intellectual content. MG made substantial contributions to the conception and design of the statistical analysis plan, critically revised the manuscript for important intellectual content, and has greed to be accountable for all aspects of the work. The PARITY Investigators contributed to the design, conduct, and overall data collection for the PARITY trial. All authors have read and approved the final manuscript to be published.
Research grants to conduct this research were received from the following funding sources: Canadian Cancer Society Research Institute (CCSRI) Innovation and Innovation to Impact Grants (PI: M. Ghert), Canadian Institutes of Health Research (CIHR) Operating Grant (PI: M. Ghert, M. Bhandari), Canadian Orthopedic Foundation (COF) J. Édouard Samson Award, Orthopedic Research and Education Foundation/Musculoskeletal Tumor Society (OREF/MSTS) Clinical Research Grant (PI: M. Ghert), and Physicians’ Services Incorporated (PSI) Health Research Grant (PI: M. Ghert). The funding sources had no role in the design or conduct of the study; the collection, management, analysis, or interpretation of the data; or the preparation, review, or approval of the manuscript. Dr. M. Ghert had full access to all of the study data and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Availability of data and materials
The final dataset will be shared through an open access data repository once all analyses are completed.
Ethics approval and consent to participate
Ethics approval, including Informed Consent Form approval, was granted for the Methods Centre at McMaster University by the Hamilton Integrated Research Ethics Board (HiREB No. 12-009), as well as at each participating clinical site as per their local ethics committee.
Consent for publication
The authors declare that they have no competing interests.
1 Department of Surgery, McMaster University, Hamilton, ON L8V 1C3, Canada. 2 Department of Health Research Methods, Evidence and Impact, McMaster University, Hamilton L8S 4L8, ON, Canada. 3 Juravinski Hospital and Cancer Centre, Hamilton Health Sciences, 711 Concession Street, B3 169A, Hamilton L8V 1C3, ON, Canada.
Expressão de neurotrofinas e de seus receptores no osteossarcoma primário.
Expression of neurotrophins and their receptors in primary osteosarcoma.
Bruno Pereira Antunes 1,2; Ricardo Gehrke Becker 1,3; André Tesainer Brunetto 3; Bruno Silveira Pavei 1; Caroline Brunettode Farias 3; Luís Fernandoda Rosa Rivero 4; Julie Francine Cerutti Santos 3; Bruna Medeirosde Oliveira 3; Lauro José Gre Gianin 3,5; Rafael Roesler 3,6,7; Algemir Lunardi Brunetto 3; Fernando Pagnussato 8; Carlos Roberto Galia 1,2.
Objetivo: determinar a expressão de neurotrofinas e seus receptores tirosina quinases em pacientes com osteossarcoma (OS) e sua correlação com desfechos clínicos.
Métodos: biópsias de tumores primários de pacientes com OS tratados em uma única instituição, consecutivamente, entre 2002 e 2015, foram analisados através de imuno-histoquímica para expressão de receptores de tirosina quinase A e B (TrKA e TrKB), fator de crescimento neural (NGF) e fator neurotrófico derivado do cérebro (BDNF). De forma independente, dois patologistas classificaram os marcadores de imuno-histoquímica como negativos (negativos e focais fracos) ou positivos (moderado focal/difuso ou forte focal/difuso).
Resultados: foram analisados dados de 19 pacientes (10 do sexo feminino e 9 do masculino) com mediana de idade de 12 anos (5 a 17,3 anos). Dos tumores, 83,3% estavam localizados em membros inferiores e 63,2% dos pacientes eram metastáticos ao diagnóstico. A sobrevida global em cinco anos foi de 55,3%. BDNF foi positivo em 16 pacientes (84%) e NGF em 14 pacientes (73%). TrKA e TrKB apresentaram coloração positiva em quatro (21,1%) e oito (42,1%) pacientes, respectivamente. A análise de sobrevida não demonstrou diferença significativa entre receptores TrK e neurotrofinas.
Conclusão: amostras de OS primário expressam neurotrofinas e receptores TrK através de imuno-histoquímica. Estudos futuros podem auxiliar na identificação do papel das mesmas na patogênese do OS e determinar se há possível correlação prognóstica.
Descritores: Osteossarcoma. Fatores de Crescimento Neural. Fator Neurotrófico Derivado do Encéfalo. Receptor trkA. Receptor trkB.
Osteossarcoma (OS) é um tumor ósseo maligno encontrado preferencialmente em indivíduos entre dez e 25 anos de idade. Ao diagnóstico, até 30% dos pacientes apresentam metástases, o que é considerado o principal fator relacionado ao prognóstico. O desenvolvimento de
quimioterapia em altas doses aumentou a sobrevida consideravelmente. No entanto, desde o início dos anos 2000, esta opção terapêutica atingiu seu platô. De modo geral, não houve progresso significativo quanto ao tratamento do OS durante este período1-6. Consequentemente, a procura por terapias baseadas no perfil molecular do tumor tem crescido consideravelmente.
Neurotrofinas e seus receptores de tirosina quinase (Trk) são responsáveis pela modulação sináptica do sistema nervoso central. Estudos recentes têm demonstrado em amostras de sarcomas, a expressão do receptor TrkA e do seu ligante, fator de crescimento nervoso (NGF), que
tem sido usado como um potencial marcador de prognóstico e tratamento. O aumento da expressão de NGF pode eventualmente estar associado com o estágio tumoral e risco de metástases em certas neoplasias. A sinalização de TrkA tem também sido descrita como promotora de atividade mitótica e antiapoptótica em osteoblastos de diferentes linhagens celulares8-10.
O receptor de Trk B (TrkB) apresenta afinidade pelo fator neurotrófico derivado do cérebro (BDNF).
O aumento da expressão do BDNF tem demonstrado estar intimamente relacionado à viabilidade tumoral, migração e invasão de tecidos saudáveis em diversas malignidades. Amostras diferentes de leiomiossarcoma uterino humano têm demonstrado expressão de BDNF e TrkB, e o aumento da expressão de TrkB e de seus ligantes nesta neoplasia mesenquimal tem sido associada com resistência a múltiplos agentes quimioterápicos. O aumento da expressão de TrkB e seus ligantes também tem sido associado a desfechos clínicos desfavoráveis em alguns tumores de origem neuroectodérmica11-18.
OS é um tumor ósseo de origem mesenquimal que pode compartilhar algumas características comuns a outros sarcomas e tumores que metastatizam para os ossos. Estudos correlacionando OS com TrkA, TrkB e neurotrofinas (NGF e BDNF) são raros7 e ainda não incluem seres humanos. No presente estudo, analisamos a expressão de TrkA, TrkB e seus ligantes (NGF e BNDF) em amostras de tumores de pacientes com OS.
Pacientes e amostras histológicas
O estudo foi aprovado e registrado pelo Conselho de Revisão institucional do Hospital de Clínicas de Porto Alegre através do Escritório de Pesquisa e Pós-Graduação (IRB no 00000921) com o número de referência 15-0499.
Os participantes elegíveis foram todos os pacientes com OS tratados em uma única instituição de acordo com as diretrizes do Grupo Brasileiro de Tratamento do Osteossarcoma (BOTG) e do Grupo Latino-Americano de Tratamento de Osteossarcoma (GLATO) em dois protocolos de tratamento consecutivos (BOTG V, GCBTO 2006) entre 2002 e 2015. Todos os pacientes assinaram um termo de consentimento livre e esclarecido previamente à sua inclusão nos protocolos BOTG V e GCBTO de 20063,19.
Pacientes sem tratamento prévio, metastáticos ou não, que tiveram suas biópsias analisadas no Departamento de Patologia do Hospital de Clínicas de Porto Alegre, foram incluídos no estudo. Detalhes dos regimes quimioterápicos foram publicados anteriormente3,19. Os critérios de
exclusão foram presença de material insuficiente para a realização dos testes imuno-histoquímicos e não ser incluído no protocolo de tratamento citado.
Todos os pacientes incluídos tiveram suas biópsias analisadas por imuno-histoquímica para os seguintes marcadores tumorais: BNDF, NGF, TrkA e TrkB. Informações médicas e cirúrgicas foram extraídas dos registros médicos dos pacientes. O grau de necrose tumoral foi graduado de acordo com a classificação Huvos-Ayala20.
Todas as amostras foram fixadas em parafina, cortadas em lâminas de 4μm de espessura, incubadas e re-hidratadas em álcool. A recuperação antigênica foi realizada em um forno de micro- ondas. A atividade da peroxidase endógena foi bloqueada pela incubação das lâminas em peróxidode hidrogênio, e os sítios de ligação não específicos foram bloqueados com soro normal. As lâminas foram incubadas com o anticorpo primário, com uma diluição de 1:50, por 12 horas a 4oC, depois foram imunomarcadas com o complexo estreptavidina-biotina-peroxidase (LSAB, Dako) e desenvolvidos com tetracloridrato de diaminobenzidina (DAB Kit, Dako). Os anticorpos primários foram anti-NGF policlonal de coelho (sc-33603; Biotecnologia Santa Cruz), anti-BDNF (sc-20981, Biotecnologia Santa Cruz), TrkB policlonal de camundongo (sc-377218,
Biotecnologia Santa Cruz) e anti-TrkA policlonal de cabra (sc-20539, Biotecnologia Santa Cruz).
As lâminas foram avaliadas independentemente por dois especialistas em patologia cirúrgica e imuno-histoquímica para a expressão de NGF, BDNF, TrkB e TrkA. Em caso de discordância entre os patologistas, as lâminas eram revisadas em conjunto até o consenso ser alcançado. A coloração imuno-histoquímica foi pontuada de acordo com a intensidade em uma escala de 0 a 3, onde 0 indica ausência de coloração (negativa); 1, coloração fraca; 2, coloração moderada; e 3, coloração forte. Com relação à porcentagem de células imuno positivas, 1 indicava menos de 10% das células coradas (focal) e 2 indicava mais de 10% das células coradas (difusas). Os pacientes foram divididos em dois grupos: negativo e focal fraco (negativo); focal moderado/difuso e focal forte/difuso (positivo).
As variáveis foram expressas em frequências absolutas e relativas, com exceção da idade, expressa em média e desvio padrão. As diferenças entre os grupos foram avaliadas pelo teste t de Student para idade e pelo teste exato de Fisher para todas as outras variáveis. O teste log-rank foi utilizado para comparar as curvas de sobrevida global e sobrevida livre de doença, estimadas pelo método de Kaplan-Meier. Um P=0,05 foi considerado significativo. Os dados foram analisados no SPSS versão 18.0 e no Epi Info.
Características e marcadores dos pacientes
De um total de 28 pacientes tratados por OS nesse Serviço, foram excluídos quatro por amostra insuficiente ou indisponível para realização do estudo imuno-histoquímico e cinco que não entraram no protocolo do BOTG V e GCBTO 2006 (por escolha do oncologista, do paciente ou condições clínicas que impossibilitassem realizar o tratamento).
Os pacientes que apresentaram má resposta à quimioterapia e necessitaram trocar a linha de tratamento durante o estudo não foram descartados. Analisamos, portanto, os dados de 19 pacientes (9 homens e 10 mulheres; com uma média de idade de 12 anos) de uma única instituição que foram incluídos nos estudos BOTG V e GCBTO 2006. As características da amostra foram salientadas na tabela 1.
A mediana de idade foi de 12 anos (variação de 5 a 17,3 anos) e o tempo mediano de acompanhamento foi de 2,7 anos (amplitude de variação de 0,6 a 14 anos). Quinze tumores (78,9%) localizavam-se nas extremidades inferiores, dois (10,5%) nas extremidades superiores, um
(5,3%) na pelve e um (5,3%) não teve registro de localização. Doze (63,2%) pacientes eram metastáticos e sete (36,8%) tinham tumor localizado. O principal sítio metastático foi o pulmão (83%), seguido por ossos (17%). A resposta à quimioterapia foi ruim em cinco pacientes,
boa em seis e não registrada em oito pacientes.
Quatro (21,1%) pacientes apresentaram progressão de doença, sendo dois locais e duas recorrências sistêmicas. Dos 19 pacientes, 17 foram operados, dos quais nove (52,9%) submetidos a procedimentos que permitiram a conservação do membro e oito (47,1%) à amputação.
O BDNF foi considerado positivo em 16 (84,2%) pacientes, enquanto o NGF foi positivo em 14 (73,7%) (Figura 1). Por outro lado, TrkA e TrkB foram mais frequentemente negativos do que positivos, apresentando quatro (21,1%) e oito (42,1%) casos de coloração positiva, respectivamente, dentre os 19 pacientes (Figura 2). Houve diferença significativa entre os marcadores positivos e negativos da amostra (P<0,001) (Tabela 2).
A sobrevida global em cinco anos para todos os 19 pacientes foi de 55,3%. A sobrevida acumulada em cinco anos foi de 40% para doença metastática e 60% para doença localizada (Relação de risco [RR]=2,04; P=0,39). Apesar de não significativa, a sobrevida acumulada em cinco
anos foi de apenas 25% (hazard ratio [HR]=3,02) para pacientes com recidiva da doença, sendo de 72,5% para pacientes sem recidiva (P=0,17).
A sobrevida cumulativa de cinco anos foi maior nos pacientes que apresentaram coloração positiva para BDNF e NGF do que para aqueles com coloração negativa (61,3 vs. 33,3%; 66,7 vs. 33,3, respectivamente). A RR para pacientes com coloração positiva para BDNF e NGF foi de 0,51
(intervalo de confiança de 95% [IC 95%]: 0,09- 2,8), 0,69 (IC 95%: 0,13-3,81), respectivamente. No entanto, a sobrevida foi maior em pacientes com coloração negativa para TrkA e TrkB do que para aqueles com coloração positiva (58,2 versus 37,5%; 56,3 versus 54,7%, respectivamente). Não houve diferença significativa entre neurotrofina e receptores Trk na análise de sobrevivência. A RR para pacientes com coloração positiva para TrkA e TrkB foi de 3,48 (IC 95%: 0,62 a 19,70) e 1,84 (IC 95%: 0,37 a 9,20), respectivamente. Tanto o receptor TrkA quanto as características de recorrência foram submetidos à análise ajustada ao risco, pois o valor de P foi menor que 20%; entretanto, nenhum deles apresentou diferenças estatisticamente significantes associadas ao risco de morte (Tabela 3).
Apesar da conhecida afinidade entre TrkA e NGF, os tumores coraram positivamente para ambos os marcadores apenas em três (15,7%) casos, e negativos para ambos os marcadores em quatro (21,1%). Com relação à ligação TrkB-BDNF, os tumores coraram positivamente para ambos
os marcadores em oito (42,1%) casos e negativos para ambos os marcadores em três (15,7%). As combinações entre ligação positiva de TrkA-NGF, ligação negativa de TrkA-NGF, ligação positiva de TrkB-BDNF e ligação negativa de TrkB-BDNF não foram significativamente associadas à sobrevivência.
Este é o primeiro estudo a avaliar a expressão de neurotrofinas e seus receptores TrK em tumores primários de OS em humanos. Nós encontramos coloração positiva por imuno-histoquímica para BDNF, NGF, TrkA e TrkB em 19 amostras de tumores de pacientes com OS tratados
em uma única instituição dentro dos ensaios BOTG V e GCBTO 2006 conduzidos por Petrilli e tal. 3,19. Além disso, o grupo analisado apresentou características semelhantes às relatadas nos estudos BOTG V e GCBTO 2006, com alta prevalência de metástase e taxa de sobrevida de aproximadamente 55%19. As taxas de sobrevida em relação à presença de metástases, recidiva e sexo (masculino), seguiram o padrão e valores aproximados aos já constatados em outros estudos, apesar de não se mostrarem estatisticamente significativos (provavelmente
devido ao pequeno tamanho da amostra)3,19. Da mesma forma, a expressão dos marcadores avaliados por imuno-histoquímica não foi significativamente associada à sobrevida, presença de metástases, recorrência ou resposta à quimioterapia. Porém houve tendência de associação entre sobrevida, recidiva e presença de TrkA positivo (p<0.20).
Embora a ligação TrkA-NGF esteja frequentemente presente no reparo e proliferação do tecido ósseo, estudos que investigam essas proteínas em células de OS são raros. Um estudo em células caninas de OS demonstrou que o bloqueio in vitro da ligação TrkA-NGF pode induzir apoptose e inibir a proliferação celular.
Além disso, os receptores TrkA foram identificados em tumores primários e metástases pulmonares7. Encontramos coloração positiva para TrkA em 21% dos casos e para o seu ligante NGF em 73%. Nosso estudo sugere uma menor sobrevida com expressão positiva de TrkA quando comparada a expressão negativa (37% versus 58% em cinco anos, respectivamente). Ao analisar as amostras de NGF, observou-se maior sobrevida em pacientes sem expressão de NGF (66% versus 33% aos cinco anos, respectivamente). Embora os resultados não sejam estatisticamente significativos, talvez devido ao tamanho da amostra, a ligação de TrkA-NGF pode ter um papel potencial no prognóstico de OS.
A sinalização do TrkB, através do seu ligante BDNF, tem sido relacionada ao prognóstico de Antunes Expressão de neurotrofins e de seus receptores no osteossarcoma primário. 7 Rev Col Bras Cir 46(2):e2094 certas malignidades. No neuroblastoma, a presença de BDNF e TrkB demonstrou maior resistência à quimioterapia e agressividade do tumor local. Da mesma forma, em casos de leiomiossarcoma uterino, ensaios in vitro indicam que a sinalização endógena da via TrkB está associada ao crescimento do tumor. Consequentemente, o TrkB e o BDNF
foram investigados em maior detalhe devido à sua possível relevância como alvos para a terapia antineoplásica17,18,21-28.
No presente estudo, TrkB e BDNF foram expressos em 42% e 84% das amostras de OS, respectivamente. Entre os pacientes positivos para TrkB, 80% tiveram uma baixa taxa de necrose e 25% apresentaram recidiva do tumor. Entre os pacientes negativos para TrkB, apenas 16% tiveram uma baixa taxa de necrose e 18% apresentaram recorrência do OS. Os pacientes positivos para TrkB tendem a mostrar menor sobrevida do que os pacientes com coloração negativa, embora a diferença não tenha sido estatisticamente significativa (P=0,44). Por outro lado, os pacientes com células que expressam BDNF apresentaram maior taxa de necrose, apresentaram menor frequência de metástases e maior sobrevida do que aqueles com coloração negativa (P=0,42). Mais estudos são necessários para investigar se o BDNF-TrkB desempenha um papel na patogênese do OS.
Os dados na literatura sobre a expressão dos receptores de neurotrofina e Trk são muito limitados e, até onde sabemos, somos os primeiros a relatar a expressão desses receptores em amostras de OS em humanos. Nossos achados mostraram uma prevalência considerável desses marcadores. Acreditamos que estudos com amostras maiores e estudos moleculares associados, bem como, a estratificação de pacientes por critérios de morbidade, possam contribuir para um melhor uso desses marcadores em pacientes com OS.
Os autores agradecem ao Instituto do Câncer Infantil (ICI-RS), ao Fundo de Pesquisa e Promoção de Eventos (FIPE/HCPA) e ao Hospital de Clínicas de Porto Alegre (HCPA) pelo apoio contínuo.
Objective: to determine the expression of neurotrophins and their tyrosine-kinase receptors in patients with osteosarcoma (OS) and their correlation with clinical outcomes.
Methods: we applied immunohistochemistry to biopsy specimens of patients consecutively treated for primary OS at a single institution between 2002 and 2015, analyzing them for expression receptors of tyrosine kinase A and B (TrKA and TrKB), neural growth factor (NGF) and brain derived neurotrophic factor (BDNF). Independently, two pathologists classified the immunohistochemical markers as negative (negative or weak focal) or positive (moderate focal/diffuse or strong focal/diffuse).
Results: we analyzed data from 19 patients (10 females and 9 males), with median age of 12 years (5 to 17.3). Tumors’ location were 83.3% in the lower limbs, and 63.2% of patients had metastases at diagnosis. Five-year overall survival was 55.3%. BDNF was positive in 16 patients (84%) and NGF in 14 (73%). TrKA and TrKB presented positive staining in four (21,1%) and eight (42,1%) patients, respectively. Survival analysis showed no significant difference between TrK receptors and neurotrophins.
Conclusion: primary OS samples express neurotrophins and TrK receptors by immunohistochemistry. Future studies should explore their role in OS pathogenesis and determine their prognostic significance in larger cohorts.
1. Sarman H, Bayram R, Benek SB. Anticancer drugs with chemotherapeutic interactions with thymoquinone in osteosarcoma cells. Eur Rev Med Pharmacol Sci. 2016;20(7):1263-70.
2. Robl B, Botter SM, Pellegrini G, Neklyudova O, Fuchs B. Evaluation of intraarterial and intravenous cisplatin chemotherapy in the treatment of metastatic osteosarcoma using an orthotopic xenograft mouse model. J Exp Clin Cancer Res. 2016;35(1):1-14.
