Ewing sarcoma and osteosarcoma are high-grade sarcomas of bone and bone/soft tissue, with peak incidence in adolescents and young adults. Although metastatic disease currently remains the most meaningful prognostic indicator, efforts are underway to better delineate biologic subgroups associated with treatment response and resistance in both Ewing sarcoma and osteosarcoma. Novel risk-stratified treatment approaches for bone sarcomas are emerging and will inform disease management and future clinical trial enrollment.¹ Molecular profiling is an essential component of the emerging proposals for bone sarcoma risk stratification, similar to other pediatric (eg, Wilms tumor, neuroblastoma, soft tissue sarcomas) and adult solid tumors (eg, breast cancer, lung adenocarcinoma) for which molecular biomarker status now guides standard-of-care therapy.^2–7 Indeed, international clinical guidelines for bone sarcoma now recommend the routine acquisition of snap frozen and fresh tissue for clinical molecular studies.^8–10 To achieve this vision for patients with Ewing sarcoma and osteosarcoma, the field must ensure standard clinical practices for the expert procurement, processing, and evaluation of tumor biomaterials for all patients.

However, patients diagnosed with Ewing sarcoma and osteosarcoma receive cancer care at a variety of institutions, including adult and pediatric hospitals and academic and community centers, and care is delivered by pediatric and medical oncology specialists.¹¹ In fact, the diagnostic center may differ from the facility where the patient receives treatment. Significantly more errors and complications have been reported when biopsies are performed outside of the treatment center.¹² Decentralized and fragmented care threatens consistency in procurement and processing practices of diagnostic biopsy/biomaterials. Variability in the amount and usability of material acquired from biopsies of primary bone tumors limits advances in clinical care and research. Furthermore, sample processing requirements of bone tumors relative to other tumor types heighten the need for a standardized approach to tissue collection. Although open/incisional biopsies are considered the gold standard technique, image-guided core needle biopsies are increasingly used for the acquisition of diagnostic biopsy material for bone tumors, and in many centers have become the dominant approach to biopsy.¹³ Given the potential for variable amounts of tissue procured through this approach, components of clinical care, including pathologic assessment, diagnostic accuracy, and molecular evaluations, along with additional research studies with leftover tissue (following informed consent), can be negatively impacted if inadequate tissue is obtained. Indeed, a recent review of Ewing sarcoma and osteosarcoma specimens in the Children’s Oncology Group (COG) biorepository identified quality assurance failures due to diagnostic discordance (4% of cases) or lack of viable tumor (7% of cases). Among cases with diagnostic concordance, variable volumes of tumor were present, including cases with scant viable tumor tissue available for additional testing.¹⁴ In addition to the variability noted in the amount of viable tumor acquired at diagnosis, there is often variability in tissue processing and allocation practices between institutions, resulting in the unintended procurement of nondiagnostic, acid-degraded, or insufficient volumes of Ewing sarcoma and osteosarcoma tumor material. The allocation and processing of tumor biopsy material affect the available options for subsequent clinical and research analyses, importantly including quality molecular analysis.

To address these points, the COG Bone Tumor Committee convened a multidisciplinary group of experts to evaluate available literature and institutional practices to generate the following guidance for the acquisition of Ewing sarcoma and osteosarcoma biopsy material. Current and near-future biopsy tissue requirements in the field are highlighted, with a lens toward ensuring that patients maintain access to clinical trial opportunities, which is a standard-of-care offering for patients with Ewing sarcoma and osteosarcoma. This guidance may require practice changes at institutions, with the understanding that clinical decisions about tissue acquisition at the individual case level must be based on tumor location and patient condition.

Biopsy Planning for Suspected Ewing Sarcoma and Osteosarcoma

When Ewing sarcoma or osteosarcoma is suspected, prebiopsy planning with pediatric oncology, radiology, interventional radiology, pathology, and orthopedic oncology or pediatric surgery is necessary to ensure that the necessary tissue specimens are obtained and delivered to pathology promptly (ie, coordinating with the on-call pathologist or arranging a STAT courier to minimize warm ischemia time) while prioritizing patient safety (Figure 1). This may result in practice or protocol changes at individual institutions. Given the importance of multidisciplinary planning for patients with Ewing sarcoma and osteosarcoma, referral to specialty institutions is recommended.¹² As with any procedure, patients or family must provide informed consent for the invasive collection of tumor material. The recommended tissue volumes described in the following discussion ensure equivalency regardless of biopsy approach and tissue collection necessary for modern tumor assessment and emerging disease stratification. Of note, efforts to obtain adequate clinical specimens should be differentiated from those of research-only biopsies, for which the tissue is not processed as a diagnostic specimen and which require separate informed consent for enrollment on specific Institutional Review Board (IRB)–approved research protocol(s). Collection of residual tissues for biobanking or submission to tissue repositories likewise requires IRB-approved informed consent. Recently the Fight Osteosarcoma Through European Research (FOSTER) and EURO EWING Consortium (EEC) provided guidance on biologic sample collection to specifically advance research and highlight patients’ appetite for participating in these studies.¹⁵