3. Petrilli AS, de Camargo B, Filho VO, Bruniera P, Brunetto AL, Jesus-Garcia R, Camargo OP, Pena W, Péricles P, Davi A, Prospero JD, Alves MT, Oliveira CR, Macedo CR, Mendes WL, Almeida MT, Borsato ML, dos Santos TM, Ortega J, Consentino E; Brazilian Osteosarcoma Treatment Group Studies III and IV. Results of the Brazilian Osteosarcoma Treatment Group Studies III and IV: prognostic factors and impact on survival. J Clin Oncol. 2006;24(7):1161-8.
4. Liu MH, Cui YH, Guo QN, Zhou Y. Elevated ASCL2 expression is associated with metastasis of osteosarcoma and predicts poor prognosis of the patients. Am J Cancer Res. 2016;6(6):1431-40.
5. Ram Kumar RM, Boro A, Fuchs B. Involvement and clinical aspects of MicroRNA in osteosarcoma. Int J Mol Sci. 2016;17(6):877.
6. Ding L, Congwei L, Bei Q, Tao Y, Ruiguo W, Heze Y, et al. mTOR: an attractive therapeutic target for osteosarcoma? Oncotarget. 2016;7(31):50805-13.
7. Becker RG, Galia CR, Morini S, Viana CR. Immunohistochemical expression of vegf and her-2 proteins in osteosarcoma biopsies. Acta Ortop Bras. 2013;21(4):233-8.
8. Aubert L, Guilbert M, Corbet C, Génot E, Adriaenssens E, Chassat T, et al. NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib. Oncotarget. 2015;6(12):9807-19.
9. Yue XJ, Xu LB, Zhu MS, Zhang R, Liu C. Over- expression of nerve growth factor-ß in human cholangiocarcinoma QBC939 cells promote tumor progression. PLoS One. 2013;8(4):e62024.
10. Astolfi A, Nanni P, Landuzzi L, Ricci C, Nicoletti G, Rossi I, et al. An anti-apoptotic role for NGF receptors in human rhabdomyosarcoma. Eur J Cancer. 2001;37(13):1719-25.
11. Cameron HL, Foster WG. Developmental and lactational exposure to dieldrin alters mammary tumorigenesis in Her2/neu transgenic mice. PLoS One. 2009;4(1):e4303.
12. Shi J. Regulatory networks between neurotrophins and miRNAs in brain diseases and cancers. Acta Pharmacol Sin. 2015;36(2):149-57.
13. Pinski J, Weeraratna A, Uzgare AR, Arnold JT, Denmeade SR, Isaacs JT. Trk receptor inhibition induces apoptosis of proliferating but not quiescent human osteoblasts. Cancer Res. 2002;62(4):986-9.
14. Jin W, Yun C, Kim HS, Kim SJ. TrkC binds to the bone morphogenetic protein type II receptor to suppress bone morphogenetic protein signaling. Cancer Res. 2007;67(20):9869-77.
15. Martens LK, Kirschner KM, Warnecke C, Scholz H. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene. J Biol Chem. 2007;282(19):14379-88.
16. Lin CY, Chen HJ, Li TM, Fong YC, Liu SC, Chen PC, et al. ß5 integrin up-regulation in brain-derived neurotrophic factor promotes cell motility in human chondrosarcoma. PLoS One. 2013;8(7):e67990.
17. Makino K, Kawamura K, Sato W, Kawamura N, Fujimoto T, Terada Y. Inhibition of uterine sarcoma cell growth through suppression of endogenous tyrosine kinase B signaling. PLoS One. 2012;7(7):e41049.
18. Heinen TE, Dos Santos RP, da Rocha A, Dos Santos MP, Lopez PL, Silva Filho MA, et al. Trk inhibition reduces cell proliferation and potentiates the effects of chemotherapeutic agents in Ewing sarcoma. Oncotarget. 2016;7(23):34860-80.
19. Petrilli AS, Brunetto AL, Cypriano Mdos S, Ferraro AA, Donato Macedo CR, Senerchia AA, Almeida MT, Costa CM, Lustosa D, Borsato ML, Calheiros LM, Barreto JH, Epelman S, Carvalho E, Alves MT, Petrilli Mde T, Penna V, Pericles P, de Camargo OP, Garcia- Filho On Behalf Of The Brazilian Osteosarcoma Treatment Group RJ. Fifteen years’ experience of the Brazilian Osteosarcoma Treatment Group (BOTG): a contribution from an emerging country. J Adolesc Young Adult Oncol. 2013;2(4):145-52.
20. Huvos AG. Bone tumors: diagnosis, treatment, and prognosis. 2nd ed. Philadelphia: WB Saunders; 1991.
21. Eggert A, Grotzer MA, Ikegaki N, Zhao H, Cnaan A, Brodeur GM, et al. Expression of the neurotrophin receptor TrkB is associated with unfavorable outcome in Wilms’ tumor. J Clin Oncol. 2001;19(3):689-96. Antunes Expressão de neurotrofinas e de seus receptores no osteossarcoma primário. 9 Rev Col Bras Cir 46(2):e2094
22. Zhang Y, Fujiwara Y, Doki Y, Takiguchi S, Yasuda T, Miyata H, et al. Overexpression of tyrosine kinase B protein as a predictor for distant metastases and prognosis in gastric carcinoma. Oncology. 2008;75(1-2):17-26.
23. Desmet CJ, Peeper DS. The neurotrophic receptor TrkB: a drug target in anti-cancer therapy? Cell Mol Life Sci. 2006;63(7-8):755-9.
24. Ho R, Eggert A, Hishiki T, Minturn JE, Ikegaki N, Foster P, et al. Resistance to chemotherapy mediated by TrkB in neuroblastomas. Cancer Res. 2002;62(22):6462-6.
25. Jaboin J, Kim CJ, Kaplan DR, Thiele CJ. Brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy- induced apoptosis via phosphatidylinositol 3’-kinase pathway. Cancer Res. 2002;62(22):6756-63.
26. Li Z, Jaboin J, Dennis PA, Thiele CJ. Genetic and pharmacologic identification of Akt as a mediator of brain-derived neurotrophic factor/TrkB rescue of neuroblastoma cells from chemotherapy-induced cell death. Cancer Res. 2005;65(6):2070-5.
27. Matsumoto K, Wada RK, Yamashiro JM, Kaplan DR, Thiele CJ. Expression of brain-derived neurotrophicfactor and p145TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells. Cancer Res. 1995;55(8):1798-806.
28. Nakagawara A, Azar CG, Scavarda NJ, Brodeur GM. Expression and function of TRK-B and BDNF in human neuroblastomas. Mol Cell Biol. 1994;14(1):759-67.
What is the impact of local control in Ewing sarcoma: analysis of the first Brazilian collaborative study group – EWING1
Ricardo G. Becker¹*, Lauro J. Gregianin²,³, Carlos R. Galia⁴, Reynaldo Jesus-Garcia Filho⁵, Eduardo A. Toller⁶,
Gerardo Badell⁷, Suely A. Nakagawa⁸, Alexandre David⁹, André M. Baptista¹⁰, Eduardo S. Yonamime¹¹, Osvaldo A. Serafini¹², Valter Penna¹³, Julie Francine C. Santos¹⁴, Algemir L. Brunetto¹⁵, and On behalf of the Brazilian Collaborative Study Group of Ewing Family of Tumors – EWING1 and the Brazilian Society of Pediatric Oncology – SOBOPE
Background: Relapse in localized Ewing sarcoma patients has been a matter of concern regarding poor prognosis. Therefore, we investigated the impact of local control modality (surgery, surgery plus radiotherapy, and radiotherapy) on clinical outcomes such as survival and recurrence in patients with non-metastatic Ewing sarcoma treated on the first Brazilian Collaborative Group Trial of the Ewing Family of Tumors (EWING1).
Methods: Seventy-three patients with localized Ewing sarcoma of bone aged < 30 years were included. The treating physicians defined the modality of local control based on the recommendations of the coordinating center and the patient and tumor characteristics. Possible associations of local control modality with local failure (LF), disease-free survival (DFS), event-free survival (EFS), overall survival (OS), and clinical characteristics were analyzed.
Results: Mean patient age was 12.8 years (range, 2 to 25 years) and median follow-up time was 4.5 years (range, 2. 3 to 6.7 years). Forty-seven patients underwent surgery, 13 received radiotherapy, and 13 received both. The 5-year EFS, OS, and DFS for all patients was 62.1%, 63.3%, and 73.1%, respectively. The 5-year cumulative incidence (CI) of LF was 7.6% for surgery, 11.1% for radiotherapy, and 0% for postoperative radiotherapy (PORT) (p = 0.61). The 5-year EFS was 71.7% for surgery, 30.8% for radiotherapy, and 64.1% for PORT (p = 0.009).
Conclusions: There was a significant effect of local control modality on EFS and OS in the study. Surgery and PORT modalities yielded very close results. The group treated with radiotherapy alone had considerably worse outcomes. This may be confounded by greater risk factors in these patients. There was no significant effect of local control modality on the CI of LF and DFS.
Keywords: Ewing sarcoma, Local control, Radiation oncology, Surgery, Bone tumors, Orthopedics.
Ewing sarcoma (ES) is a small round cell malignancy of bone and soft tissue that usually occurs in individuals aged 5 to 20 years. Five-year overall survival (OS) for patients with localized disease ranges from 65 to 75%, while disease relapse after local control reduces survival to less than 25% [1–8]. Multicenter trials have demonstrated the importance of aggressive chemotherapy treatment and local control of the primary tumor. Successful local control rates have improved to 74–93% with the introduction of a multidisciplinary and collaborative approach [9–12].
Current ES treatment includes induction chemotherapy, local control of the primary tumor, and consolidation chemotherapy. Surgery alone or in combination with radiation has traditionally been considered a good choice for resectable ES, while most unresectable tumors have been treated with radiation alone. However, recent studies have reported worse local recurrence and survival rates in patients treated with radiotherapy alone compared to surgery and postoperative radiotherapy (PORT). These findings have been associated with risk factors that are present in irradiated patients [12–19].
For the first time in Brazil, data on local control of ES were analyzed within a single multicenter protocol. We used a cohort of patients with localized ES treated on the EWING1 trial (first Brazilian Collaborative Group Trial for treatment of Ewing sarcoma family of tumors [ESFT])  to evaluate different local control strategies and their association with risk factors, relapse, and survival.
The study was approved by the institutional review board of Hospital de Clínicas de Porto Alegre through the Office of Research and Graduate Studies (IRB No. 00000921). All patients signed an informed consent form prior to their inclusion in the EWING1 trial from 2003 to 2010 (original trial, IRB No. 03363, date: October 15, 2003). Patients with localized ES of bone treated between 2003 and 2010 according to the EWING1 trial were eligible for the study. Patients were allocated to low-risk (LRG) or high-risk (HRG) groups, where high-risk patients were defined as those with unresectable tumors, tumors of the pelvis, and lactate dehydrogenase (LDH) levels ≥ 1.5 times the upper limit of normal (x ULN). Tumor size was assessed on magnetic resonance imaging (MRI) and computed tomography (CT) scans before starting induction chemotherapy and categorized into ≤ 8 cm (small tumors) and > 8 cm (large tumors). Chemotherapy response was defined as good or poor according to the necrosis index (> 95% or ≤ 95%, respectively) [21, 22].
Patients were treated at 15 centers located in 6 states in Brazil, and one in Uruguay. Each center’s institutional review board approved the treatment protocols, and written informed consent was obtained for all patients at enrollment.
In the EWING1 trial, the induction chemotherapy consisted of two courses of ifosfamide/carboplatin/etoposide (ICE) and two courses of vincristine/doxorubicin/cyclophosphamide (VDC), followed by local control. After local treatment, LRG patients received 10 additional alternating cycles of ifosfamide/etoposide (IE) with VDC, while HRG patients received two additional cycles of ICE at the end of the consolidation therapy. Details of the treatment plan and timing of local control have been published previously .
Local control modality was defined based on the experience of treating physicians within each participating institution; however, the coordinating center established some criteria based on the patient and tumor characteristics to standardize the choice of local control. Patients with tumors that were amenable to resection with adequate margins, regardless of size, response to chemotherapy, or location, should be treated surgically. Cases with positive surgical margins, in which wide resection was not possible due to high morbidity, should receive
PORT. The dose of PORT was defined as 45 Gy for marginal resections and 55.8 Gy for intralesional resections. The presence of necrotic tissue, even in the absence of viable ES cells, was considered incomplete resection and treated with 55.8 Gy. Patients with tumors of the ribs, with a pleural effusion contiguous to a primary lesion, should also receive PORT.
Definitive radiation was given to patients when wide resection could cause high morbidity or mutilation, and in unresectable tumors. Radiation was planned according to the X-ray, CT, and MRI when available. Radiotherapy was delivered to the original tumor volume with a 2-cm margin and a total dose of 55.8 Gy at 1.8 Gy/fraction started during week 11. At the end of treatment, it was established that patients would be followed up every 3 months during the first 2 years, then every 6 months for 5 years, and annually thereafter.
Recurrence was classified as local or systemic. For analysis purposes, any local recurrence was defined as local failure (LF) and systemic recurrence as distant failure (DF). Combined recurrences were included in the systemic group. The classification of the local control modality received by each patient was determined according to all interventions performed at the local tumor site up to and including the start of consolidation therapy. Local control was classified into one of three procedures: surgery, radiotherapy, or surgery plus radiotherapy. Overall survival (OS), event-free survival (EFS), and diseasefree survival (DFS) were defined as the time from the end of all local control measures until a respective event occurs or last patient contact, at which time the patient was censored. Patients who experienced disease progression, second malignant neoplasm, or death were scored as having experienced an event.
The outcome measures were OS, EFS, DFS, and cumulative incidence (CI) of LF and DF timed from the completion of local control therapy, as calculated by the Kaplan-Meier method. The CI of each type of event was calculated for each method of local control and compared by the log-rank test. Associations between categorical variables were analyzed using Pearson’s chi-square test. The Mann-Whitney test was used to compare medians for radiation dose. The association between local control modality and event risk was analyzed using univariate and multivariate Cox proportional-hazards regression models. The hazard ratio (HR) and 95% confidence interval (95% CI) were used as the measure of effect.
Patients selection and characteristics
Data from 73 patients (45 males and 28 females, mean age of 12.8 years) with localized bone disease submitted to local control were selected from a total of 175 patients (96 with localized bone and extraosseous ES and 79 with metastatic bone and extraosseous ES) of the EWING1 trial. The median follow-up time of patients in this study was 4.5 years (range, 2.3 to 6.7 years). Forty-three tumors (58.9%) were located in the extremities, 10 (13.7%) in the pelvis, 10 (13.7%) in the chest wall, 6 (8.2%) in the spine, and 4 (5.5%) in other sites (p > 0.001). Thirty-eight
(52.1%) patients were allocated as LRG and 35 (47.9%) patients as HRG (p < 0.001). Pelvic tumors were relatively more likely to receive radiotherapy than surgery alone. On the other hand, non-pelvic tumors were more frequently treated with surgery (p = 0.012). Tumor size ≤ 8 cm vs > 8 cm was not significantly associated with the local control modality performed (p = 0.12). The response to chemotherapy was poor (necrosis index ≤ 95%) in 56% and good (> 95%) in 44% of patients. Of 68 patients with complete LDH records, only 15 (22%) had LDH ≥ 1.5 x
ULN and were more likely to have a surgical procedure (66.6%) than radiotherapy alone (33.3%) (p = 0.05). The median radiation dose was 50.4 Gy for both groups (range, 45.0 to 55.9 Gy).
Of 43 patients with tumors of the extremities, almost all underwent surgical treatment (n = 41, 95.4%), while only 2 (4.6%) received radiotherapy alone. Of 16 patients with tumors of the pelvis and spine, only 6 (37.5%) underwent surgery, while 10 (62.5%) received radiotherapy alone (p < 0.001) (Table 1).
The estimated 5-year EFS, OS, and DFS for all 73 patients was 62.1%, 63.3%, and 73.1%, respectively. The 5-year CI of LF and DF was 6.9% and 14.7%, respectively. Sixty-eight patients had complete information on local or distant recurrence. Only 4 had isolated LF, and 11 had DF combined or not with LF (Table 2; Figs. 1, 2, and 3).
The 5-year EFS was not statistically different according to tumor size ≤ 8 cm vs > 8 cm at presentation (61.1% vs 58.1%, HR = 1.07; P = 0.89), pelvic location (41.1% vs 66.7%, HR = 1.47; p = 0.44), LDH levels < 1.5 vs ≥ 1.5 x ULN (63.1% vs 51.3%, HR = 1.11; p = 0.83), or radiation dose (HR = 0.99; p = 0.56). LRG and HRG patients had EFS rates of 73.7% and 48.2% and LF rates of 5.6% and 8.3%, respectively (p = 0.16) (Table 2).
On multivariate analysis, definitive radiotherapy, age > 15 years and HRG were not associated with a higher risk of any event (Table 3);
Local control analysis
The 5-year EFS was 30.8% for patients submitted to definitive radiotherapy (13 patients), 64.1% for surgery plus
radiotherapy (13 patients), and 71.7% for surgery alone (47 patients) (p = 0.009). There was no significant difference
in LF rates by local control modality (p = 0.61), and the LF rates were the same at 2 and 5 years of follow-up:
7.6% in the surgery group, 11% in the radiotherapy group, and 0% in the PORT group (p = 0.62). Considering
all 15 patients with local or systemic recurrence, the CI of LF and DF at both 2 and 5 years was 11% for radiotherapy alone, 16.7% for surgery plus radiotherapy, and 25% for surgery alone (p = 0.64). The local disease control rate was 78%.
Small round cells tumors such as ES are usually good responders to irradiation. Consequently, radiotherapy has been an important option for local control either alone or with surgery. However, radiotherapy is not free from complications at the primary tumor sites. Soft tissue fibrosis, osteonecrosis, impaired long-bone growth, secondary malignancies, and up to 35% rate of local recurrence have been related to high-dose irradiation [5, 9–13, 23].
On the other hand, development of orthopedic endoprostheses has enabled surgeons to perform non-mutilating procedures with adequate margins in ES patients. Continuous advances have introduced structural auto and allografts in surgical reconstructions, thus offering more biological treatment options. Therefore, amputation has become extremely infrequent in ES [24, 25].
The presence of marginal or contaminated margins is still the main indication for PORT in the treatment of ES. Conversely, PORT has been routinely used in patients with poor response to chemotherapy as well as in large volume tumors in European centers. The current consensus on the type of local treatment of ES follows criteria based on the patient and tumor characteristics and, not less important, on the level of experience of treating physicians [12–19].
The heterogeneity of clinical factors may be a source of confusion when following the guidelines for local treatment in ES [6, 26]. Yock et al. evaluated the impact of the local control modality for localized ES in a non-randomized study including 75 patients with pelvic bone disease. There was no difference in recurrence rates or survival between the different local control methods. However, patients with larger tumors were more likely to receive combined surgery plus radiotherapy (p = 0.013) . Similarly, in the EWING1 trial, there was no difference in recurrence rate
(LF) between the different treatment modalities, and larger tumors were more likely to receive surgery and PORT than radiotherapy (p = 0.12). Nevertheless, we believe that the limited size of the sample and the inability to control for confounding factors may be reflected in the results.
Surgery is reserved for situations in which the tumor can be resected with adequate margins, that is, with no evidence of residual disease. Although based on observational studies, local recurrence and survival have shown better results in patients submitted to neoadjuvant chemotherapy and surgery compared to patients submitted to neoadjuvant chemotherapy and radiotherapy [16, 27, 28]. DuBois et al. analyzed using propensity scores the risk of LF and survival in 465 patients with localized ES of bone and found that radiotherapy had a higher
risk of local recurrence and death than surgery alone . In the EWING1 trial, radiation therapy showed worse results in terms of EFS (p = 0.009) than surgery and PORT. These findings should be analyzed with caution because 70% (9/13) of the patients subjected to radiation had unresectable tumors; 10 patients had tumors located in the spine and pelvis and 3 developed secondary myeloproliferative neoplasms at the beginning of the follow-up period. Due to the small number of local recurrences (n = 4), there was no significant difference
in LF rates by local control modality.
Several studies included only patients with pelvic ES to investigate possible associations between local control modality and treatment failure [19, 29–31]. Raciborska et al. found that survival was higher in patients treated with surgery and PORT than in those treated with radiotherapy alone (81% and 78% vs 36% at 3 years, respectively) . In the present study, 10 patients had pelvic tumors, and 50% of these patients were treated with definitive radiotherapy (p = 0.012). As expected, survival was considerably lower in patients with pelvic compared to non-pelvic tumors (41.1% vs 66.7%, p = 0.44). There was no difference in the incidence of LF and survival between the different local control measures in the pelvis.
Nowadays, definitive radiation is an almost exclusive indication for unresectable tumors and for patients with poorer prognosis for whom surgical procedures may be exceptionally mutilating. Advances in radiation technology and multidisciplinary approach have enhanced local control and decreased complications in healthy tissues surrounding tumors. Studies analyzing the use of radiation alone reported 5-year local control rates ranging from 53 to 86% with doses between 45 and 65 Gy [9–11, 26, 32, 33].
The EWING1 trial demonstrated that most patients with unresectable tumors and tumors located in the spine and pelvis were treated with definitive radiotherapy. Considerably worse results were obtained in patients treated with radiotherapy alone than in those treated with surgery and PORT. This may be due to high disease morbidity, suboptimal local control with radiotherapy alone, or a combination of these and other factors. The differing clinical characteristics of the radiotherapy group precluded a perfectly reliable comparison between the different local treatment modalities.
Moreover, EWING1’s sample was characterized by patients with many risk factors associated with poor prognosis. Forty-eight percent were in the HRG, and more than half had tumors >8 cm and were poorer responders to chemotherapy.
These worse characteristics suggest a delay in ES diagnosis probably related to social and economic issues from a developing country. Furthermore, higher resistance to chemotherapy could be related to both larger tumors and a specific resistance profile of the patients. Despite all this, for 73 patients included in the current study, the remission rate was 78%.
In summary, we observed similar results to those published by large international cooperative groups [5, 16, 19, 34]. Every effort made to provide training to local investigators, gather data, and monitor the progress of the first Brazilian protocol for ES has allowed us to describe the different local control strategies used in the treatment of ES in a country of continental size like Brazil. The great economic, cultural and social diversity of patients as well as the different levels of knowledge of health professionals on the topic make clear the importance of a collaborative
approach for a study of this magnitude.
The EWING1 trial found no significant difference in local or systemic disease recurrence between different treatment modalities. However, regarding survival, there was a significant difference between surgery, radiotherapy, and PORT.
The Brazilian Collaborative Study Group for treatment of ESFT has now been incorporated into the newly formed Latin American Pediatric Oncology Group (GALOP) and a second ESFT study was activated in 2011 . The next step is intended to analyze and report the impact of local control in the second ESFT study.
95% CI: 95% confidence interval; CI: Cumulative incidence; CT: Computed tomography; DF: Distant failure; EFS: Event-free survival; ES: Ewing sarcoma; ESFT: Ewing sarcoma family of tumors; HR: Hazard ratio; HRG: High-risk group; ICE: Ifosfamide, carboplatin, and etoposide; IE: Ifosfamide and etoposide; LDH: Lactate dehydrogenase; LF: Local failure; LRG: Low-risk group; MRI: Magnetic resonance imaging; OS: Overall survival; ULN: Upper limit of normal; VDC: Vincristine, doxorubicin, and cyclophosphamide.
This work was financially supported by the Children’s Cancer Institute and Rafael Accordi Foundation, Porto Alegre, RS, Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
RGB, LJG, RJF, and ALB contributed to the analysis and interpretation of the patient data regarding local control modalities and were involved in drafting the manuscript. CRG and RGB are the heads of the Department of Orthopedic Research and revised the manuscript critically for important intellectual content. JFCS contributed to the acquisition, analysis and interpretation of data. RJF, EAT, GB, SAN, AD, AMB, ESY, OAS, VP, RGB, and LJG contributed to the conception and design of the study and included more than 5 patients from their institutions. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was approved by the institutional review board of Hospital de Clínicas de Porto Alegre through the Office of Research and Graduate Studies (IRB No. 00000921). All patients signed an informed consent form prior to their inclusion in the EWING1 trial from 2003 to 2010.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
¹Service of Orthopedics and Traumatology, Hospital de Clínicas de Porto Alegre (HCPA), Rua Ramiro Barcelos, 2350, Bairro Santa Cecilia, Porto Alegre, RS, 90035-903, Brazil.
²Department of Pediatrics, HCPA, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil.
³Department of Pediatrics, Hospital São Lucas, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
⁴Service of Orthopedics and Traumatology, HCPA, Porto Alegre, RS, Brazil.
⁵Support Group for Children and Adolescents with Cancer (GRAACC), Universidade Federal de São Paulo
(UNIFESP), São Paulo, SP, Brazil.
⁶Fundação Pio XII, Hospital de Câncer Infantojuvenil, Barretos, SP, Brazil.
⁷Centro Hospitalario Pereira Rossell, Montevideo, Uruguay.
⁸Orthopedics Service, Hospital A.C. Camargo Cancer Center, São Paulo, SP, Brazil.
⁹Service of Orthopedics and Traumatology, Santa Casa de Misericórdia de Porto Alegre, Porto Alegre, RS, Brazil.
¹⁰Orthopedic Trauma Institute, Hospital das Clínicas de São Paulo, School of Medicine, Universidade de São Paulo (USP), São Paulo, SP, Brazil.
¹¹Department of Orthopedics and Traumatology, Santa Casa de Misericórdia de São Paulo (HSCSP), São Paulo, SP, Brazil.
¹²Service of Orthopedics and Traumatology, Hospital São Lucas, Pontifícia Universidade Católica do Rio
Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
¹³Hospital das Clínicas de Botucatu, School of Medicine, Universidade Estadual Paulista (UNESP),
Botucatu, SP, Brazil.
¹⁴Instituto do Câncer Infantil, Porto Alegre, RS, Brazil. 15Instituto do Câncer Infantil, Porto Alegre, RS, Brazil.
1. Grier HE. The Ewing family of tumors. Ewing’s sarcoma and primitive neuroectodermal tumors. Pediatr Clin N am. 1997;44:991–1004.
2. Ferrari S, Sundby Hall K, Luksch R, Tienghi A, Wiebe T, Fagioli F, et al. Nonmetastatic Ewing family tumors: high-dose chemotherapy with stem cell rescue in poor responder patients. Results of the Italian sarcoma group/ Scandinavian sarcoma group III protocol. Ann Oncol. 2011;22:1221–7.
3. Gupta AA, Pappo A, Saunders N, Hopyan S, Ferguson P, Wunder J, et al. Clinical outcome of children and adults with localized Ewing sarcoma: impact of chemotherapy dose and timing of local therapy. Cancer. 2010; 116:3189–94.
4. Gaspar N, Hawkins DS, Dirksen U, Lewis IJ, Ferrari S, Le Deley MC, et al. Ewing sarcoma: current management and future approaches through collaboration. J Clin Oncol. 2015;33:3036–46.
5. Grier HE, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, Pritchard DJ, et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing’s sarcoma and primitive neuroectodermal tumor of bone. N Engl J med. 2003;348:694–701.
6. Paulussen M, Craft AW, Lewis I, Hackshaw A, Douglas C, Dunst J, et al. Results of the EICESS-92 study: two randomized trials of Ewing’s sarcoma treatment–cyclophosphamide compared with ifosfamide in standard-risk patients and assessment of benefit of etoposide added to standard treatment in high-risk patients. J Clin Oncol. 2008;26:4385–93.
7. Oberlin O, Rey A, Desfachelles AS, Philip T, Plantaz D, Schmitt C, et al. Impact of high-dose busulfan plus melphalan as consolidation in metastatic Ewing tumors: a study by the Societe Francaise des cancers de l’Enfant. J Clin Oncol. 2006;24:3997–4002.
8. Delattre O, Zucman J, Melot T, Garau XS, Zucker JM, Lenoir GM, et al. The Ewing family of tumors–a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J med. 1994;331:294–9.
9. Burgert EO Jr, Nesbit ME, Garnsey LA, Gehan EA, Herrmann J, Vietti TJ, et al. Multimodal therapy for the management of nonpelvic, localized Ewing’s sarcoma of bone: intergroup study IESS-II. J Clin Oncol. 1990;8:1514–24.
10. Nesbit ME Jr, Gehan EA, Burgert EO Jr, Vietti TJ, Cangir A, Tefft M, et al. Multimodal therapy for the management of primary, nonmetastatic Ewing’s sarcoma of bone: a long-term follow-up of the first intergroup study. J Clin Oncol. 1990;8:1664–74.
11. Craft A, Cotterill S, Malcolm A, Spooner D, Grimer R, Souhami R, et al. Ifosfamide-containing chemotherapy in Ewing’s sarcoma: the second United Kingdom Children’s Cancer study group and the Medical Research Council Ewing’s tumor study. J Clin Oncol. 1998;16:3628–33.
12. Schuck A, Ahrens S, Paulussen M, Kuhlen M, Konemann S, Rube C, et al. Local therapy in localized Ewing tumors: results of 1058 patients treated in the CESS 81, CESS 86, and EICESS 92 trials. Int J Radiat Oncol Biol Phys. 2003;55:168–77.
13. DuBois SG, Krailo MD, Gebhardt MC, Donaldson SS, Marcus KJ, Dormans J, et al. Comparative evaluation of local control strategies in localized Ewing sarcoma of bone: a report from the Children’s oncology group. Cancer. 2015;121:467–75.
14. Foulon S, Brennan B, Gaspar N, Dirksen U, Jeys L, Cassoni A, et al. Can postoperative radiotherapy be omitted in localised standard-risk Ewing sarcoma? An observational study of the euro-E.W.I.N.G group. Eur J Cancer. 2016;61:128–36.
15. Bacci G, Forni C, Longhi A, Ferrari S, Donati D, De Paolis M, et al. Long-term outcome for patients with non-metastatic Ewing’s sarcoma treated with adjuvant and neoadjuvant chemotherapies. 402 patients treated at Rizzoli between 1972 and 1992. Eur J Cancer. 2004;40:73–83.
16. Shankar AG, Pinkerton CR, Atra A, Ashley S, Lewis I, Spooner D, et al. Local therapy and other factors influencing site of relapse in patients with localised Ewing’s sarcoma. United Kingdom Children’s Cancer study group (UKCCSG). Eur J Cancer. 1999;35:1698–704.
17. Donaldson SS. Ewing sarcoma: radiation dose and target volume. Pediatr Blood Cancer. 2004;42:471–6.
18. Hosalkar HS, Dormans JP. Limb sparing surgery for pediatric musculoskeletal tumors. Pediatr Blood Cancer. 2004;42:295–310.
19. Yock TI, Krailo M, Fryer CJ, Donaldson SS, Miser JS, Chen Z, et al. Local control in pelvic Ewing sarcoma: analysis from INT-0091–a report from the Children’s oncology group. J Clin Oncol. 2006;24:3838–43.
20. Brunetto AL, Castillo LA, Petrilli AS, Macedo CD, Boldrini E, Costa C, et al. Carboplatin in the treatment of Ewing sarcoma: results of the first Brazilian collaborative study group for Ewing sarcoma family tumors-EWING1. Pediatr Blood Cancer. 2015;62:1747–53.
21. Krasin MJ, Rodriguez-Galindo C, Davidoff AM, Billups CA, Fuller CE, Neel MD, et al. Efficacy of combined surgery and irradiation for localized Ewing’s sarcoma family of tumors. Pediatr Blood Cancer. 2004;43:229–36.
22. Picci P, Bohling T, Bacci G, Ferrari S, Sangiorgi L, Mercuri M, et al. Chemotherapy-induced tumor necrosis as a prognostic factor in localized Ewing’s sarcoma of the extremities. J Clin Oncol. 1997;15:1553–9.
23. Nesbit ME Jr, Perez CA, Tefft M, Burgert EO Jr, Vietti TJ, Kissane J, et al. Multimodal therapy for the management of primary, nonmetastatic Ewing’s sarcoma of bone: an intergroup study. Natl Cancer Inst Monogr. 1981:255–62.
24. Zahlten-Hinguranage A, Bernd L, Ewerbeck V, Sabo D. Equal quality of life after limb-sparing or ablative surgery for lower extremity sarcomas. Br J Cancer. 2004;91:1012–4.
25. Germain MA, Mascard E, Dubousset J, Nguefack M. Free vascularized fibula and reconstruction of long bones in the child–our evolution. Microsurgery. 2007;27:415–9.
26. Donaldson SS, Torrey M, Link MP, Glicksman A, Gilula L, Laurie F, et al. A multidisciplinary study investigating radiotherapy in Ewing’s sarcoma: end results of POG #8346. Pediatric oncology group. Int J Radiat Oncol Biol Phys. 1998;42:125–35.
27. Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, Grier HE, et al. Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children’s oncology group. J Clin Oncol. 2012;30:4148–54.
28. Bacci G, Ferrari S, Bertoni F, Rimondini S, Longhi A, Bacchini P, et al. Prognostic factors in nonmetastatic Ewing’s sarcoma of bone treated with adjuvant chemotherapy: analysis of 359 patients at the Istituto Ortopedico Rizzoli. J Clin Oncol. 2000;18:4–11.
29. Raciborska A, Bilska K, Rychlowska-Pruszynska M, Drabko K, Chaber R, Pogorzala M, et al. Internal hemipelvectomy in the management of pelvic Ewing sarcoma – are outcomes better than with radiation therapy? J Pediatr Surg. 2014;49:1500–4.
30. Scully SP, Temple HT, O’Keefe RJ, Scarborough MT, Mankin HJ, Gebhardt MC. Role of surgical resection in pelvic Ewing’s sarcoma. J Clin Oncol. 1995; 13:2336–41.
31. Carrie C, Mascard E, Gomez F, Habrand JL, Alapetite C, Oberlin O, et al. Nonmetastatic pelvic Ewing sarcoma: report of the French society of pediatric oncology. Med Pediatr Oncol. 1999;33:444–9.
32. Ahrens S, Hoffmann C, Jabar S, Braun-Munzinger G, Paulussen M, Dunst J, et al. Evaluation of prognostic factors in a tumor volume-adapted treatment strategy for localized Ewing sarcoma of bone: the CESS 86 experience. Cooperative Ewing sarcoma study. Med Pediatr Oncol. 1999;32:186–95.
33. Dunst J, Jurgens H, Sauer R, Pape H, Paulussen M, Winkelmann W, et al. Radiation therapy in Ewing’s sarcoma: an update of the CESS 86 trial. Int J Radiat Oncol Biol Phys. 1995;32:919–30.
34. Dunst J, Sauer R, Burgers JM, Hawliczek R, Kurten R, Winkelmann W, et al. Radiation therapy as local treatment in Ewing’s sarcoma. Results of the cooperative Ewing’s sarcoma studies CESS 81 and CESS 86. Cancer. 1991;67:2818–25.
Tiago Elias Heinen¹ ², Rafael Pereira dos Santos¹ ², Amanda da Rocha¹ ², Michel Pinheiro dos Santos³, Patrícia Luciana da Costa Lopez¹ ², Marco Aurélio Silva Filho¹ ², Bárbara Kunzler Souza¹ ², Luís Fernando da Rosa River⁴, Ricardo Gehrke Becker⁵, Lauro José Gregianin¹ ⁶ ⁸, Algemir Lunardi Brunetto⁷, André Tesainer Brunetto⁷, Caroline Brunetto de Farias¹ ⁷, Rafael Roesler¹ ²
¹Cancer and Neurobiology Laboratory, Experimental Research Center, Clinical Hospital (CPE-HCPA), Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
²Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
³Faculty of Health Sciences, UniRitter Laureate International Universities, Porto Alegre, RS, Brazil
⁴Departament of Pathology, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
⁵Department of Orhopaedics and Traumatology, Clinical Hospital, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
⁶Department of Pediatrics, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
⁷Children’s Cancer Institute (ICI), Porto Alegre, RS, Brazil
⁸Pediatric Oncology Service, Clinical Hospital, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
Correspondence to: Rafael Roesler, e-mail: firstname.lastname@example.org
Keywords: TrkA, TrkB, neurotrophin, neurotrophin receptor, Ewing sarcoma
Received: October 05, 2015 Accepted: April 10, 2016 Published: April 26, 2016
Ewing sarcoma (ES) is a highly aggressive pediatric cancer that may arise from neuronal precursors. Neurotrophins stimulate neuronal devlopment and plasticity. Here, we found that neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as their receptors (TrkA and TrkB, respectively) are expressed in ES tumors. Treatment with TrkA (GW-441756) or TrkB (Ana-12) selective inhibitors decreased ES cell proliferation, and the effect was increased when the two inhibitors were combined. ES cells treated with a panTrk inhibitor,
K252a, showed changes in morphology, reduced levels of β-III tubulin, and decreased mRNA expression of NGF, BDNF, TrkA and TrkB. Furthermore, combining K252a with subeffective doses of cytotoxic chemotherapeutic drugs resulted in a decrease in ES cell proliferation and colony formation, even in chemoresistant cells. These results indicate that Trk inhibition may be an emerging approach for the treatment of ES.
Tumors of the Ewing sarcoma (ES) family are aggressive childhood cancers . ES remains the second most common primary bone malignancy in the pediatric population, with an annual incidence of almost 3 cases per million people in the USA . These tumors are characterized by highly aggressive, small round blue cells of the bone and soft tissue, genetically marked by gene
fusions involving, most commonly, the EWS gene and a gene of the ETS family (primarily FLI-1) [1, 3]. The malignant properties of ES have been attributed to EWS/FLI1 proteins acting as aberrant transcription factors .
Before chemotherapy became available, only about 10% of patients with ES survived . Advances in multimodality therapy, including aggressive neoadjuvant and adjuvant chemotherapy combined with surgery and/or radiation therapy, have improved long-term survival
dramatically, with the 5-year survival of patients with localized disease reaching 70% [3, 5]. Unfortunately, almost 20% of patients have refractory or recurrent disease and approximately one-quarter to one-third present with metastatic disease at diagnosis . Despite many attempts to
intensify treatments, survival remains poor in these patients
Chemotherapy resistance has long been an assiduous challenge for oncologists treating patients with bone sarcomas . Disease recurrence or progression due to treatment resistance of the primary tumor accounted for 60.3% of ES deaths among long-term (≥5-year) survivors in North America who were followed for 20 years posttreatment . However, attempts to attack ES
with a higher chemotherapy dose-intensity have produced great morbidity in patients . Therefore, many recent studies have focused on resolving the mechanisms of ES resistance [9–12].
Elucidation of the mechanisms of ES resistance, however, has been impeded by the elusiveness of the cellular origin of ES. Substantial evidence supports a neural cell origin [13–17], while other evidence supports a mesenchymal stem cell origin [18–20]. Analyzing the expression and function of tropomyosin receptor kinase (Trk) family receptors, which are highly expressed in cells of neural origin , on ES cells may inform the development of targeted ES therapies.
The endogenous ligands for Trks are neurotrophins, secreted proteins that play a major role in the survival, differentiation, and maintenance of neuronal populations . Neurotrophins also mediate physiological actions outside of the nervous system, including regulating cardiac
development, neovascularization, and immune system homeostasis . The four known human neurotrophins — nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) — exert their effects by binding Trk subtypes A, B and C, or binding neurotrophin receptor p75NTR, a member of the tumor necrosis factor receptor superfamily . Trk receptors have been identified as prognostic factors
in pediatric malignancies of diverse origins, including neuroblastoma and medulloblastoma . In addition, recent studies have shown that neurotrophins and their receptors are involved in the proliferation, invasiveness, angiogenesis, and drug resistance in various tumor types [25–29].
The potential involvement of neurotrophin receptors in ES has been suggested [21, 30–35], but remains poorly understood. Here, we verified whether Trk receptor inhibition can display anticancer activities in ES cells.
Neurotrophin and Trk mRNA expression in cell lines and protein content in tumor samples.
Reverse transcriptase polymerase chain reaction (RT-PCR) experiments confirmed detectable levels of mRNA transcripts for both NGF and BDNF, as well as for the TrkA and TrkB receptors in SK-ES-1 and RDES cell lines (Figure 1A, 1B). Subsequent analyses of immunohistochemically labelled NGF, BDNF, TrkA, and TrkB proteins in a set of seven tumor samples from seven patients with ES revealed heterogenous expression of these proteins across tumor samples (Figure 1C–1G).
BDNF was detected in all seven samples, involving, on average, 41.5% of imaged tumor cells. TrkB and NGF proteins were observed on average in 37% and 47% of imaged cells, respectively, in six of the seven samples. TrkA protein was detected in only two samples, in 40% of cells on average. Detailed reporting of the incidence (number of tumors) and distribution (percentage of tumor
cells) of labelling for each antigen according to labeling strength/density are reported in detail in Table 1.
Inhibition of TrkA or TrkB reduce ES cell proliferation
Cell counting after 72-h treatments of RD-ES and SK-ES-1 cells with a range of doses of BDNF and NGF (0.1, 1, 10, 100, 200 ng/ml) revealed no effects on cell proliferation (Figure 2A, 2B). The lack of effect of BDNF and NGF was also observed under quiescent conditions (data not shown). When SK-ES-1 cells were exposed to the selective BDNF inhibitor Ana-12, there was a significant reduction in cell proliferation, relative to controls, at the doses of 5 μM (p < .05), 10 μM (p < .01), and 15 μM (p < .001; IC50 = 23.28 μM) (Figure 2D). Only the 15 μM dose of Ana-12 (p < .05) reduced cell proliferation of RD-ES cells significantly (IC50 = 20.89 μM) (Figure 2C).