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Prebiopsy multidisciplinary discussion recommendations. A multidisciplinary discussion for the management of patients undergoing biopsy of a suspected Ewing sarcoma or osteosarcoma is needed to coordinate appropriate preoperative imaging, future oncologic/local control surgical planning, pathology workflow including timely tissue processing and allocation (eg, histology, molecular and research studies), clinical trial eligibility considerations, and acquisition of biopsy tissue specimen reagents (eg, liquid nitrogen, dimethyl sulfoxide [DMSO], media).

Citation: Journal of the National Comprehensive Cancer Network 23, 1; 10.6004/jnccn.2024.7063

Establishing the quantity of tumor tissue material required prior to the procedure is instrumental in ensuring that all desired tissue-based analyses can be performed. This is especially important when using image-guided core needle biopsy techniques, because they typically yield smaller tumor samples compared with open or incisional biopsies. In both North America and Europe, there is consensus that bone sarcomas with an associated large soft tissue component are generally safe to biopsy, and for such cases, procuring diagnostic material is typically quite feasible, as has been demonstrated in soft tissue sarcomas.^16,17 However, there are cases in which tumors arise in anatomically sensitive locations and biopsy can carry greater risk. For example, for a patient with Ewing sarcoma arising from a vertebral body or rib, it may not be possible or safe to increase the amount of tumor material obtained. Although studies of soft tissue sarcomas have shown equivalent rates of local recurrence when comparing core versus open biopsies,¹⁸ the rates of biopsy tract seeding are much lower with core needle biopsies. We still advocate for careful biopsy site planning, including limiting to one biopsy site, with the tract ideally placed in the same location as the planned incision for eventual definitive resection or in a location where the tract can be safely resected to reduce the risk of local recurrence. The specific anatomy of the biopsy tract will vary based on whether the primary tumor is in the extremity or the trunk, and should be explicitly discussed in a multidisciplinary fashion. These cases underscore the need for prebiopsy planning and coordination with pathology to ensure specimens that can be obtained undergo appropriate processing and allocation to maximize specimen utility.

Image-Guided Core Needle Biopsies

Image-guided techniques for the acquisition of biopsy material of suspected Ewing sarcoma and osteosarcoma are often performed by interventional radiologists.¹⁹ The safety of image-guided core needle biopsy has been established in prospective and retrospective studies without additional safety events.^20–22 When core biopsy is used for sample collection, the volume of viable tissue obtained for future use is determined by the number of cores collected, gauge of needle used,²³ length of the core (>10 mm superior to <5 mm²⁴), and viability of tumor in the area sampled. However, this method of tissue acquisition lacks standardization. Discussions with members of the COG Bone Tumor Committee revealed differences across institutions in sampling techniques, such as location sampled and the number and length of biopsy cores obtained. Standard recommendations are needed to ensure this diagnostic approach results in sufficient tissue for the patient’s clinical needs as well as desired research purposes.

Ideally, specimens are acquired from an area presumed to contain viable tumor tissue. Intraoperative tumor viability checks are not available at every institution, in which case viability is assessed by pathology postprocedurally (see later section on “Pathology/Specimen Processing Plan”). Intraprocedure imaging is often used to guide sampling of different portions of the tumor to increase the likelihood of acquiring viable material.¹⁹ Given that fine-needle aspirates (FNAs) are inadequate alone for the diagnosis of osseous lesions,^25,26 we do not recommend using FNAs for the diagnosis of suspected Ewing sarcoma or osteosarcoma.