The specific TrkA receptor inhibitor GW 441756 reduced proliferation of SK-ES-1 cells at all doses tested [0.1 μM, (p < .01), 1 μM (p < .001), 5 μM (p < .001), 10 μM (p < .001), and 15 μM (p < .001; IC50 = 1.13 μM)] (Figure 2F) and reduced proliferation of RD-ES cells at all but the lowest dose [1 μM (p < 0.05), 5 μM (p < 0.01), 10 μM (p < .001), and 15 μM (p < .001)(IC50 = 1.94 μM)] (Figure 2E). It is noteworthy that the IC50 values were more than ten times greater for the TrkB receptor inhibitor than for the TrkA receptor inhibitor in both cell lines, indicating higher sensitivity to the TrkA receptor inhibitor.
Inhibition was even more pronounced in both cellswith the pan-Trk receptor inhibitor K252a. After 72 h of treatment, SK-ES-1 cell proliferation was decreased, compared to controls, at K252a doses of 100 nM (K100) (p< .001) and 1000 nM (K1000) (p < .001) (IC50 = 61.27 nM) (Figure 2H). In the RD-ES line, reductions in proliferationwere also observed with 100 nM (p < .001) and 1000 nM (p < .001) K252a (IC50 = 48.57 nM) (Figure 2G). K252a exhibited an inhibition potency that was almost 20 times higher than that of the TrkA receptor inhibitor GW 441756, which was the more potent selective inhibitor.
When SK-ES-1R cells were exposed to K252a (Figure 2I–2K), the K100 and K1000 groups had reduced cell proliferation, relative to controls, in cells resistant to Doxo (IC50 = 60.75 nM), VP-16 (IC50 = 48.66 nM), and VCR (IC50 = 66.73 nM)(all p < .001). The results were similar to those obtained in non-resistant cells, demonstrating that sensitivity to Trk receptor inhibition was retained in the chemoresistant cells.
Figure 1: (Continued) Neurotrophin and Trk mRNA expression in cell lines and protein content in tumor samples. A. and B.
Expression of NGF/TrkA and BDNF/TrkB mRNA transcripts in both examined ES cell lines, RD-ES and SK-ES-1. C. Representative example of HE-stained section of ES tumor sample demonstrating the small round blue cells that are characteristic of this tumor. D-G. Representative
photomicrographs of ES sections immunolabeled for BDNF, TrkB, NGF, and TrkA, respectively. Arrows indicate labelled cells.
Figure 2: (Continued) Inhibition of TrkA or TrkB reduces ES cell proliferation. A, B. Cell proliferation after 72-h treatmentwith BDNF or NGF (0.1, 1, 10, 100, and 200 ng/mL) in RD-ES and SK-ES-1 cells (n = 3). C-J. Dose-response study of the TrkB-specific inhibitor Ana-12 (μM) (C, D) the TrkA-specific inhibitor GW 441756 (μM) (E, F) and the pan-Trk inhibitor K252a (nM) G-K. on tumor cell proliferation in human ES RD-ES, SK-ES-1, and SK-ES-1R cell lines. The IC50 for each drug was determined by trypan blue counting assay after 72 h treatments. Cell proliferation was assessed in triplicate, in at least three independent experiments. Effect (fraction affected
vs. control) is plotted on the y-axis versus dose on the x-axis. The linear correlation coefficient r of the median-effect plot was >0.90 for all tested agents, ensuring measurement accuracy and conformity to mass-action. Positive controls (100% cell viability) are denoted as ‘0’ effect on the y-axis. L. Cell counts following combination treatments of Ana-12 with GW 441756 (0.1 and 1 μM, 72 h; n = 3). * vs. control; # vs. respective Ana-12 dose; & vs. respective GW 441756 dose. Single, double, and triple symbols represent p < .05, p < .01, p < .001, respectively.
Combined treatment of Ana-12 and GW 441756 produced more robust inhibition of cell roliferation at 0.1 μM and 1 µM than either inhibitor alone at the same doses in both cell lines (Figure 2L). These results are consistent with the observation of greater effectiveness of the pan-Trk receptor inhibitor K252a compared to selective TrkA and TrkB receptor inhibitors.
SK-ES-1 cells are affected by specific inhibitors of main pathways activated by Trks.
The Trk-activated phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and phospholipase C-gamma (PLCγ)/protein kinase C (PKC) intracellular signaling pathways are involved in vital cell growth and survival processes . As shown in Figure 3, treatment of ES cells with inhibitors of PI3K (LY294002; p < .05), MAPK (UO 126; p < .05), or PLCγ/PKC (Gö 6983; p < .01) for 72 h resulted in significant reductionsin proliferation.
Cell cycle, morphological, and mRNA expression changes in cells treated with K252a.
Flow cytometry cell-cycle analysis after K252a treatment of SK-ES-1 cells for 24 h showed that at
100 nM, but not 1 nM, K252a increased the proportion of G1 cells and decreased the proportion of cells in Sphase. Doxo was used as a positive control (Figure 4A). Morphological changes, with possible neurite extensions, were observed in cells exposed to 1000-nM K252a for 48 h (Figure 4B). Moreover, Trk inhibition led to a decrease in the protein expression of β-III tubulin, a neural
differentiation marker associated with aggressiveness in tumors. K252a at 100 and 1000 nM induced a mean decrease of 18% and 67% respectively in β-III tubulin relative to controls (Figure 4C–4E).
Significant decreases in the mRNA expression of NGF (p < .05), TrkA (p < .01), BDNF (p < .001), and (p < .01) were observed in SK-ES-1 cells treated with 100 nM K252a for 24 h (Figure 4F).
Antitumor effects of citotoxic chemotherapeutic agents in human ES cell lines.
RD-ES, SK-ES-1, and SK-ES-1R cells were exposed to increasing concentrations of standard clinical
chemotherapeutic agents, namely vincristine (VCR) (1–5 nM), etoposide (VP-16) (0.1–0.4 μM), and doxorubicin (Doxo) (10–50 nM), for 72 h and trypan blue counting assays were performed (dose-response curves and IC50 values are shown in Figure 2). SK-ES-1R cells — in which chemoresistance was induced by the stepwise method (see Materials and Methods) — had significantly higher IC50 values (Figure 5C, 5F, and 5I) than non-resistant lines for
all three of these chemotherapeutic agents.
Trk inhibition results in a synergistic enhancement of the antiproliferative effects of chemotherapeutic agents in ES cells.
Addition of K252a to a 72-h treatment with a cytotoxic chemotherapeutic agent (VCR, Doxo, or VP16) resulted in lower cell counts compared to treatments with each chemotherapeutic agent alone (Figure 6). For example, at a 1 nM dose, neither K252a nor VCR affected proliferation significantly. However, when a combined VCR + K252a treatment was used, cell numbers were reduced significantly, with the resultant cell counts being 55% and 25% for SK-ES-1 and RD-ES respectively, of the numbers of cells observed after individual treatments (Table 2).
Figure 3: Specific Trk pathway inhibitors reduce SK-ES-1 cell growth. Cell proliferation, accessed by cell counting (n = 3), was reduced after 72-h treatment with 20 μM LY294002 (PI3K inhibitor; p < .05), UO 126 (MAPK inhibitor p < .05), or Gö 6983 (PLCγ/ PKC inhibitor; p < .01) compared to controls.
Figure 4: (Continued) Analyses of cell cycle, morphological changes, neuronal differentiation, and mRNA expression of cells treated with K252a. A. SK-ES-1 cells were treated with K252a at doses of 1 (K1) and 100 nM (K100) for 24 h. D40 represents 40 nM doxorubicin, positive control. G2-phase and S-phase proportions of cells were increased and decreased, respectively with 100 nM, but not 1 nM, K252a (n =3). B. Image displaying morphological differences in SK-ES-1 cells treated with 1000 nM of K252a for 48 h. C. Trk inhibition decreases expression of the neural differentiation marker β-III tubulin. Protein levels of β-III tubulin were evaluated by immunoblotting (IB). Relative densitometric analyses were normalized by β-actin and corrected based on vehicle controls. K252a at 100 and 1000 nM induced a mean decrease of 18% and 67% respectively in β-III tubulin relative to controls (p < .001; n=3). D. Representative Western blot replicate of β-III tubulin levels after 48h of treatment with K252a. β-actin was used for loading control. E. Morphology of cells treated with 100 or 1000 nM K252a for 48 h. F. The mRNA expression levels of NGF (p < .05), TrkA (p < .01), BDNF (p < .001), and TrkB
(p < .01) were reduced in SK-ES-1 cells treated with 100 nM K252a for 24 h (K) relative to levels in non-treated (NT) control cells (n = 3).
Trk inhibition enhances the antiproliferative effect of chemotherapeutic agents synergistically in ES chemoresistant cells.
The antiproliferative effect of K252a in chemoresistant cells was similar to that seen in nonresistant
cells (Figure 2). Notably, administration of K252a in combination with each of the tested chemotherapeutic agents (VCR, VP-16, and Doxo) also produced a synergistic antiproliferative effect in chemoresistant cells, indicating that Trk receptor inhibition can re-sensitize cells to chemotherapy (Figure 7, Table 3).
Treatment with chemotherapeutics plus K252a co-treatment reduces ES cell colony formation
Analyses of wells plated with cells that had been treated with drugs and allowed to grow for 10–14 days showed that treatments of K252a combined with either Doxo (Figure 8A, 8B) or VCR (Figure 8C, 8D) reduced the number of colonies formed and the total area occupied by colonies in both SK-ES-1 and RD-ES cell lines. Combined treatment of K252a with VP-16 had a reducing effect on the area occupied by colonies, but not colony number (Figure 8F).
In this report, we showed that the pan-Trk inhibitor K252a can change ES cell morphology, leading to decreased expression of NGF, TrkA, BDNF, and TrkB. K252a reduced the proliferation and survival of ES cells, and produced a synergistic effect when used in combination with chemotherapeutic agents at low doses, even in chemoresistant cells.
Conventional cytotoxic chemotherapy is ineffective in a quarter of patients with localized ES, and in threequarters of patients with metastatic disease . First-line therapy for localized disease consists of neoadjuvant chemotherapy, which entails a combination of four to six drugs (e.g VCR, Doxo, VP-16, cyclophosphamide, ifosfamide, and dactinomycin) followed by local interventions with surgery and/or radiotherapy when Figure 4: (Continued) Analyses of cell cycle, morphological changes, neuronal differentiation, and mRNA expression of cells treated with K252a. A. SK-ES-1 cells were treated with K252a at doses of 1 (K1) and 100 nM (K100) for 24 h. D40 represents 40 nM doxorubicin, positive control. G2-phase and S-phase proportions of cells were increased and decreased, respectively with 100 nM, but not 1 nM, K252a (n =3). B. Image displaying orphological differences in SK-ES-1 cells treated with 1000 nM of K252a for 48 h. C. Trk inhibition decreases expression of the neural differentiation marker β-III tubulin. Protein levels of β-III tubulin were evaluated by immunoblotting (IB). Relative densitometric analyses were normalized by β-actin and corrected based on vehicle controls. K252a at 100 and 1000 nM induced a mean decrease of 18% and 67% respectively in β-III tubulin relative to controls (p < .001; n=3). D. Representative
Western blot replicate of β-III tubulin levels after 48h of treatment with K252a. β-actin was used for loading control. E. Morphology of cells treated with 100 or 1000 nM K252a for 48 h. F. The mRNA expression levels of NGF (p < .05), TrkA (p < .01), BDNF (p < .001), and TrkB (p < .01) were reduced in SK-ES-1 cells treated with 100 nM K252a for 24 h (K) relative to levels in non-treated (NT) control cells (n = 3). www.impactjournals.com/oncotarget 9 Oncotarget appropriate. Multimodal treatment can improve overall survival (up to 60–70%) in localized disease ,
however this improvement seems to have plateaued. These therapies are being administered at a maximum tolerated intensity. Therefore, raising cure rates may require a more biologically targeted approach, such as one that enhances the effectiveness of current modalities without worsening
side effects . Furthermore, relapsed/refractory ES remains uniformly fatal and novel approaches are urgently needed to deal with such cases .
Neurotrophins and their receptors play several roles in cancer. Neuroblastoma patients whose tumors have elevated TrkA  or TrkC [40, 41] expression have a better prognosis, than those who do not, whereas those with higher TrkB and BDNF levels have a particularly poor prognosis . TrkB expression is also associated with a bad prognosis in patients diagnosed with Wilms
tumor , but a favorable prognosis in medullary thyroid carcinoma .
Some studies have shown that tumor cells treated with BDNF are less sensitive to cytotoxic drugs [26, 45]. Moreover, neurotrophin signaling pathways may function as endogenous systems that protect neurons after biochemical insults, transient ischemia, or physical injury [45, 46]; in other studies, however, BDNF showed anti-cancer potential . Neither BDNF nor NGF alone affected cell proliferation at any of the doses tested here. It is possible that ligand-independent eurotrophin
receptor signaling occurs in ES. Alternatively, secretion of endogenous BDNF and NGF by the cells may be enough to activate neurotrophin signaling at optimal levels.
When low doses of selective TrkA and TrkB inhibitors were combined, we observed an increase in
antiproliferative effects relative to either inhibitor alone. Also, a similar effect could be reached with nanomolar doses of the pan-Trk receptor inhibitor K252a and was observed in both chemoresistant and non-resistant ES cells. These findings indicate that the combined inhibition of TrkA and TrkB shows higher efficacy compared to inhibiting either receptor alone.
Figure 5: Reduced antitumor effects of VCR (nM) A, B, C. VP-16 (μM) D, E, F. and Doxo (nM) G, H, I. in chemoresistant ES cell line (SK-ES-IR) relative to two non-resistant cell lines. Dose-effect IC50 concentration curves with drug effect (fraction affected vs. control) represented on the y-axis and dose shown on the x-axis. Cell proliferation was assessed with the trypan blue counting assay after 72-h drug treatments in triplicate, in at least three independent series of experiments. Positive controls corresponding to 100% cell viability are denoted as ‘0’ effect on the y-axis. The linear correlation coefficient r of the median-effect plot was >0.90 for all tested agents, indicating that the measurements were accurate and had conformity to mass-action. Doxo = doxorubicin; VCR = vincristine; VP-16 = etoposide.
Figure 6: Trk inhibition enhanced the antiproliferative effects of Doxo, VP-16, and VCR synergistically in ES cells. A, B, C. Proliferation of cells treated with the chemotherapeutic agents VP-16 [0.01 (VP-16 0,01) and 0.1 (VP-16 0,1) μM], VCR [1 (VCR1) and 2 (VCR2) nM], and Doxo [10 (D10) and 20 (D20) nM] alone or in combination with the Trk inhibitor K252a [1 (K1) and 100 (K100) nM]. All combination treatments produced significant decreases compared to controls; some showed further differences versus single treatment. * vs. control; # vs. respective K252a dose; & vs. respective chemotherapeutic dose. Single, double, and triple symbols represent p < .05, p < .01, p < .001, respectively. See Table 2 for additional related data. SK-ES-1 treated with VCR (n = 4), VP-16 (n = 3), Doxo (n = 5). RD-ES treated with VCR (n = 3), VP-16 (n = 3), Doxo (n = 4), where n is number of independent experiments contributing to mean data shown. Doxo = doxorubicin; VCR = vincristine; VP-16 = etoposide.
of TrkA and TrkB shows higher efficacy compared to inhibiting either receptor alone.
Previous studies in other solid tumor types have indicated that blocking either TrkA or TrkB may
produce antitumor effects [29, 36]. For example, Lee and colleagues  showed that colorectal tumors positive for TrkA expression presented NTRK1 rearrengements. In addition, proliferation of NTRK1-rearranged patientderived cells was profoundly inhibited by entrectinib, a pan-TRK inhibitor, and such inhibition was associated with inactivation of TrkA, and down-regulation of
downstream signaling pathways.
Low doses of chemotherapeutics with differing mechanisms of action had which had no effect when given alone, but reduced cell proliferation when used together with K252a. A similar effect was seen in a recent study evaluating the efficacy of combining Doxo with an AXL receptor inhibitor (another tyrosine kinase) , wherein it was suggested that the synergistic effect depends on the dose and drugs used. Importantly, the fact that similar results were obtained with chemoresistant cells in our
study suggests that K252a may be able to subvert general mechanisms of tumor resistance in ES.
Thompson and Levin  showed that the morphology of RGC-5 cells (transformed cells Expressing surface markers characteristic of neuronal precursor cells similar to ES cells) is changed following treatment with 1000 nM K252a. Similar to the present study, they observed neurite extension following treatment. The fact that this observation occurred in both normal and
Figure 7: Trk inhibition enhances the antiproliferative effects of VCR, VP-16 and doxorubicin synergistically in chemoresistant ES cells. A-C. Cells were treated and analyzed as in Figure 6. All adjuvant treatments produced significant reduction in cell proliferation compared to non-treated controls; some differed significant from single treatments. * vs. control; # vs. respective K252a dose; & vs. respective chemotherapeutic dose. Single, double, and triple symbols represent p < .05, p < .01, p < .001, respectively. See Table 3 for additional related data. SK-ES-1R treated with VCR (n = 4), VP-16 (n = 3), Doxo (n = 3), where n is number of independent
experiments contributing to mean data shown. Doxo = doxorubicin; VCR = vincristine; VP-16 = etoposide.
Figure 8: (Continued) K252a co-treatment with Doxo, VP-16, or VCR reduces ES cell colony formation. A, B. K252a and Doxo. C, D. K252a and VCR. E, F. K252a and VP-16. Cells were treated as in Figure 6. Colony count data are shown in (A, C) and (E); colony area data are shown in (B, D) and (F). Mean percentages of three independent experiments are shown. *, **, and *** represent p < .05, p < .01 and p < .001, respectively, vs. control; # , ##, and ### represent p < .05, p < .01, and p < .001, respectively, vs. respective K252a dose; &, &&, and &&& represent p < .05, p < .01, and p < .001, respectively, vs. respective chemotherapeutic dose. G. Representative images from cell survival experiments with SK-ES-1 cells treated with Doxo [10 (D10) and 20 (D20) nM] and K252a [1 (K1) and 100 (K100) nM]. Doxo = doxorubicin; VCR = vincristine; VP-16 = etoposide.
chemoresistant cells could be critical to understanding the synergy between K252a and chemotherapeutic agents in that differentiated cells can become more sensitive. There may be some specificity to ES cells with respect to cell differentiation given that no morphological changes
were observed in fibroblast or feocromacitomas cell lines treated with K252a . However, neuronal differentiation in our study was not confirmed by measuring the content of β-III tubulin, a marker of neuronal differentiation . In fact, a decrease in β-III tubulin levels was observed
in cells treated with K252a. Interestingly, increased β-III tubulin has been associated with aggressiveness, resistance to chemotherapy, and poor clinical outcomes in solid tumors [51–54]. β-III tubulin confers dynamic properties to microtubules, likely contributing to resistance to microtubule-targeting chemotherapy . Thus, the decrease in β-III tubulin observed in our study
might be related to restoring sensitivity to VCR and K252-induced phenotypic alterations associated with reduced aggressiveness. Further studies are warranted to investigate this interesting possibility.
Activation of Trks leads to stimulation of downstream mediators (i.e. MAPK, PLCγ and PI-3
kinase pathways) important for growth, differentiation, metastasis, and cell survival . BDNF-induced stimulation of TrkB results in increased expression of a wide range of genes, and these alterations are blocked by K252a. In addition, signaling mediated by MAPK is a universal requirement for gene transcription alterations related to Trk activation . Our results indicate that Trk activity regulates the gene expression of Trk receptors as well as their ligands, given that K252a reduced the mRNA levels of NGF, TrkA, BDNF, and TrkB. This transcription inhibition of Trk pathway components represents a likely candidate mechanism involved in the antiproliferative
effects of Trk inhibitors.
Only one previous study investigated the immunohistochemical expression of Trk receptors in ES
tumors . The authors used a pan-Trk receptor antibody in tumor samples from 5 patients, and found that all samples were positive for at least one Trk receptor. The present study was the first to discriminate between TrkA and TrkB receptor expression, in addition to showing the expression of Trk receptor ligands.
The mechanisms by which ES cells become resistant to chemotherapy are likely multiple and may involve cancer stem cells, proliferative intracellular pathways, and new mutations that allow the tumor cells to escape the effects of chemotherapy . K252a was able to subvert these resistance mechanisms, and produced an excellent long-term response when used in conjunction with chemotherapeutic agents (Figure 8). K252a, an alkaloid-like compound isolated from Nocardwpsis, was characterized originally as an inhibitor of PKC and cyclic nucleotide-dependent kinases . It is a potent and selective inhibitor of the tyrosine protein kinase activity of the Trk family of oncogenes and neurotrophin receptors . The pegylated form or K252a (CT 327) has already
been tested as a potential treatment for psoriasis in a clinical trial (NCT00995969), and synthetic derivatives of K252a (e.g. CEP-701) have been examined in phase I and phase II studies for leukemia and neuroblastoma [59, 60]. The prior clinical application of these drugs increases the
chances that they can be used to treat ES.
Prospective assessment of TrkA and TrkB receptor expression might be used to identify tumors that are likely to respond to Trk receptor inhibitors, either alone or in combination with conventional agents. Indeed, very recent studies have shown that co-administration of Trk receptor inhibitors with traditional chemotherapeutic agents, specific small-interfering RNAs, or radiation enhanced the tumor response greatly in both in vitro and in vivo models [61–65], supporting the notion that such an approach is a promising avenue for the future of anticancer therapy.
In conclusion, the present results showed that Trk inhibition inhibits ES cell proliferation, particularly when delivered in combination with low-dose chemotherapeutic agents, even in chemoresistant cells. These findings provide the first evidence indicating that Trk pathway
inhibition can improve treatment efficacy in ES.
MATERIALS AND METHODS
Cell lines and treatments
Human cell lines (SK-ES-1 and RD-ES) were obtained from the American Type Culture Collection
(ATCC; Rockville, MD) within six months before the beginning of the experiments and were authenticated using morphology, karyotyping, and PCR based approaches according to standard ATCC procedures. Cells were grown in RPMI-1640 medium (Gibco-BRL, Carlsbad, CA), containing 0.1% Fungizone (250 mg/kg; Invitrogen, São Paulo, Brazil), 100 U/l gentamicin (4 mg/ml; Nova
Pharma, Jardim Anápolis, Brazil), 50 mg/ml ampicilin (Nova Pharma, Jardim Anápolis, Brazil), and 10% fetal bovine serum (Invitrogen, São Paulo, Brazil), at 37 °C in a humidified incubator under 5% CO2 . Exponentially growing cells were detached with trypsin 0% EDTA, transferred to culture dishes, and treated accordingly to experimental group designations.
To induce chemoresistance, SK-ES-1 cells were cultured with stepwise escalation of concentrations of VCR (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 nM), Doxo (1, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 nM), and VP-16 (0.01, 0.05, 0.10, 0.20, 0.25, 0.30, 0.35 and 0.4 μM) over 5 months . Cells were exposed to each dose for 15 days. After the highest dose was reached, dose-effect curves were established while the cells were still exposed to the highest dose. The resultant resistant cells were referred to as SK-ES-1R.
Cellular proliferation assay
Cells were seeded at a density of 2 × 104 cells/well (SK-ES-1) or 2.5 × 104 cells/well (RD-ES and SKES-1R) in 24-well plates (TPP, Switzerland). After 24 h, they were treated with K252a (Sigma-Aldrich, St. Louis, MO), VCR, VP-16, Doxo, Ana-12 (Sigma-Aldrich, St. Louis, MO), GW 441756 (Tocris Bioscence, Bristol, UK), BDNF (Sigma-Aldrich, St. Louis, MO) and NGF (SigmaAldrich,
St. Louis, MO) alone or combined. The medium was removed 72 h after experimental treatments, and the cells were washed with Hanks’ balanced salt solution (Invitrogen, São Paulo, Brazil), detached with 0.25% trypsin solution, and counted the trypan blue exclusion method in a hemocytometer . Previous studies from our group [26, 68] have indicated that 72 h is the most
appropriate exposure time to assess proliferation in cancer cells treated with K252a. The mean of at least three experiments for each dose, was used to calculate IC50 and combination index values.
Colony formation assay
SK-ES-1 and RD-ES cells were seeded in 6-well plates (500 cells/well) and treated with different doses of K252a, VP-16, Doxo, and VCR, alone or in combination, for 24 h. Subsequently, the drug solution was removed and the cells were placed in a treatment-free medium. After being incubated for 10–14 days, the cells were fixed in 70% ethanol and counterstained with 0.5% crystal violet.
Images of each plate were obtained with a desktop scanner (L-pix Chemi Molecular Imaging, Loccus Biotecnologia). Each plate was placed in the same position on the light table by aligning it with the center of the preview exposed light window. For analysis of the clonogenic assay images,
optimized digital colony counts were performed with ImageJ software (version 1.37 for Windows) as described by dos Santos et al. . Drug effects were expressed as surviving fraction of the colonies (SF; according to the formula below) and area occupied by colonies. The area occupied by colonies was analyzed in addition to colony number because colony size can vary substantially. All
measures were calculated by ImageJ software to ensure
SF =No. colonies of treated cells/No. untreated control colonies ×100uniformity of results.
SK-ES-1 (chemoresistant and non-resistant cells) and RD-ES cells were cultured in normal RT-PCR
medium. Total RNA was extracted with Trizol reagent (Invitrogen, São Paulo, Brazil) in accordance with the manufacturer’s instructions and reverse transcribed with superscripttm III First-Strand Synthesis supermix (Invitrogen, São Paulo, Brazil). Human β-actin, BDNF, TrkB, NGF, and TrkA primers were designed according to the corresponding GenBank sequences (Table 4).
Semiquantitative RT-PCR conditions were optimized to determine the number of cycles that would
allow product detection within the linear phase of mRNA transcript amplification. Experiments were performed with 1.5 mM MgCl2 , 0.1 μM for each primer, 0.2 mM DNTPs, 1U Taq Platinum, and 2 μl cDNA template. Expression of β-actin was measured as an internal control. The PCR conditions for β-actin, BDNF, TrkB, NGF, and TrkA experiments were: 2.5 mM MgCl2 , 0.1
μM for each primer, 0.2 mM DNTPs, 1U Taq Platinum, and 1 μl cDNA template. All assays were carried out in a total volume of 15 μl with 35 amplification cycles that consisted of 1 min at 95 °C, denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and primer extension at 72 °C for 45 s, followed by a final extension at 72 °C for 10 min. The products were electrophoresed in 1.0% agarose gels containing ethidium bromide (Biotium, Hayward, USA) and visualized with ultraviolet light. Fragment lengths were confirmed by reference to a Low DNA Mass Ladder (Invitrogen, São Paulo, Brazil) and relative gene expression was determined by densitometry in ImageJ 1.37 for Windows. Each experiment was performedin triplicate with RNA isolated from independent cell
cultures, and representative findings are shown. A negative control was included in each PCR set. Semiquantitative data are shown as percentage changes relative to β-actin (the lowest value among triplicates in the control group was taken as 100%).
Flow cytometry cell cycle analysis
SK-ES-1 cells were seeded at the density of 2 × 105 in 6-well plates. The next day, cells were treated with K252a (1 nM or 100 nM) or Doxo (40 nM) as positive control for 24 h. The medium was removed, and the cells were washed with Hanks’ balanced salt solution and detached with 0.25% trypsin solution. Cells were centrifuged and re-suspended in 50 μg/mL propidium iodide (Sigma-Aldrich, St. Louis, USA) and 0.1% Triton X-100 in 0.1% sodium citrate solution (appropriate
volume to maintain 1 × 106 cells per ml ratio in solution). Cells were incubated on ice for 15 min prior to analysis in an Attune® Acousting Focusing Cytometer (Applied Biosystem, Life Technologies).
Paraffin blocks of tumors from 7 patients with ES were obtained from the Hospital de Clínicas de Porto Alegre Pathology Department. Four-micron-thick sections were mounted on organosilane-coated slides and dried overnight at 37 °C. The sections were deparaffinized in a stove, rehydrated in graded alcohols, and washed with distilled water. Antigenic recuperation was performed in a
microwave, endogenous peroxidases were inactivated by immersion in hydrogen peroxide, and cross-reactivity was blocked with normal serum. Primary antibody [polyclonal rabbit anti-NGF (sc-33603; Santa Cruz Biotechnology), anti-BDNF (sc-20981, Santa Cruz Biotechnology), -TrkB (sc 377218, Santa Cruz Biotechnology), and polyclonal goat anti-TrkA (sc-20539, Santa Cruz Biotechnology)] diluted 1:50 in phosphate-buffered saline was applied for 12 h at 4 °C, followed by biotin streptavidin-biotin peroxidase complex (LSAB, Dako). Immunlabelling was visualized by reaction with diamino-9-benzidine tetrahydrochloride (DAB Kit, Dako). Cell nuclei were
counterstained lightly with hematoxylin-eosin (HE).
A pathologist (LFRR), who was blind to the group designations scored the immunolabelling semiquantitatively according to intensity and distribution, as described by Scott et al. : for stain intensity, 0 = none; 1 = weak; 2 = moderate, and 4 = strong; and for staining distribution, 1 = focal, <10% of cells and 3 = diffuse, >10% of cells. Tumor sections were considered negative if the sum (intensity + distribution) score was ≤1, weak positive if the sum score was 2–4 (weak diffuse, moderate or strong focal), and strong positive if the sum score was ≥ 5 (moderate
or strong diffuse).
Western blot analysis of β-III tubulin levels
SK-ES-1 ES cells were homogenized in radioimmunoprecipitation assay buffer containing complete
Protease Inhibitors (Roche) and quantified using a colorimetric protein assay (Bradford, Bio-rad, CA, USA). 20 μg of total protein lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and blotted with antibodies against β-III tubulin (D71G9 – Cell Signaling Technology, MA, USA) and anti-β-actin
(A2228, Sigma Aldrich, MO, USA) used as loading control. Incubation with appropriate horsedish peroxidaseconjugated secondary antibody (Santa Cruz, TX, USA) for 1h at RT was performed. Chemiluminescence was detected using ECL Western Blotting substrate (EMD Millipore, DE) and analyzed by ImageQuant LAS500 (GE Healthcare Life Sciences, UK). Densitometric analyses
were performed using Image J software (NIH, MD, USA). Relative Densitometric Units (RDU) in controls were expressed as 1 arbitrary unit. Three individual replicates were performed.
Median dose-effect analysis
The combination index, a measure of synergism and antagonism, was calculated by Chou and Talalay’s method with CalcuSyn software version 2.11 for Windows (Biosoft, Ferguson, MO). This method takes into account both potency and dose-effect curve shape. Synergy, additivity, and antagonism were defined as CI < 0.9, CI = 0.9–1.1, and CI > 1.1, respectively. A CI value ≤0.3 and
≤0.1 was interpreted as strong and very strong synergism, respectively .
All data are shown as means ± standard errors of the mean (SEM) of 3–5 independent experiments. Differences between mean values were evaluated by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test in SPSS, version 16.0. P-values < .05 were considered
This research was supported by the National Council for Scientific and Technological Development
(CNPq; grant numbers 484185/2012-8 and 303276/2013- 4 to R.R.); PRONON/Ministry of Health, Brazil (number 25000.162.034/2014-21 to C.B.F); the Rafael Koff Acordi Research Fund, Children’s Cancer Institute (ICI); and the Clinical Hospital institutional research fund (FIPE/
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
1. Balamuth NJ, Womer RB. Ewing’s sarcoma. Lancet Oncol. 2010; 11:184–192.
2. Esiashvili N, Goodman M, Marcus RB Jr. Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: Surveillance Epidemiology and End Results data. J Pediatr Hematol Oncol. 2008; 30:425–430.
3. Ross KA, Smyth NA, Murawski CD, Kennedy JG. The biology of Ewing sarcoma. ISRN Oncol. 2013; 2013:759725.
4. Granowetter L, Womer R, Devidas M, Krailo M, Wang C, Bernstein M, Marina N, Leavey P, Gebhardt M, Healey J, Shamberger RC, Goorin A, Miser J, et al. Dose-intensified compared with standard chemotherapy for non metastatic Ewing sarcoma family of tumors: a Children’s Oncology
Group Study. J Clin Oncol. 2009; 27:2536–41.
5. Grier HE, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, Pritchard DJ, Gebhardt MC, Dickman PS, Perlman EJ, Meyers PA, Donaldson SS, Moore S, Rausen AR, et al. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing’s sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med. 2003; 348:694–701.
6. Limin Z, Madonna MM, Dennis PMH. Understanding the biology of bone sarcoma from early initiating events through late events in metastasis and disease progression. Front Oncol. 2013; 3:230.
7. Ginsberg JP, Goodman P, Leisenring W, Ness KK, Meyers PA, Wolden SL, Smith SM, Stovall M, Hammond S, Robison LL, Oeffinger KC. Long-term survivors of childhood Ewing sarcoma: report from the childhood cancer survivor study. J Natl Cancer Inst. 2010; 102:1272–83.
8. Ginsberg JP, Goodman P, Leisenring W, Ness KK, Meyers PA, Wolden SL, Smith SM, Stovall M, Hammond S, Robison LL, Oeffinger KC. Long-term survivors of childhood Ewing sarcoma: report from the childhood cancer survivor study. J Natl Cancer Inst. 2010; 102:1272–83.
9. Jiang X, Gwye Y, Russell D, Cao C, Douglas D, Hung L, Kovar H, Triche TJ, Lawlor ER. CD133 expression in chemo-resistant Ewing sarcoma cells. BMC Cancer. 2010; 10:116.
10. May WA, Grigoryan RS, Keshelava N, Cabral DJ, Christensen LL, Jenabi J, Ji L, Triche TJ, Lawlor ER, Reynolds CP. Characterization and drug resistance patterns of Ewing’s sarcoma family tumor cell lines. PLoS One. 2013; 8.
11. Garofalo C, Mancarella C, Grilli A, Manara MC, Astolfi A, Marino MT, Conte A, Sigismund S, Carè A, Belfiore A, Picci P, Scotlandi K. Identification of common and distinctive mechanisms of resistance to different antiIGF-IR agents in Ewing’s sarcoma. Mol Endocrinol. 2012; 26:1603–16.
12. Nakatani F, Ferracin M, Manara MC, Ventura S, Del Monaco V, Ferrari S, Alberghini M, Grilli A, Knuutila S, Schaefer KL, Mattia G, Negrini M, Picci P, Serra M, Scotlandi K. miR-34a predicts survival of Ewing’s sarcoma patients and directly influences cell chemo-sensitivity and
malignancy. J Pathol. 2012; 226:796–805.
13. Cavazzana AO, Miser JS, Jefferson J, Triche TJ. Experimental evidence for a neural origin of Ewing’s sarcoma of bone. Am J Pathol. 1987; 127:507–518.
14. Knezevich SR, Hendson G, Mathers JA, Carpenter B, Lopez-Terrada D, Brown KL, Sorensen PH. Absence of detectable EWS/FLI1 expression after therapy-induced neural differentiation in Ewing sarcoma. Hum Pathol. 1998; 29:289–294.
15. O’Regan S, Diebler MF, Meunier FM, Vyas S. A Ewing’s sarcoma cell line showing some, but not all, of the traits of a cholinergic neuron. J Neurochem. 1995; 64:69–76.
16. Staege MS, Hutter C, Neumann I, Foja S, Hattenhorst UE, Hansen G, Afar D, Burdach SE. DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets. Cancer Res. 2004; 64:8213–8221.
17. von Levetzow C, Jiang X, Gwye Y, von Levetzow G, Hung L, Cooper A, Hsu JH, Lawlor ER. Modeling initiation of Ewing sarcoma in human neural crest cells. PLoS One.2011; 6.
18. Miyagawa Y, Okita H, Nakaijima H, Horiuchi Y, Sato B, Taguchi T, Toyoda M, Katagiri YU, Fujimoto J, Hata J, Umezawa A, Kiyokawa N. Inducible expression of chimeric EWS/ETS proteins confers Ewing’s family tumorlike phenotypes to human mesenchymal progenitor cells. Mol Cell Biol. 2008; 28:2125–2137.
19. Riggi N, Suvà ML, Suvà D, Cironi L, Provero P, Tercier S, Joseph JM, Stehle JC, Baumer K, Kindler V, Stamenkovic I. EWS-FLI-1 expression triggers a Ewing’s sarcoma initiation program in primary human mesenchymal stem cells. Cancer Res. 2008; 68:2176–2185.
20. Tirode F, Laud-Duval K, Prieur A, Delorme B, Charbord P, Delattre O. Mesenchymal stem cell features of Ewing tumors. Cancer Cell. 2007; 11:421–429.
21. Kim MS, Kim CJ, Jung HS, Seo MR, Juhnn YS, Shin HY, Ahn HS, Thiele CJ, Chi JG. Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells. J Pathol. 2004; 202:103–112.
22. Arévalo JC, Wu SH. Neurotrophin signaling: many exciting surprises! Cell Mol Life Sci. 2006; 63:1523–1537.
23. Reichardt LF. Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci. 2006; 361:1545–64.
24. Skaper SD. The neurotrophin family of neurotrophic factors: an overview. Methods Mol Biol. 2012; 846:1–12.
25. Schulte JH, Schramm A, Klein-Hitpass L, Klenk M, Wessels H, Hauffa BP, Eils J, Eils R, Brodeur GM, Schweigerer L, Havers W, Eggert A. Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas. Oncogene. 2005; 24:165–77.
26. de Farias CB, Heinen TE, dos Santos RP, Abujamra AL, Schwartsmann G, Roesler R. BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition. Biochem Biophys Res Commun. 2012; 425:328–32.
27. Bao W, Qiu H, Yang T, Luo X, Zhang H, Wan X. Upregulation of TrkB promotes epithelial-mesenchymal transition and anoikis resistance in endometrial carcinoma. PLoS One. 2013; 8.
28. Cornelio DB, De Farias CB, Prusch DS, Heinen TE, Dos Santos RP, Abujamra AL, Schwartsmann G, Roesler R. Influence of GRPR and BDNF/TrkB signaling on the viability of breast and gynecologic cancer cells. Mol Clin Oncol. 2013; 1:148–152.
29. Roesler R, de Farias CB, Abujamra AL, Brunetto AL, Schwartsmann G. BDNF/TrkB signaling as an anti-tumor target. Expert Rev Anticancer Ther. 2011; 11:1473–5.
30. Thomson TM, Pellicer A, Greene LA. Functional receptors for nerve growth factor on Ewing’s sarcoma and Wilm’s tumor cells. J Cell Physiol. 1989; 141:60–4.
31. Nogueira E, Navarro S, Pellín A, Llombart-Bosch A. Activation of TRK genes in Ewing’s sarcoma. Trk A receptor expression linked to neural differentiation. Diagn Mol Pathol. 1997; 6:10–6.
32. Donovan MJ, Hempstead BL, Horvath C, Chao MV, Schofield D. Immunohistochemical localization of Trk receptor protein in pediatric small round blue cell tumors. Am J Pathol. 1993; 143:1560–7.
33. Sugimoto T, Umezawa A, Hata J. Neurogenic potential of Ewing’s sarcoma cells. Virchows Arch. 1997; 430:41–6.
34. Kim GJ, Kim CJ, Cho SY, et al. Activation of trkA induces differentiation and inhibits the growth of JK-GMS Askin tumor cells. Lab Invest. 2002; 82:221–9.
35. Sturla LM, Westwood G, Selby PJ, et al. Cancer Res. Induction of cell death by basic fibroblast growth factor in Ewing’s sarcoma. 2000; 60:6160–70.
36. Thiele CJ, Li Z, McKee AE. On Trk–the TrkB signal transduction pathway is an increasingly important target in cancer biology. Clin Cancer Res. 2009; 15:5962–7.
37. Amaral AT, Ordóñez JL, Otero-Motta AP, GarcíaDomínguez DJ, Sevillano MV, de Álava E. Innovative therapies in Ewing Sarcoma. Adv Anat Pathol. 2014; 21:44–62.
38. Subbiah V, Kurzrock R. Ewing’s sarcoma: overcoming the therapeutic plateau. Discov Med. 2012; 13:405–15.
39. Hawkins DS. Sarcomas gone bad: what to do about recurrent Ewing sarcoma. Pediatr Blood Cancer. 2012; 57:535–536.
40. Svensson T, Rydén M, Schilling FH, Dominici C, Sehgal R, Ibáñez CF, Kogner P. Coexpression of mRNA for the fulllength neurotrophin receptor trk-C and trk-A in favourable neuroblastoma. Eur J Cancer. 1997; 33:2058–63.
41. Rydén M, Sehgal R, Dominici C, Schilling FH, Ibáñez CF, Kogner P. Expression of mRNA for the neurotrophin receptor trkC in neuroblastomas with favourable tumour stage and good prognosis. Br J Cancer. 1996; 74:773–9.
42. Nakagawara A, Azar CG, Scavarda NJ, Brodeur GM. Expression and function of TRK-B and BDNF in human neuroblastomas. Mol Cell Biol. 1994; 14:759–67.
43. Eggert A, Grotzer MA, Ikegaki N, Zhao H, Cnaan A, Brodeur GM, Evans AE. Expression of the neurotrophin receptor TrkB is associated with unfavorable outcome in Wilms’ tumor. J Clin Oncol. 2001; 19:689–96.
44. McGregor LM, McCune BK, Graff JR, McDowell PR, Romans KE, Yancopoulos GD, Ball DW, Baylin SB, Nelkin BD. Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression. Proc Natl Acad Sci U S A. 1999; 96:4540–5.