When targeting a suspected viable tumor site, we provide the following recommendations. For bone tumors with a soft tissue component, obtain biopsies from the soft tissue whenever feasible, with the aim of collecting a total of 15 to 20 cores measuring 1.5 to 3 cm using a 16-gauge needle. We recognize that some institutions prefer alternative gauge sizes. The overarching goal of this recommendation is to acquire an equivalent volume of tissue as that acquired through open biopsies (1–3 cm³ or 1–3 g of tissue). Automated or vacuum-assisted biopsy devices are available at many institutions and can increase the speed at which multiple biopsies can be obtained.^27–29 Because many downstream testing applications require the presence of viable tumor (sometimes >50%), the recommended ≥15 cores help to achieve adequate viable tumor acquisition even when some cores are small (<1 cm) and mostly necrotic. For bone tumors without a soft tissue component (or the soft tissue component cannot be adequately accessed), we recommend collecting 5 to 7 cores for bone biopsies using a 12- to 13-gauge needle. This number of cores is feasible and does not require a second skin incision (although a second cortical hole is needed in rare cases). When osteosarcoma is suspected, acquire an additional 2 to 3 cores of the underlying osteoid using a 12- to 13-gague needle. These core totals would cover all clinical and research/clinical trial–based needs, including 2 to 4 blocks with 2 to 3 cores per block for clinical diagnostic needs, snap frozen tissue (∼0.5 g per vial), and fresh or viably frozen tumor material. Additional allocation details are provided in Figure 2. An overview of clinical and downstream uses of Ewing sarcoma and osteosarcoma biopsy tissue is provided in Figure 3. Specimens should ideally be submitted to pathology on saline-dampened gauze/Telfa or, if not available, in a dry container (no formalin). Effective coordination with pathology is crucial so that tissue does not desiccate in the container and can be promptly processed within the pathology laboratory.

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Pathology processing recommendations for Ewing sarcoma and osteosarcoma biopsies. The material should encompass all clinical needs as well as those related to consented clinical and translational research. Tissue allotment can be modified as needed to address the needs of specific institutional protocols.

Abbreviations: FFPE, formalin-fixed paraffin-embedded; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulfoxide; RPMI, Roswell Park Memorial Institute medium.

Citation: Journal of the National Comprehensive Cancer Network 23, 1; 10.6004/jnccn.2024.7063

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Clinical and downstream uses of Ewing sarcoma and osteosarcoma biopsy tissue. Tissue analysis methodologies are organized by suitable tissue preservation/processing method (FFPE, snap frozen tissue, or fresh tissue) and identified as components of initial diagnosis (yellow), expanded workup (green), or research applications (purple).

Abbreviations: decals, decalcification solutions; FISH, fluorescence in situ hybridization; FFPE, formalin-fixed paraffin-embedded; H&E, hematoxylin-eosin staining; IHC, immunohistochemistry.

^aIndicates tissue analysis methodologies where FFPE specimens are also suitable.

Citation: Journal of the National Comprehensive Cancer Network 23, 1; 10.6004/jnccn.2024.7063

Open Biopsy by Pediatric or Orthopedic Surgical Oncology

Following basic open biopsy surgical principles, an open biopsy may be performed, preferably by the treating orthopedic oncologist or pediatric surgeon. Adhering to principles for safe open biopsies is critical. For extremity tumors, a small longitudinal incision that is in line with the planned resection incision should be used to allow for resection of the biopsy tract at the time of primary tumor resection. Similarly, for chest wall tumors, an incision that overlies the planned incision for definitive resection is recommended, generally along the course of the primarily affected rib. Other safety considerations include maintenance of hemostasis, minimizing dissection, limited drain use, and avoidance of neurovascular structures. If there is no soft tissue component, creating a bone defect during biopsy can increase the risk of pathologic fracture, so this should be considered carefully during open biopsy planning. A minimum of 1 cm³ (ideally 1–3 cm³ or 1–3 g of tissue) should be obtained. Identification of viable tumor tissue by frozen section, touch preparation, or other preferred method is recommended when institutionally available.³⁰ If frozen section is used, care should be taken to minimize the amount of tissue used in order to preserve sufficient material for permanent sections or other purposes. Specimens should ideally be submitted on saline-dampened gauze/Telfa or, if not available, in a dry container (no formalin). See Figure 2 for biopsy processing and allocation recommendations.

Pathology/Specimen Processing Planning

Handling of biopsy specimens for histologic and subsequent molecular testing requires thoughtful timing, processing, and tissue prioritization. In addition, specific biospecimens are now a standard requirement for enrollment in many therapeutic clinical trials. Moreover, a growing number of registry, biomarker, and biorepository studies are available in which patients may be interested in participating.