45. Jaboin J, Kim CJ, Kaplan DR, Thiele CJ. Brain derived neurotrophic factor activation of TrkB protects neuroblastoma cells from chemotherapy induced apoptosis via phosphatidylinositol 3′- kinase pathway. Cancer Res. 2002; 62:6756–63.
46. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001; 24:677–736.
47. Nör C, de Farias CB, Abujamra AL, Schwartsmann G, Brunetto AL, Roesler R. The histone deacetylase inhibitor sodium butyrate in combination with brainderived neurotrophic factor reduces the viability of DAOY human medulloblastoma cells. Childs Nerv Syst. 2011; 27:897–901.
48. Lee SJ, Li GG, Kim ST, Hong ME, Jang J, Yoon N, Ahn SM, Murphy D, Christiansen J, Wei G, Hornby Z, Lee DW, Park JO, Park YS, Lim HY, Hong SN, Kim SH, Kang WK, Park K, Park WY, Kim KM, Lee J. NTRK1 rearrangement in colorectal cancer patients: evidence for actionable target
using patient-derived tumor cell line. Oncotarget. 2015; 6:39028–35.
49. Fleuren ED, Hillebrandt-Roeffen MH, Flucke UE, Te Loo DM, Boerman OC, van der Graaf WT, VersleijenJonkers YM. The role of AXL and the in vitro activity of the receptor tyrosine kinase inhibitor BGB324 in Ewing sarcoma. Oncotarget. 2014; 5:12753–68.
50. Thompson AF, Levin LA. Neuronal differentiation by analogs of staurosporine. Neurochem Int. 2010; 56:554–60.
51. Katsetos CD, Herman MM, Mörk SJ. Class III beta-tubulin in human development and cancer. Cell Motil Cytoskeleton. 2003; 55:77–96.
52. Hetland TE, Hellesylt E, Flørenes VA, Tropé C, Davidson B, Kærn J. Class III β-tubulin expression in advanced-stage serous ovarian carcinoma effusions is associated with poor
survival and primary chemoresistance. Hum Pathol. 2011;42:1019–1026.
53. Lebok P, Öztürk M, Heilenkötter U, Jaenicke F, Müller V, Paluchowski P, Geist S, Wilke C, Burandt E, Lebeau A, Wilczak W, Krech T, Simon R, Sauter G, Quaas A. High levels of class III β-tubulin expression are associated with aggressive tumor features in breast cancer. Oncol Lett.2016; 11:1987–1994.
54. Zhang HL, Ruan L, Zheng LM, Whyte D, Tzeng CM, Zhou XW. Association between class III β-tubulin expression and response to paclitaxel/vinorebine-based chemotherapy for non-small cell lung cancer: A meta-analysis. Lung Cancer. 2012; 77:9–15.
55. Gokce O, Runne H, Kuhn A, Luthi-Carter R. Shortterm striatal gene expression responses to brain-derived neurotrophic factor are dependent on MEK and ERK activation. PLoS One. 2009; 4:e5292.
56. Ahmed AA, Zia H, Wagner L. Therapy resistance mechanisms in Ewing’s sarcoma family tumors. Cancer Chemother Pharmacol. 2014; 73:657–63.
57. Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A., Kaneko, M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987; 142:436–440.
58. Tapley P, Lamballe F, Barbacid M. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene. 1992; 7:371–81.
59. Minturn JE, Evans AE, Villablanca JG, Yanik GA, Park JR, Shusterman S, Groshen S, Hellriegel ET, Bensen-Kennedy D, Matthay KK, Brodeur GM, Maris JM. Phase I trial of lestaurtinib for children with refractory neuroblastoma: a new approaches to neuroblastoma therapy consortium
study. Cancer Chemother Pharmacol. 2011; 68:1057–65.
60. Knapper S, Burnett AK, Littlewood T, Kell WJ, Agrawal S, Chopra R, Clark R, Levis MJ, Small D. A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-linetreatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy. Blood. 2006;
61. Aubert L, Guilbert M, Corbet C, Génot E, AdriaenssensE, Chassat T, Bertucci F, Daubon T, Magné, Le Bourhis X, Toillon RA. NGF-induced TrkA/CD44 association is involved in tumor aggressiveness and resistance to lestaurtinib. Oncotarget. 2015; 6:9807–19.
62. Li Z, Zhang Y, Tong Y, Tong J, Thiele CJ. Trk inhibitor attenuates the BDNF/TrkB-induced protection of neuroblastoma cells from etoposide in vitro and in vivo. Cancer Biol Ther. 2015; 16:477–83.
63. Iyer R, Varela CR, Minturn JE, Ho R, Simpson AM, Light JE, Evans AE, Zhao H, Thress K, Brown JL, Brodeur GM. AZ64 inhibits TrkB and enhances the efficacy of chemotherapy and local radiation in neuroblastoma xenografts. Cancer Chemother Pharmacol. 2012; 70:477–86.
64. Croucher JL, Iyer R, Li N, Molteni V, Loren J, Gordon WP, Tuntland T, Liu B, Brodeur GM. TrkB inhibition by GNF-4256 slows growth and enhances chemotherapeutic efficacy in neuroblastoma xenografts. Cancer Chemother Pharmacol. 2015; 75:131–41.
65. Zhang J, Wang LS, Ye SL, Luo P, Wang BL. Blockage of tropomyosin receptor kinase a (TrkA) enhances chemosensitivity in breast cancer cells and inhibits metastasis in vivo. Int J Clin Exp Med. 2015; 8:634–41.
66. Shien K, Toyooka S, Yamamoto H, Soh J, Jida M, Thu KL, Hashida S, Maki Y, Ichihara E, Asano H, Tsukuda K, Takigawa N, Kiura K, et al. Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. Cancer Res. 2013; 73:3051–61.
67. Flores DG, de Farias CB, Leites J, de Oliveira MS, Lima RC, Tamajusuku AS, Di Leone LP, Meurer L, Brunetto AL, Schwartsmann G, Lenz G, Roesler R. Gastrin-releasing peptide receptors regulate proliferation of C6 glioma cells through a phosphatidylinositol 3-kinase-dependent
mechanism. Curr Neurovasc Res. 2008; 5:99–105.
68. de Farias CB, Rosemberg DB, Heinen TE, Koehler-Santos P, Abujamra AL, Kapczinski F, Brunetto AL, Ashton-Prolla P, Meurer L, Reis Bogo M, Damin DC, Schwartsmann G, Roesler R. BDNF/TrkB content and interaction with gastrin-releasing peptide receptor blockade in colorectal
cancer. Oncology. 2010; 79:430–9.
69. dos Santos MP, de Farias CB, Roesler R, Brunetto AL, Abujamra AL. In vitro antitumor effect of sodium butyrate and zoledronic acid combined with traditional chemotherapeutic drugs: a paradigm of synergistic molecular targeting in the treatment of Ewing sarcoma. Oncol Rep. 2014; 31:955–68.
70. Scott N, Millward E, Cartwright EJ, Preston SR, Coletta PL: Gastrin releasing peptide and gastrin releasing peptide receptor expression in gastrointestinal carcinoid tumors. J Clin Pathol. 2004; 57:189–192.
71. Chou TC: The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Synergism and Antagonism in Chemotherapy. Chou TC and Rideout DC (eds). Academic Press, San Diego, pp61–102, 1991.
Objetivos Identificar a prevalência de HER-2 e do fator de crescimento do endotélio vascular (VEGF) em biópsias de osteossarcoma e correlacioná-los com possíveis fatores de prognóstico. Métodos: Estudo retrospectivo realizado no Hospital de Câncer de Barretos-SP incluindo 27 biópsias de osteossarcoma imuno-histoquimicamente coradas para VEGF e HER-2. Características clínico-patológicas foram coletadas dos prontuários médicos dos pacientes para correlação com marcadores. Resultados: Em 27 biópsias, quatro foram superexpressas para VEGF e três para HER-2. Dois terços dos pacientes eram não metastáticos. Quase todos pacientes com VEGF superexpresso apresentaram metástases. A superexpressão para HER-2 apresentou relação inversa à presença de metástases. Não houve associação significativa entre os marcadores e o prognóstico.
Conclusão: Identificamos baixa prevalência de VEGF e HER-2 na amostra. Não houve associação significativa entre superexpressão dos marcadores e características clínico-patológicas. A ampliação da amostra e do tempo de seguimento, além do emprego de novas técnicas laboratoriais pode determinar a real expressão de VEGF e HER-2 e seu papel em osteossarcomas. Nível de Evidência III, Estudo de Caso-controle.
Descritores: Fator A de crescimento do endotélio vascular. Genes HER-2. Osteosarcoma. Imunoistoquímica.
Objectives: To identify the prevalence of erbB-2 and vascular endothelial growth factor (VEGF) in osteosarcoma biopsies and to correlate them with possible prognosis factors. Methods:
Retrospective study conducted at the Hospital do Câncer de Barretos-SP including 27 osteosarcoma biopsies immunohistochemically stained for VEGF and erbB-2. The pathological characteristics were collected from medical records of patients to correlate with markers. Results: In 27 biopsies, four overexpressed VEGF and three overexpressed erbB-2. Two thirds of patients had no metastases. Almost all patients with overexpression of VEGF showed metastases. Overexpression of erbB-2 was inversely related to the presence of metastases. There was no significant association between markers and prognosis. Conclusion: We identified a low prevalence of erbB-2 and VEGF in the sample. There was no significant association between overexpression of markers and pathological features. A larger sample and a longer follow-up, in addition to using new labora-
tory techniques can determine the real expression of VEGF and erbB-2 and its role in osteosarcoma. Level of Evidence III, Case-Control Study.
Keywords: Vascular endothelial growth factor A. Genes, erbB-2. Osteosarcoma. Immunohistochemistry.
Osteossarcoma é um tipo de câncer ósseo agressivo de origem mesenquimal geralmente encontrado em jovens entre 10 e 25 anos. Qualquer osso do corpo humano pode ser acometido por esta neoplasia e a sobrevida geral em cinco anos é de aproximadamente 65 a 75%. A principal causa de óbito são as metástases pulmonares, diagnosticadas por tomografia computadorizada (TC)
em 35 a 45% dos pacientes.1 Diversos protocolos foram desenvolvidos nas últimas décadas incluindo quimioterapia em altas doses e novas técnicas para ressecções com margem oncológica e reconstruções preservadoras de extremidades. Apesar da evolução no campo da oncologia, a busca por melhores resultados em curvas de sobrevida ainda continua sendo um desafio, principalmente em pacientes portadores de metástases.2-5 O crescimento tumoral depende da proliferação de novos vasos sanguíneos em seu interior, os quais estão diretamente e indiretamente relacionados à liberação de diversos fatores de crescimento tanto pelo tumor primário quanto pelas lesões metastáticas decorrentes. A identificação de marcadores tumorais tem sido motivo de pesquisas com o objetivo de identificar e estratificar pacientes com alto ou baixo risco para desenvolvimento de metástases. A superexpressão do Fator de Crescimento do Endotélio Vascular (VEGF) e do Receptor tipo 2 do Fator de Crescimento Epidérmico Humano (HER-2) tem evidenciado resultados conflitantes com relação ao prognóstico em portadores de osteossarcoma.4-10
O fator de crescimento do endotélio Vascular (VEGF) é uma proteína homodimérica que aumenta a permeabilidade endotelial vascular e estimula a proliferação de células endoteliais. Esta proteína está diretamente envolvida no processo de angiogênese, que é responsável pela neovascularização, crescimento tumoral e disseminação de metástases. A neovascularização é evidente em amostras histológicas de osteossarcomas clássicos, o que reflete a agressividade e o potencial metastático deste tipo de câncer.4,9,11 Em tumores do aparelho gastrointestinal como o carcinoma gás-
trico, os tumores esofágicos e os carcinomas colorretais, a supe rexpressão de VEGF sugere relação direta com pior prognóstico. No entanto, evidências clínicas ainda não estão bem estabelecidas
entre a expressão deste marcador de angiogênese e sobrevida em portadores de osteossarcoma.2,4,5
O oncogene HER-2 (ErbB-2 ou neu) é o responsável pela codificação de uma glicoproteína transmembrana chamada HER-2, que está superexpressa em 20 a 40% dos pacientes portadores
de câncer de mama, sendo associada ao pior prognóstico.12 Este marcador biológico também tem sido relacionado à menor sobrevida, má resposta à quimioterapia e à presença de metástases em alguns estudos envolvendo o osteossarcoma.6,8-10
Esta coorte histórica tem o objetivo de identificar a prevalência de VEGF e HER-2 em biópsias de osteossarcoma anteriores à quimioterapia e correlacionar a superexpressão destes marcadores a características clínico-patológicas envolvidas com o prognóstico.
PACIENTES E MÉTODOS
Pacientes e amostras histológicas
Neste estudo retrospectivo 27 pacientes com osteossarcoma tiveram seus prontuários médicos revisados e suas biópsias coradas para VEGF e HER-2. As amostras pertenciam a pacientes diagnosticados entre os anos de 2005 e 2009. Todas as amostras foram fixadas em formalina 10%, incluídas em blocos de parafina e armazenadas no departamento de patologia do Hospital de Cân-
cer de Barretos – Brasil. Os critérios para inclusão foram pacientes de ambos os sexos, com idade entre 5 e 30 anos, diagnóstico de osteossarcoma sem tratamento prévio, além de presença ou
ausência de metástases. A violação no protocolo de tratamento foi considerada critério de exclusão do estudo. Características clínico patológicas como idade, sexo, subtipo histológico, término do protocolo, resposta à quimioterapia neoadjuvante e a presença de metástases foram analisados e tabulados. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa da Instituição (CEP #195/2009).
Técnica de imuno-histoquímica
Todas as amostras foram incluídas em blocos de parafina e submetidas a cortes de 2 a 3 micra em micrótomo padronizado e coradas imuno-histoquímicamente pelo método do complexo avidina-biotina-peroxidase. O anticorpo primário para VEGF foi um anticorpo anti-humano monoclonal de ratos em diluição de 1:50 (DakoCitomation, Denmark, A/S), e para HER-2, o anticorpo anti-humano policlonal de coelhos em diluição de 1:200 (DakoCitomation, Denmark, A/S). A recuperação antigênica foi realizada através da incubação em calor úmido em tampão de citrato (Dako – 10 mM, ph 9,0 e 90°C). A amplificação da reação foi realizada no Dakoautostainer (Universal Staining System – Dako). Os controles positivos e negativos foram utilizados para cada teste.
Dois patologistas (SM e CRV) analisaram as amostras de osteossarcomas sem qualquer informação sobre o histórico dos pacientes e utilizaram um microscópio de duas cabeças binoculares simulta-
neamente. Frequências quase similares de porcentagem de células coradas foram encontradas nas lâminas por ambos profissionais com índice kappa menor que 5%. VEGF foi considerado positivo quando corados mais de 30% tanto do citoplasma quanto da membrana das células tumorais4. HER-2 foi avaliado através de escala semi-quantitativa. As amostras foram graduadas de 0 (zero) até +3 de acordo com a proporção de células coradas tanto no citoplasma quanto na membrana. Resultados de zero a +1 foram considerados como até 30% das células coradas e, de +2 até +3, como acima de 30%. Apenas a coloração de imuno-histoquímica acima de 30% foi classificada como superexpressão de HER-2.8,13
Todas as variáveis foram descritas por frequências absolutas e relativas, exceto a idade que foi descrita por média e desvio padrão. Para comparar os grupos foi aplicado o teste t student (idade) e o teste exato de Fisher (demais variáveis). Para estimar as curvas de sobrevida, o método de Kaplan-Meier foi aplicado e para compará-las foi utilizado o teste log-rank. O cálculo do tamanho da amostra foi realizado no programa PEPI (Programs for Epidemiologists) versão 4.0 e baseado no estudo de Kaya et al.4, 2000. Para um nível de significância de 5% (p ≤ 0,05), um poder de 90%, uma proporção de sobrevida de 90% no grupo VEGF negativo e uma proporção de 20% no grupo de VEGF positivo, obteve-se um total mínimo de 22 pacientes. As análises foram realizadas no programa SPSS (Statistical Package for the Social Sciences) versão 18.0.
Os resultados deste estudo estão sumarizados nas Tabelas 1 e 2. Quinze pacientes eram do sexo masculino e 12 do sexo feminino, com média de idade de 13 anos (sete a 27 anos). Os casos foram divididos em acima (n=21) e abaixo (n=6) de 14 anos considerando como um maior ou menor risco de agressividade do tumor. Treze indivíduos concluíram o protocolo brasileiro (GBTO), nove
permaneciam em tratamento durante este estudo3 e cinco não dispunham de dados nos prontuários. Todos osteossarcomas foram estadiados em IIB e III de acordo com o Sistema de Es-
tadiamento de Enneking. 14 Oito pacientes (30%) apresentaram metástases pulmonares no momento do diagnóstico e foram classificados como estágio III. Nenhum paciente apresentou descrição de metástases não pulmonares.
Os subtipos histológicos foram descritos de acordo com a OMS – Classificação de Tumores Ósseos, 2002.15 A classificação de Huvos-Ayala foi utilizada para descrever a resposta à quimioterapia neoadjuvante como pobre (I-II) e boa (III-IV).16 Mais da metade dos pacientes (56%) apresentaram pobre resposta ao tratamento quimioterápico neoadjuvante, 22% apresentaram boa
resposta e outros 22% não apresentaram registros de exames anatomopatológicos. A Tabela 1 descreve todas variáveis clínico-patológicas analisadas. O intervalo de seguimento foi registrado desde a biópsia inicial até julho de 2009. O período mínimo de seguimento foi de seis meses.
Apenas quatro amostras (15%) apresentaram superexpressão de VEGF. Todas as amostras positivas para VEGF foram encontradas no sexo masculino, e acima de 14 anos. Três quartos (75%) dos pacientes que superexpressaram VEGF apresentaram metástases pulmonares, inferindo um risco teórico para pior prognóstico já descrito em publicações prévias.4,7 A Tabela 1 sumariza a correlação entre VEGF e as variáveis analisadas. Não encontramos correlação significativa quando realizadas análises descritiva e univariada. Curvas de sobrevida de Kaplan-Meier para VEGF estão descritas na Figura 1. A superexpressão de HER-2 foi observada em três casos (11%). Todos do sexo masculino, e dois (67%), acima de 14 anos. Metástases pulmonares foram identificadas em apenas um terço dos pacientes com HER-2 considerados positivos (>1+). As amostras coradas como po-
sitivas para HER-2 não apresentavam descrição sobre resposta à quimioterapia em nossos arquivos médicos. Não encontramos correlação significativa quando realizadas análises descritiva e univariada. Curvas de sobrevida de Kaplan-Meier para HER-2 estão descritas na Figura 2. Imuno-histoquímica de VEGF é demonstrada na Figura 3A. Imuno-histoquímica de HER-2 é demonstrada na Figura 3B.
No presente estudo, demonstrou-se a baixa prevalência de VEGF (15%) e HER-2 (11%) em biópsias de osteossarcoma analisadas através de imuno-histoquímica. Diferentes métodos para quantifi-
car a presença de marcadores biológicos teoricamente envolvidos na proliferação e disseminação de tumores têm sido descritos nas últimas décadas. Testes laboratoriais como a imuno-histoquímica,
pesquisa de DNA e de RNA estão entre os mais conhecidos. Diferenças entre protocolos de pesquisa incluindo uma grande variedade de anticorpos e de técnicas sem padronização para a identificação de genes são responsáveis pela discrepância em estudos ao redor do mundo.15,17
Desde que o DNA complementar do HER-2 foi isolado há aproximadamente 25 anos, vieram à tona importantes descobertas no mecanismo de ação dos receptores de tirosinoquinases (RTQ), que quando mutados ou alterados, tornam-se potentes oncoproteínas. Em 1985, o grupo de Ullrich da Genentech descreveu a estrutura primária completa de suposto RTQ que demonstrou alto grau de homologia com o Receptor do Fator de Crescimento Epidérmico (EGFR) humano, tendo sido denominado de human EGFR-related 2, ou HER2 (17). Dois anos mais tarde, Slamon et al.12 relataram que o HER2 encontrava-se amplificado em 30% dos casos de câncer de mama invasivos e, pela primeira vez, demonstraram correlação significativa entre a superexpressão de HER2, redução da sobrevida e recidiva tumoral aumentada.
No final dos anos 90, dois estudos sugeriram que a superexpressão de HER-2 em osteossarcoma poderia estar associada à menor sobrevida e ao desenvolvimento de metástases. Em um destes estudos, 26 amostras de osteossarcomas foram analisadas para HER-2 através de imuno-histoquímica, e a superexpressão de HER-2 foi identificada em 42% das amostras. No entanto, os
pacientes metastáticos foram incluídos na mesma amostra e os protocolos de quimioterapia não foram adequadamente descritos. Por fim, pacientes em estágios IIB e III deveriam ter sido analisados separadamente para HER-2, pois é de conhecimento geral que o prognóstico de indivíduos metastáticos (III) é inferior ao dos não metastáticos (IIB).6 Em outro estudo, 53 amostras de osteossarcomas foram analisadas, enquanto todos pacientes portadores de metástases (estágio III) foram excluídos. Nem dados à respeito da sobrevida geral, nem qualquer descrição sobre análises uni ou multivariadas foram descritos.10
Em 2002, Akatsuka et al.8 propuseram diferente explicação para a associação entre a superexpressão de HER-2 e os achados clínicos em osteossarcoma, comparando os níveis de HER-2 entre amostras de biópsias, de tumores ressecados após a quimioterapia neoadjuvante e de metástases pulmonares em 19 pacientes não metastáticos ao diagnóstico. O desaparecimento de HER-2 foi visível durante o tratamento com quimioterapia em 14 de 19 pacientes (74%) a medida que se tornaram metastáticos. Estes achados sugerem que a superexpressão de HER-2 não tem um papel importante no desenvolvimento de metástases, e que tumores onde o HER-2 é superexpresso existirá maior benefício com a quimioterapia em relação à sobrevida geral e livre de doença
quando comparados aos com HER-2 negativo.
A expressão de VEGF tem sido utilizada como um marcador mais objetivo para avaliar a importância da angiogênese em tumores sólidos como o osteossarcoma. Um estudo com 30 pacientes identificou que o mRNA de VEGF estava expresso em todas as amostras tumorais, e em 80% na isoforma de VEGF165. Apenas 20% expressava a isoforma VEGF121. 83% dos VEGF165- positivos desenvolveram metástases, enquanto o mesmo ocorreu em apenas 16% dos VEGF165-negativos. Nos mesmos grupos, VEGFR-1 e 2 (receptores tipo 1 e 2 de VEGF) foram expressos em mais da metade dos pacientes, porém a expressão não se correlacionou com o prognóstico. Os indivíduos deste estudo não haviam sido submetidos a tratamento prévio e também não apresentavam metástases ao diagnóstico, e o VEGF foi analisado através da reação em cadeia de polimerase transcriptase (RT-PCR).7,18
Em outro estudo, 63% de 27 pacientes expressaram VEGF através de imuno-histoquímica. Oitenta e dois por cento das amostras onde o VEGF foi positivo desenvolveram metástases, enquanto apenas 10% nas amostras onde VEGF foi negativo. Como esperado, a sobrevida nos indivíduos com VEGF positivo foi menor que a dos negativos.4 Um grupo de pesquisas norte-americano des-
creveu em 2004 a possível relação entre a expressão de VEGF em biópsias de osteossarcoma e o nível de permeabilidade vascular através de ressonância nuclear magnética (RNM) em 15 pacientes.
Dez amostras coraram-se positivas à imuno-histoquímica, sendo quatro com 1+, outras quatro com 2+, e mais duas com 3+. Qualquer positividade tanto em citoplasma quanto em membrana foi considerada. O único achado significativo entre VEGF superexpresso e alto coeficiente de permeabilidade foi possível quando as amostras foram estratificadas, ou seja, avaliadas separadamente (0, 1+, 2+, 3+) de acordo com o número de cruzes.19
Uma metanálise incluindo 387 pacientes em 11 publicações identificou taxa de risco de 2,84 para VEGF positivos em relação ao risco de óbito quando comparados aos VEGF negativos. Estes
achados, apesar da heterogeneidade das publicações incluídas, sugerem pior prognóstico para biópsias onde VEGF apresentou-se superexpresso.20
Em nosso estudo, além da baixa prevalência dos marcadores imunológicos, foi apresentada de maneira descritiva uma tendência à presença de metástases em portadores de osteossarcoma com
VEGF positivo e HER-2 negativo. Não foi encontrada correlação significativa entre HER-2 e VEGF quanto à sobrevida geral e livre de doença, assim como das variáveis clínico-patológicas. Apesar
de ainda não existir consenso na literatura, a positividade de VEGF e a negatividade de HER-2 têm sugerido piores resultados na evolução desta doença.
A superexpressão das proteínas de VEGF e HER-2 demonstrou baixa prevalência, sendo de apenas 11% e 15%, respectivamente. Apesar da adequada metodologia empregada, estes resultados encontram-se no limite inferior das publicações vigentes. Nossos achados, apesar da limitada significância, colocam em discussão a real prevalência de VEGF e HER-2 e sua possível associação com o prognóstico. O aumento do tamanho da amostra e do período de seguimento podem fornecer mais informações a respeito da relevância destes marcadores em portadores de osteossarcoma.
OBJETIVO: Demonstrar a experiência de uma única instituição em hemipelvectomias internas sem reconstrução. Avaliar as cirurgias pélvicas preservadoras e as amputações interílio-abdominais e seu prognóstico.
MÉTODOS: 21 pacientes com tumores primitivos pélvicos submetidos à hemipelvectomia com ou sem preservação de membro. Sete foram tratados com hemipelvectomias externas (amputação) e 14 com internas, entre junho de 2004 e julho de 2009. A classificação cirúrgica utilizada foi a de Enneking para tumores pélvicos. O método de avaliação funcional foi o escore de ISOLS/MSTS.
RESULTADOS: A sobrevida dos pacientes em dois anos foi de 63,9%. A média de sobrevida do grupo todo foi de 43 meses. A avaliação funcional demonstrou que as hemipelvectomias preservadoras com ressecção do osso inominado obtiveram 12,5%, 62,5% e 25% de resultados ruins, bons e excelentes, respectivamente. Nos casos em que o osso inominado foi preservado, os resultados foram 16,7% e 83,3% bons e excelentes, respectivamente.
CONCLUSÕES: A hemipelvectomia é procedimento pouco usual e causador de importante limitação funcional e comorbidades. A alternativa de ressecar a hemipelve sem reconstrução tem demonstrado resultados tão bons quanto a não-reconstrução. Os elevados custos médicos, além das possíveis complicações com uso de enxerto e próteses justificam a técnica empregada neste artigo. Nível de Evidência IV, Estudo de caso-controle.
Descritores: Hemipelvectomia. Neoplasias. Amputação. Tumores de partes moles. Taxa de sobrevida.
Os ossos da região pélvica são localização comum de tumores malignos primitivos e lesões metastáticas.1 Os tumores malignos mais comumente encontrados nessa região são, em ordem de freqüência, o condrossarcoma, o sarcoma de Ewing e o osteossarcoma.2 Sarcomas primários da pelve são considerados de pior prognóstico quando comparados aos localizados em ossos longos.3
Com o advento de novas drogas quimioterápicas, radioterapia e novos métodos de diagnóstico, como a tomografia computadorizada, a ressonância magnética, e as novas técnicas cirúrgicas, houve aumento do número de pacientes que foram submetidos às cirurgias com preservação de membros.2,4,5
A hemipelvectomia externa, também conhecida com amputação interílioabdominal, é o tratamento clássico para lesões pélvicas e está historicamente associada a um pobre resultado funcional e psicológico. A literatura descreve um risco em torno de 50% a 80% de complicações relacionadas ao método, ou à doença, no seguimento da hemipelvectomia externa.6
O objetivo principal da cirurgia é a ressecção do tumor primário com margem oncológica, porém as cirurgias da região pélvica, apesar de todo o avanço na forma de abordagem e tratamento cirúrgico dos tumores malignos, apresentam uma taxa de recidiva em torno de 27% após o tratamento cirúrgico.7,8
A indicação de preservação de membro só é possível quando oferecer margem cirúrgica adequada, sem aumentar as chances de recidiva se comparada à amputação interilio-abdominal.2
A classificação cirúrgica de Enneking modificada para ressecções dos tumores pélvicos, utilizada neste artigo, é baseada na região do osso inominado ressecado, de posterior para anterior, dividindo as ressecções pélvicas em quatro tipos: Tipo I – ressecção do ilíaco, Tipo II – ressecção periacetabular, Tipo III – ressecção dos arcos anteriores, Tipo IV – ressecção em bloco de todo o ilíaco, também chamada Tipo I estendida. Os resultados funcionais distinguem-se quando a articulação fêmuro-acetabular é preservada ou ressecada. Cada tipo de hemipelvectomia é subdividido em quatro categorias, de acordo com a extensão da sua ressecção.3,4
O objetivo deste estudo foi descrever a experiência do grupo de ortopedia oncológica do Hospital de Câncer de Barretos no tratamento dos tumores pélvicos, avaliar o prognóstico dos pacientes submetidos à hemipelvectomia, estratificar em resultados funcionais nos diferentes tipos de ressecção e determinar a morbidade e mortalidade associados ao método.
MATERIAIS E MÉTODOS
Foi realizado estudo retrospectivo através de análise de prontuários de 21 pacientes entre novembro de 2004 e julho de 2009. Todos apresentavam em comum tumores pélvicos e haviam sido submetidos a hemipelvectomia interna ou externa na mesma instituição. Os casos de hemipelvectomia interna foram realizados sem reconstrução. Sete pacientes foram tratados com hemipelvectomia externa (clássica) e 14 com hemipelvectomia interna. As 21 peças de ressecção foram encaminhadas para análise anatomopatológica e apresentaram margens cirúrgicas livres.
O atendimento aos pacientes foi realizado por equipe assistencial multidisciplinar do Hospital de Câncer de Barretos.
Os pacientes portadores de osteossarcoma foram submetidos a tratamento quimioterápico pré e pós-operatório quando indicado, conforme o protocolo do Grupo Brasileiro para Tratamento do Osteossarcoma (GBTO). A maioria dos indivíduos portadores de condrossarcoma foram submetidos apenas à cirurgia com margem ampla, sem quimioterapia, devido às peculiaridades da histologia tumoral. Os portadores de fibrohistiocitoma maligno ósseo foram incluídos em protocolos semelhantes aos de osteossarcoma, porém com períodos de tratamento e doses menores de quimioterapia de acordo com a faixa etária.
A avaliação funcional foi baseada no escore da MSTS como proposta por Enneking et al.4 O escore é baseado em seis variáveis (dor, função, aceitação emocional, uso de sustentação como bengalas ou muletas, deambulação e marcha) sendo a cada um atribuídos no máximo 5 pontos. O somatório total pode ir até 30 pontos. O número de pontos do paciente é então dividido pelo valor máximo (30 pontos). Encontra-se, então, uma porcentagem que é expressa da seguinte forma: excelente (67% -100%), bom (50%-66%) e ruim (<50%) de acordo com um seguimento de no mínimo seis meses de pós-operatório. Todos pacientes foram orientados a permanecerem sem carga no pós-operatório por período entre 60 a 90 dias e incluídos em programa de reabilitação motora e proprioceptiva.
A análise dos dados foi realizada utilizando o software SPSS (Statistical Package for the Social Sciences) versão 17.0. As variáveis contínuas foram descritas através de média e desvio padrão e as variáveis categóricas foram descritas através de frequências absolutas e relativas. Para comparar as variáveis contínuas foi utilizada a Análise de Variância (ANOVA) one-way e para comparar as variáveis categóricas foi aplicado o teste qui-quadrado de Pearson. Para estimar a probabilidade de sobrevivência foi utilizado o método de Kaplan-Meier e para a comparação das curvas de sobrevida, o teste qui-quadrado de log-rank foi aplicado. O nível de significância estatística considerado foi de 5% (p < 0,05).
A amostra foi constituída por 21 pacientes com tumores da bacia e idade média de 38,1 anos (± 18,4) variando de 13 a 68 anos. A preponderância foi do sexo masculino (65%), nos estágios IIB e III (66,7%) e diagnóstico de Osteossarcoma (47,6%). O tempo médio de seguimento foi de 24,8 meses (±15,1) com variação entre 2 e 60 meses.
Apenas cinco pacientes (23,8%) apresentaram complicações pós-cirúrgicas, como necrose de pele e infecção superficial. Entre estes, apenas dois necessitaram reintervenção cirúrgica com debridamento e limpeza.
Do ponto de vista oncológico, as hemipelvectomias internas foram realizadas com intenção curativa. De acordo com a classificação cirúrgica de Enneking para hemipelvectomias internas, as ressecções foram do tipo I em quatro pacientes, do tipo II em três pacientes, duas ressecções foram do tipo III, três do tipo IV e duas do tipo I + II. O tempo médio de cirurgia foi de três horas e quinze minutos (90 – 300 minutos). A caracterização da amostra é apresentada na Tabela 1.
A Tabela 2 apresenta a situação dos pacientes na amostra total e por tipo de cirurgia. Observamos pior prognóstico nas hemipelvectomias externas, e melhor nas internas do tipo I e III.
Agrupamos as cirurgias em três grupos com intuito de identificar os resultados funcionais baseados em critérios anatômicos de ressecção envolvendo ou não o osso inominado. Os grupos foram divididos da seguinte forma: hemipelvectomias do tipo I e III formaram o primeiro grupo, onde a articulação fêmuro-acetabular foi preservada; as hemipelvectomias do tipo II, IV e I+II formaram o segundo grupo; e as hemipelvectomias externas o terceiro grupo. A caracterização dos grupos está descrita na Tabela 3.
Observamos diferença estatisticamente significativa entre os tipos de hemipelvectomia e a progressão da doença (p=0,030) e óbito (p=0,020). Em termos de prognóstico, ou seja, considerando-se como desfecho óbito e atividade da doença, as hemipelvectomias internas do tipo I e III foram melhor que as externas.
Dos 14 pacientes submetidos à hemipelvectomia interna, apenas dois foram a óbito por progressão ou atividade da doença. O primeiro óbito foi registrado seis meses após a cirurgia e o segundo após dois anos. Doze pacientes estão vivos (85%) e 10 deles permanecem em remissão oncológica (71,4%). Apenas dois pacientes necessitaram de UTI após a cirurgia (4,3%), sendo que um apresentava doença não oncológica pulmonar, e o outro, previamente hígido, porém evoluiu com infecção do trato urinário alto e febre.
O resultado funcional dos pacientes com cirurgia preservadora de membro foi avaliado após dois meses da ressecção, repetido ao completar seis meses e, finalmente, quando atingido um ano. Foi baseado no Sistema de Avaliação Funcional padronizado por Enneking et al.9 e validado pela Musculoskeletal Tumor Society (MSTS).
Pacientes submetidos às hemipelvectomias internas com ressecção do osso inominado, como as do tipo IV, II e I+II, apresentaram resultados funcionais inferiores às que preservaram a articulação coxo-femoral. A avaliação funcional demonstrou que as hemipelvectomias internas com ressecção do osso inominado obtiveram 12,5%, 62,5% e 25% de resultados ruins, bons e excelentes, respectivamente. (Figuras 1 e 2) Nos casos onde o osso inominado foi preservado, os resultados foram 16,7% e 83,3% bons e excelentes, respectivamente. (Figura 3) Reiteramos que não foram realizadas reconstruções com próteses ou enxerto estrutural.
Apesar de não realizarmos nenhum tipo de reconstrução, obtivemos resultados funcionais animadores para as ressecções pélvicas. Os indivíduos submetidos às cirurgias mais limitantes funcionalmente como às hemipelvectomias internas do tipo I+II, II e IV surpreenderam positivamente, retornando às atividades diárias com boa aceitação emocional.
A indicação de amputação interílioabdominal foi baseada nas dimensões do tumor, invasão de partes moles e feixe vásculo-nervoso. Pacientes em que os exames de imagem e a análise clínica não oferecerem margem de segurança para preservação foram amputados. (Figura 4) Os sete casos amputados apresentavam inviabilidade de preservação, sendo que os resultados em termos de prognóstico também foram piores. Dois pacientes amputados necessitaram de UTI (28 %) no pós-operatório imediato devido a complicações cardiorrespiratórias.
A curva de sobrevida, através do método de Kaplan-Meier, da amostra total de pacientes é apresentada na Figura 5. A chance de sobrevida dos pacientes foi de 85,4% (IC 95%: 70,1% a 100%) e 63,9% (IC 95%: 42,3% a 85,5%) em 12 e 24 meses, respectivamente. A média de sobrevida do grupo todo foi de 43 meses (IC 95%: 32,9 a 53,2). Observamos diferenças importantes entre os grupos hemipelvectomias que devem ser destacadas. No grupo de hemipelvectomias do tipo I / III a chance de sobrevida foi de 100%, não sendo possível calcular o intervalo de confiança, pois não houve nenhum óbito neste grupo. No grupo de cirurgias dos tipos II, IV e I+II, a chance de sobrevida em um ano foi de 87,5% (IC 95%: 64,6% a 100%), em dois anos de 72,9% (IC 95%: 40,6% a 100%) e em cinco anos a probabilidade foi a mesma dos dois anos. No entanto, no grupo de hemipelvectomias externas a chance de sobrevida em um ano foi de 71,4% (IC 95%: 37,9% a 100%), decaindo para 28,6% (IC 95%: 0% a 62,1%) em dois anos. A probabilidade em cinco anos também foi idêntica a dos dois anos. (Figura 6) Quando avaliado o tempo de sobrevida pela atividade da doença (Figura 7), houve diferença estatisticamente significativa (p<0,001). Os pacientes com a doença ativa, também chamada de progressão da doença, tiveram chance de sobrevida de 66,7% (IC 95%: 35,9% a 97,4%) e 22,2% (IC 95%: 0% a 49,4%) em 12 e 24 meses, respectivamente. Em cinco anos novamente a chance de sobrevida foi a mesma dos dois anos. Estes valores já eram esperados, pois é consenso na literatura mundial que o prognóstico está intimamente relacionado à progressão da doença.
A hemipelvectomia interna oferece ao paciente portador de lesão pélvica melhor função e menor prevalência de complicações pós-cirúrgicas, quando comparada à hemipelvectomia clássica. A opção de reconstruir ou não o anel pélvico com uso de enxerto estrutural ou endoprótese depende da experiência do cirurgião e de sua equipe. Não há consenso na literatura quanto ao que seria melhor em termos de resultados funcionais e complicações quando comparados os métodos de ressecção com e sem reconstrução. O’Connor e Sim et al.10 compararam o uso de técnica de artrodese a não reconstrução do anel pélvico e encontraram melhores resultados a favor da artrodese. No entanto, Hillmann et al.11 encontraram 37% de maus resultados em reconstruções com endopróteses e amputações contra 79% de bons resultados quando não realizada reconstrução. O custo do tratamento também tem se mostrado elevado em casos onde se opta por reconstrução devido às complicações e preço de implantes. Complicações frequentemente identificadas em reconstruções são a fratura do enxerto ósseo, infecção, soltura do implante protético e pseudoartroses. Nos pacientes submetidos à hemipelvectomia sem reconstrução observamos maior discrepância de membros quando realizada ressecção do osso inominado que em média atinge 6 a 10 cm como descrito na maioria dos artigos.12
Os resultados funcionais de nosso grupo nas hemipelvectomias sem reconstrução encorajam o cirurgião ortopedista oncológico a executar o procedimento. Identificamos baixas taxas de infecção (23,8%) e, sem dúvidas, menor custo operacional. O tempo de execução da cirurgia sem a necessidade de reconstruir também foi menor, em torno de 3 horas e 15 minutos de média. As complicações decorrentes de reconstruções com autoenxertos, aloenxertos, placas e endopróteses não justificam o ganho funcional ou psicológico em nossa opinião.
A sobrevida dos pacientes portadores de tumores pélvicos em nossa instituição é muito semelhante aos demais trabalhos publicados em nível nacional.12 A peculiaridade de nosso hospital em receber pacientes de estados do Brasil que não oferecem adequado suporte assistêncial à saúde piora o prognóstico e dificulta a indicação de cirurgias preservadoras. Por este motivo, 1/3 dos nossos pacientes foram submetidos à desarticulação da hemipelve.
Somos favoráveis às hemipelvectomias sem reconstrução, independente do envolvimento do osso inominado pela doença. Os nossos resultados falam a favor desta afirmação, sendo que o objetivo de demonstrar as vantagens da hemipelvectomias sem reconstrução foram atingidos.
1. Malawer MM, Sugarbaker PH. Musculoskeletal cancer surgery. Treatment of sarcomas and allied diseases. Dubai: Kluwer Academic Publishers; 2001.
2. Lopes A, Penna V, Rossi BM, Chung WT, Tanaka MH. Hemipelvectomia total interna no tratamento dos tumores malignos da região pélvica. Rev Bras Ortop. 1994;29:11-2.
3. Campanacci M, Capanna R. Pelvic resections: the Rizzoli Institute experience. Orthop Clin North Am. 1991;22:65-86.
4. Enneking WF. Limb salvage in musculoskeletal oncology. New York: Churchill-Livingstone; 1987.
5. Nielsen HK, Veth RP, Oldhoff J, Koops HS, Scales JT. Resection of a periacetabular chondrosarcoma and reconstruction of the pelvis. A case report. J Bone Joint Surg Br. 1985;67:413-5.
6. Beck LA, Einertson MJ, Winemiller MH, DEPompolo RW, Hoppe KM, Sim FF. Functional outcomes and quality of life after tumor-related hemipelvectomy. Phys Ther. 2008;88:916-27.