Decalcification Recommendations for Bone Sarcoma Diagnostic Biopsies

Decalcification is used to make hard, mineralized tissues more amenable to subsequent cutting and analysis. Soft tissue biopsies generally do not need to undergo decalcification, and not all bone biopsies require decalcification. The feasibility of cutting a bone core or open biopsy should be checked prior to placing a specimen in decalcification solution. Pure acid decalcification (eg, hydrochloric acid or formic acid) should be avoided in biopsy samples because these agents can affect the histology for primary diagnosis, interfere with immunogenicity of tissue, and denature nucleic acids, thus rendering the material less useful for molecular assays or downstream research.³³ “Soft decals” such as ethylenediaminetetraacetic acid (EDTA) or EDTA/formic acid combinations (ie, Formical 2000TM [Fisher Scientific] or similar products) are critically important in the age of molecular profiling, because nucleic acids are better preserved through this processing method.³⁴

We suggest decalcifying tissue only if necessary. In the case of an open biopsy, a portion of the tissue may be able to be separated and soft enough to not require decalcification. Every effort should be made to generate fresh and frozen samples before placing biopsy material in either formalin or a decalcifying agent. If specimens are overly calcified or ossified and require decalcification for sampling, we recommend submitting at least one block of tissue in formalin, followed by a “soft decalcification” solution (and consider submitting the entire specimen). It is important to first “fix” the tissue in formalin before placing the tissue in decalcification solution. Tissues should be checked regularly and ideally should not remain in decalcification solution for longer than 2 to 3 hours (small samples may require only 30 to 60 minutes). Even with the use of “soft” decalcification methods, prolonged decalcification may affect the histology and/or preservation of nucleic acids. To help clearly identify potential downstream material issues, we recommend clearly stating the type of decalcifying agent used in the gross pathology report.

Future Directions

It is anticipated that future Ewing sarcoma and osteosarcoma clinical trials will incorporate molecular biomarkers into treatment risk stratification, and some, if not all, of these proposed biomarkers will require prospective validation on future clinical trials.¹ Identification of requisite tumor biopsy volumes and harmonization of pathology processing is paramount to prospective efforts to advance bone sarcoma clinical care and research. Furthermore, novel therapeutic agents are routinely introduced with companion biomarkers, thus it is expected that molecular biomarker testing will become the standard of care. Beyond diagnostic biopsy approaches, image-guided core needle biopsy and/or liquid biopsies may be increasingly used for on-therapy tumor response evaluations, already a common practice for early-phase clinical trials in adult-onset cancers.^35,36 Biological correlate analyses exploring tumor evolution, response, and resistance are not commonly conducted in the pediatric setting. However, as targeted therapies are developed and evaluated, it is anticipated that implementation of on-therapy biopsies will yield clinically actionable results. Although on-treatment biopsy is not on the immediate horizon, developing consensus recommendations for biopsy tissue sampling, as well as for pathology tissue processing and preservation, is immediately relevant for diagnostic tissue in bone sarcoma. These same tissue acquisition principles should also be considered with resection specimens, including both local control and surgical metastatic control, and when biopsies or resections are performed at the time of suspected disease relapse.

Conclusions

The COG Bone Tumor Committee recognizes the great need for safely improving the amount and usability of diagnostic biopsy material obtained from patients with Ewing sarcoma and osteosarcoma to continue to advance the field and improve care. Importantly, this patient population is cared for by both pediatric and medical oncologists in academic and community settings, underscoring the need to improve their decentralized care through collaboration. The clinical use of less invasive, safe, and accurate diagnostic biopsy techniques will continue to grow, as will the required tumor tissue volume to meet clinical and research needs. The diagnostic biopsy and processing recommendations for clinical management of bone sarcomas described herein reflect the perspective of clinical and scientific experts in the field in North America and aim to serve as a reference to facilitate harmonization in tissue acquisition and processing algorithms for Ewing sarcoma and osteosarcoma specimens (Figure 4).

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Quick reference checklist for Ewing sarcoma and osteosarcoma biopsies.

Abbreviation: FFPE, formalin-fixed paraffin-embedded.

^aGoal if tumor site and patient condition permits to acquire approximately equivalent tissue volumes regardless of approach.

Citation: Journal of the National Comprehensive Cancer Network 23, 1; 10.6004/jnccn.2024.7063