7. Enneking WF, Dunham WK. Resection and reconstruction for primary neoplasms involving the innominate bone. J Bone Joint Surg Am. 1978;60:731-46.
8. Healey JH, Lane JM, Marcove RL, Duane K, Otis JC. Resection and reconstruction of periacetabular malignant and aggressive tumors. In: Yamamuro T. New developments for limb salvage in musculoskeletal tumors. Tokyo: Springer Verlag; 1989. p. 443-50.
9. Enneking WF, Dunham W, Gebhardt MC, Malawar M, Pritchard DJ. A system for the functional evaluation of reconstructive procedures after surgical treatment of tumors of the musculoskeletal system. Clin Orthop Relat Res. 1993;(286):241-6.
10. Babis GC, Sakellariou VI, O’Connor MI, Hanssen AD, Sim FH. Proximal femoral allograft-prosthesis composites in revision hip replacement: a 12-year follow-up study. J Bone Joint Surg Br. 2010;92:349-55.
11. Hillmann A, Hoffmann C, Gosheger G, Rödl R, Winkelmann W, Ozaki T. Tumors of the pelvis: complications after reconstruction. Arch Orthop Trauma Surg. 2003;123:340-4.
12. Lackman RD, Crawford EA, Hosalkar HS, King JJ, Ogilvie CM. Internal hemipelvectomy for pelvic sarcomas using a T-incision surgical approach. Clin Orthop Relat Res. 2009;467:2677-84.
OBJETIVO: As endopróteses parciais de joelho para as ressecções em sarcomas ósseos demonstram serem boa solução para o tratamento de pacientes com imaturidade esquelética. O objetivo deste estudo é avaliar o escore funcional, as vantagens, as desvantagens e indicações para esta técnica cirúrgica em quatorze pacientes em um protocolo brasileiro de osteossarcoma e sarcoma de Ewing.
MÉTODOS: Análise retrospectiva realizada para identificar a evolução funcional e as possíveis complicações do procedimento. 14 pacientes com idade entre 10 e 22 anos avaliados funcionalmente pelos critérios de Enneking/ISOLS (International Society of Limb Salvage), sendo todos operados na mesma Instituição e pelo mesmo cirurgião. Foram utilizadas endopróteses parciais das extremidades distal do fêmur e proximal da tíbia com reconstrução ligamentar.
ReSULTADOS: A análise do escore funcional de Enneking/ISOLS demonstrou 78,6 % de excelentes resultados e 21,4% de bons. Dos 14 pacientes, todos portadores de tumores primitivos ósseos em protocolo de quimioterapia, nove não apresentaram nenhum tipo de complicação e cinco indivíduos evoluíram com complicações relacionadas ao procedimento, sendo que houve relação estatística positiva entre os maus resultados e a presença de complicações (p=0,027).
CONCLUSÃO: As endopróteses parciais de joelhos são menos prejudiciais ao estoque ósseo de pacientes com esqueleto imaturo. As críticas sobre os maus resultados funcionais estão sendo suplantadas pelas novas técnicas de reconstrução, corretos protocolos de reabilitação, qualidade e tecnologia dos implantes, e o aumento da curva de aprendizado. Essa opção de tratamento per-mite a preservação do estoque ósseo e a possibilidade de revisão da artroplastia não convencional de modo menos agressivo.
Descritores: Joelho; Sarcoma de Ewing; Osteossarcoma; Prótese do joelho; Estudos retrospectivos
Os tumores ósseos primários malignos mais comuns na infância e da adolescência são o osteossarcoma e o sarcoma de Ewing. Apresentam como um dos principais sítios de localização a extremidade distal do fêmur e proximal da tíbia. Estas localizações comprometem muitas vezes a articulação do joelho, necessitando, de cirurgias preservadoras com a substituição dos segmentos por endopróteses. Diversos modelos de endopróteses estão disponíveis para as mais variadas indicações cirúrgicas nas ressecções de tumores ósseos do joelho(1). No entanto, em casos onde o tumor não respeita os limites da cartilagem de crescimento, invadindo a epífise dos ossos longos do joelho, sem invasão articular, é possível indicar a ressecção com substituição por endoprótese parcial. Esta técnica permite a ressecção em bloco da extremidade distal do fêmur ou proximal da tíbia, preserva a epífise adjacente articular e substitui apenas o segmento afetado pelo implante fixado no fêmur ou na tíbia.
A utilização de endopróteses parciais se restringe a pacientes portadores de tumores com as características descritas acima e com imaturidade esquelética na faixa etária entre 10 a 16 anos. Indivíduos com idade entre 17 e 22 anos também se beneficiam das endopróteses parciais devido à preservação do estoque ósseo e ao crescimento residual até próximo do limite etário de 22 anos(2). Pacientes muito jovens, que não iniciaram o segundo estirão de crescimento, submetidos à substituição por implantes no membro inferior, com o passar dos anos apresentarão discrepâncias incompatíveis com a funcionalidade da extremidade inferior. Na opinião dos autores, nesses casos é mais prudente a cirurgia radical (amputação). Já em indivíduos que completaram o crescimento, as vantagens de preservar a região epifisária são muito menores, sendo mais indicada a endoprótese total de joelho(3,4).
As ressecções com margem oncológica para tumores ósseos primários da infância e adolescência em passado não muito distante eram sinônimo de amputação de membro. O desenvolvimento de novas técnicas cirúrgicas, melhores condições hospitalares, a introdução da quimioterapia neo-adjuvante com protocolos bem definidos, o aperfeiçoamento dos tipos de implantes cirúrgicos e a curva de aprendizado dos cirurgiões ortopedistas proporcionaram mais segurança e qualidade de vida aos portadores dessas enfermidades(3,5-9).
O aumento da sobrevida livre de doença e cura em tumores como o sarcoma de Ewing e o osteossarcoma trouxe a preocupação da vida útil do implante utilizado(10). Implantes como as endopróteses totais de joelho em pacientes jovens apresentam a desvantagem de necessitar de ressecção ou osteotomia femoral/tibial para fixação do implante no segmento adjacente, e por conseguinte, remover a região de crescimento meta-epifisária. Isso implica em discrepância de crescimento das extremidades inferiores, diminuição do estoque ósseo e complicações futuras para revisão do implante devido à cimentação e a ressecção de osso não acometido por tumor.
Com o objetivo de reduzir complicações como às descritas e avaliar a funcionalidade e características dos pacientes submetidos a esta indicação de exceção, analisamos os casos onde foram utilizados implantes parciais não articulados (endopróteses parciais) de joelho em pacientes jovens, associados a reconstrução ligamentar, nas ressecções com margem oncológica na extremidade distal do fêmur e proximal da tíbia.
Todos os pacientes foram operados pelo grupo de oncologia ortopédica do Hospital de Câncer de Barretos, SP. Foram avaliados de modo retrospectivo 14 pacientes incluídos nos Protocolos Brasileiros de Osteossarcoma e Ewing, com idade entre 10 e 22 anos, submetidos a ressecção da extremidade distal do fêmur ou proximal da tíbia devido a tumores ósseos primários com substituição por endoprótese parcial de joelho não articulada e reconstrução ligamentar.
A indicação cirúrgica foi baseada em características morfológicas do tumor no joelho, ou seja, tumores localizados no fêmur distal ou tíbia proximal com invasão da cartilagem de crescimento e epífise, mas sem comprometimento articular visível à Ressonância Magnética. A presença de metástases pulmonares não foi critério de exclusão.
Todos os casos foram operados com critérios oncológicos no período entre fevereiro de 2003 e fevereiro de 2008 na mesma Instituição e pelo mesmo cirurgião. Os 14 pacientes apresentaram margens cirúrgicas livres no exame anátomo-patológico.
Os implantes de escolha foram: Endoprótese para extremidade distal do fêmur Parcial Não Articulada (Impol®) para tumores da extremidade distal do fêmur e Endoprótese de extremidade distal da tíbia Parcial Não Articulada (Impol®) para as neoplasias da extremidade proximal da tíbia. O implante é constituído de uma liga metálica de cromo-cobalto-molbidênio em sua superfície articular, reduzindo ao mínimo o atrito com a cartilagem normal do segmento adjacente. O corpo da endoprótese foi fabricado em polietileno de ultra alto peso molecular e a haste femoral ou tibial confeccionada em liga de titânio. A fixação do implante ao osso foi feita com cimento ósseo radiopaco.
A avaliação funcional foi baseada no escore como proposto por Enneking et al (11). O escore é baseado em seis variáveis (dor, função, aceitação emocional, uso de sustentação como bengalas ou muletas, deambulação e marcha) sendo a cada uma atribuídos no máximo 5 pontos. O somatório total pode ser de até 30 pontos. O número de pontos do paciente é então dividido pelo valor máximo (30 pontos). Encontra-se, então, uma porcentagem que é expressa da seguinte forma: excelente (67% -100%), bom (50%-66% ) e ruim (<50%) de acordo com um seguimento de no mínimo seis meses de pós-operatório. Todos pacientes foram orientados a permanecerem com um tutor no pós-operatório por período entre 60 a 90 dias sem carga e, após, foram incluídos, pelos autores, em protocolo de reabilitação para ganho de arco de movimento, propriocepção e reforço muscular(12,13).
As variáveis quantitativas foram descritas por meio de média ± desvio padrão e as variáveis qualitativas por freqüências absolutas e relativas.
Para avaliar possíveis associações entre as variáveis categóricas foi utilizado o teste exato de Fisher. Para comparar médias, foi utilizado o teste t de student.
O nível de significância adotado foi de 5% (p<0,05) e as análises foram realizadas no programa SPSS (Statistical Package for the Social Sciences) versão 13.0.
Nos 14 pacientes avaliados, a média de idade foi de 13,5±3,5 anos. Quanto ao sexo, 10 dos pacientes (71,4%) eram do sexo feminino e quatro (28,6%) do masculino. Neste grupo, osteossarcoma aparecia em doze dos pacientes (85,7%), enquanto um (7,1%) pertencia ao grupo dos sarcomas de Ewing, e um (7,1%) teve como diagnóstico fibrohistiocitoma maligno (protocolo de tratamento igual ao osteossarcoma). Nove dos tumores (64,3%) se localizava na extremidade distal do fêmur cinco (35,7%) na extremidade proximal da tíbia. A maioria dos pacientes (57,1%) eram procedentes do Estado de São Paulo.
O estudo foi aprovado pelo Comitê de Ética em Pesquisa do Hospital de Câncer de Barretos – Fundação Pio XII.
A análise dos dados obtidos demonstrou que onze dos pacientes (78,6%) obtiveram escore de Enneking excelente, e que três (21,4 %) apresentaram resultado bom. A presença de complicações foi baixa, todas resolvidas no pós-operatório recente. Dos 14 pacientes, nove não apresentaram qualquer complicação em relação à artroplastia; um apresentou infecção superficial; um apresentou instabilidade articular, com subluxação, provavelmente devido a retirada muito precoce da imobilização na cidade de origem; outros três evoluíram com complicações como: úlcera de pressão e ruptura do tendão patelar na prótese tibial. Dos pacientes com escore bom, 100% apresentaram complicações pós-operatórias (p=0,027), portanto as complicações no transcorrer do tratamento diminuíram a funcionalidade do joelho (Gráfico 1).
O tempo médio de imobilização foi de 9,76±3,3 semanas (máximo 16,4 e mínimo 3,8 semanas) e o tempo médio de seguimento dos pacientes foi de 23,1±15,8 meses (máximo 68,2 e mínimo 2,2 meses). Verificamos que o grupo com maior tempo de imobilização apresentou melhor escore funcional (p=0,048) (Gráfico 2).
Não identificamos correlação estatística entre a idade dos pacientes e o escore funcional. Os indivíduos mais jovens não obtiveram melhor funcionalidade do joelho.
A localização do tumor ou na extremidade proximal da tíbia ou distal do fêmur não foi responsável por variação no escore de Enneking com significância estatística.
No seguimento dos pacientes, identificamos onze (78,6%) que estão vivos e três (21,4 %) que foram a óbito devido à progressão da doença (Tabela 1).
A introdução da quimioterapia neoadjuvante nos anos 80 aumentou muito a possibilidade de ressecção tumoral com preservação de membro. Mais de 80% dos pacientes com osteossarcoma de extremidade se tornaram candidatos a cirurgia preservadora de membro(3). A cirurgia preservadora para tumores ósseos primários muito volumosos necessita de grandes ressecções no nível do joelho e criam defeitos segmentares importantes que necessitam de algum tipo de substituição que conserve a funcionalidade articular (Figuras 1 e 2). Os métodos de substituição podem ser os mais variados, entre eles estão alternativas biológicas, como o uso de enxerto livre ou vascularizado do próprio paciente ou de outros indivíduos (aloenxerto). Outras opções incluem as endopróteses distais do fêmur e proximais da tíbia. Entre as diferentes endopróteses comercializadas podemos descrever as articuladas fixas, rotatórias e as do tipo artrodese. Algumas utilizam cimento para a fixação femoral e outras são fixadas pelo método press-fit(4).
A indicação cirúrgica para tumores localizados na extremidade distal do fêmur e proximal da tíbia depende da relação anatômica da neoplasia com as estruturas que fazem parte do joelho normal. Tumores que invadem a articulação do joelho tornam o paciente candidato ressecção extra-articular com ou sem artrodese, e conseqüentemente, restrição funcional parcial ou total. As neoplasias que não invadem a articulação, mas que comprometem a cartilagem de crescimento e a epífise, limitam as alternativas cirúrgicas e obrigam o cirurgião ortopedista a lançar mão de certos procedimentos mais específicos. Entre eles, o aloenxerto osteoarticular foi descrito como boa alternativa em trabalho realizado por Muscolo et al(14) com 80 pacientes portadores de tumores na extremidade distal do fêmur submetidos a esse método e com seguimento de cinco a 10 anos. Essa alternativa, no entanto, é descrita por muitos autores apresentando complicações como fratura do enxerto, pseudoartrose, infecção, osteoartose secundária a osteonecrose condilar(15-17).
Uma alternativa que é usada há muito tempo para tumores localizados no joelho de jovens são as endopróteses totais articuladas para extremidade distal do fêmur e proximal da tíbia. Este implante fornece estabilidade, retorno mais rápido às atividades e melhor qualidade de vida ao portador de tumores ósseos. No entanto, em pacientes esqueleticamente imaturos seu uso compromete a epífise do osso adjacente , resultando em diminuição do estoque ósseo e piora da discrepância entre os membros inferiores. A indicação deste tipo de implante se encaixa melhor em indivíduos que não apresentam mais cartilagem de crescimento aberta ou que já estejam encerrando, pelo menos, o segundo estirão de crescimento(2-4,18).
Optamos nesse estudo por um implante que substituiu apenas a extremidade distal do fêmur ou a extremidade proximal da tíbia (Figuras 3, 4 e 5). Todos pacientes apresentavam invasão tumoral da cartilagem de crescimento, mas sem penetrar na cavidade articular ou extensão para ligamentos cruzados. O implante permitiu a preservação da epífise do osso adjacente (tíbia ou fêmur), reduzindo o risco de discrepância e problemas futuros com o pouco estoque ósseo nas revisões da prótese. Foi necessária a reconstrução ligamentar dos cruzados e colaterais; além do tendão patelar nas substituições da extremidade proximal da tíbia. Apesar da necessidade de reconstrução dos ligamentos, o tempo cirúrgico permaneceu muito semelhante ao das endopróteses totais de joelho, pois não se perdeu tempo com osteotomia do segmento adjacente.
Não há artigos que descrevam este implante parcial sendo avaliado isolado das endopróteses totais de joelho. O método apresenta uma série de vantagens em relação aos implantes articulados em pacientes com imaturidade esquelética. Clinicamente, a avaliação dinâmica da marcha se assemelha muito às artroplastias não convencionais totais(19,20). Apesar de haver certo grau de hiperlaxidão estática, não há instabilidade durante o ortostatismo ou deambulação. A ação muscular nas fases de apoio e balanço mantém o joelho estável. Esse estudo de 14 pacientes demonstra excelentes resultados na avaliação funcional, sendo que os indivíduos que evoluíram com alguma complicação acabaram apresentando escore inferior. A análise biomecânica na marcha in vivo de endopróteses parciais necessita ainda investigação mais aprimorada, sendo esse estudo realizado no Hospital de Câncer de Barretos um dos primeiros passos para a afirmação dessa técnica cirúrgica.
As endopróteses parciais de joelho proporcionam ao ortopedista e ao paciente um método preservador da extremidade com excelente funcionalidade, manutenção do estoque ósseo para revisão, e redução de discrepâncias em indivíduos esqueleticamente imaturos.
1. Kawai A, Muschler GF, Lane JM, Otis JC, Healey JH. Prosthetic knee replacement after resection of a malignant tumor of the distal part of the femur: medium to long-term results. J Bone Joint Surg Am. 1998;80(5):636-47.
2. Behrman K, Jenson. N. Tratado de pediatria. 17°ed. Rio de Janeiro: Elsevier; 2005.
3. Futani H, Minamizaki T, Nishimoto Y, Abe S, Yabe H, Ueda T. Long term follow-up after limb salvage in skeletally immature children with a primary malignant tumor of the distal end of the femur. J Bone Joint Surg. 2006;88(3):595-603.
4. Krepler P, Dominkus M, Toma CD, Kotz R. Endoprosthesis management of the extremities of children after resection of primary malignant bone tumors. Orthopade. 2003;32(11):1013-9.
5. Almeida MTA. Tratamento combinado do Sarcoma de Ewing: análise do Protocolo Ewing –II –94 [tese]. São Paulo: Faculdade de Medicina da Universidade de São Paulo; 2000.
6. Croci AT, Camargo OP, Oliveira NRB. Tratamento cirúrgico do sarcoma de Ewing: avaliação oncológica funcional. Rev Bras Ortop. 1996;31(11):909-18.
7. Penna V, Lopes A, Tanaka MH, Wu TC, Melaragno R, Epelman S. Osteossarcoma: tratamento multidisciplinar. Rev Bras Ortop. 1993;28(11/12):791-4.
8. Petrilli AS, Macedo CRD. Tumores ósseos malignos na criança e no adolescente. Pediatria Moderna.1999; 35:600-8.
9. Próspero JD. Tumores ósseos. São Paulo: Roca; 2001.
10. Rougraff BT, Simon MA, Kneisl JS, Greenberg DB, Mankin HJ. Limb salvage compared with amputation for osteosarcoma of the distal end of the femur. A long-term oncological, functional, and quality-of-life study. J Bone Joint Surg Am.1994; 76(5):649-56.
11. Enneking WF, Dunham W, Gebhardt MC, Malawar M, Pritchard DJ. A system for the functional evaluation of reconstructive procedures after urgical treatment of tumors of the musculoskeletal system. Clin Orthop Relat Res. 1993;(286):241-6.
12. Schultz K, Souza RV. Fisioterapia. Kowalski LP. Manual de condutas diagnósticas e terapêuticas em oncologia. 2ª. ed. São Paulo: Âmbito; 2002.
13. Tsai LY, Jesus-Garcia Filho R, Petrilli AS, Korukian M, Viola DCM, Petrilli M, et al. Protocolo fisioterapêutico em pacientes submetidos à endoprótese não convencional de joelho por osteossarcoma: estudo prospectivo. Rev Bras Ortop. 2007;42(3):64-70.
14. Muscolo DL, Ayerza MA, Aponte-Tinao LA, Ranalletta M. Use of distal femoral osteoarticular allografts in limb salvage surgery. Surgical technique. J Bone Joint Surg Am. 2006;88(Suppl 1 Pt 2):305-21
15. Harrington KD, Johnston JO, Kaufer HN, Luck JV Jr, Moore TM. Limb salvage and prosthetic joint reconstruction for low-grade and selected high-grade sarcomas of bone after wide resection and replacement by autoclaved autogenic grafts. Clin Orthop Relat Res. 1986;(211):180-214.
16. Nishida J, Shimamura T. Methods of reconstruction for bone defect after a tumor excision: a review of alternatives. Med Sci Monit. 2008;14(8):RA107-13.
17. Komiya K, Nasuno S, Uchiyama K, Takahira N, Kobayashi N, Minehara H, et al. Status of Bone Allografting in Japan – Nation-Wide Survey of Bone Grafting Performed from 1995 through 1999. Cell Tissue Bank. 2003;4(2-4):217-20.
18. Roberts P, Chan D, Grimer RJ, Sneathe RS, Scales JT. Prosthetic replacement of the distal femur for primary bone tumors. J Bone Joint Surg Br. 1991;73(5):762-9.
19. Kabo JM, Yang RS, Dorey FJ, Eckardt JJ. In vivo rotational stability of the kinematics rotating hinge knee prosthesis. Clin Orthop Relat Res 1997;(336):166-76.
20. Dennis DA, Komistek RD, Scuderi GR, Zingde S. Factors affecting flexion after total knee arthroplasty. Clin Orthop Relat Res. 2007;(464):53-60.