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Protocol for the Examination of Specimens From Pediatric and Adult Patients With Osseous and Extraosseous Ewing Sarcoma Family of Tumors, Including Peripheral Primitive Neuroectodermal Tumor and Ewing Sarcoma

2005; American Medical Association; Volume: 129; Issue: 7 Linguagem: Inglês

10.5858/2005-129-866-pfteos

1543-2165

David Carpentieri, Stephen J. Qualman, Jay Bowen, Thomas Krausz, Alberto M. Marchevsky, Paul S. Dickman,

Management of metastatic bone disease

The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the "Surgical Pathology Cancer Case Summary (Checklist)" portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice.The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with the document. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of this document.This protocol applies to osseous and extraosseous peripheral primitive neuroectodermal tumor/Ewing sarcoma (PNET/ES) only. Other tumors of the bone and soft tissue are excluded. The American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) TNM classification system for the staging of bone and soft tissue tumors is recommended.Ewing sarcoma family of tumors includes both peripheral primitive neuroectodermal tumor and Ewing sarcoma, which can occur in children and adults. The malignancy may occur in bone and soft tissue sites (including unusual sites such as skin or leptomeninges).12 Because PNET/ES can occur in bone and soft tissue, AJCC/UICC staging systems for both are included.First priority should always be given to formalin-fixed tissues for morphologic evaluation. Special studies (eg, reverse transcriptase polymerase chain reaction [RT-PCR]) are critical to the molecular workup of PNET/ES and require at least 100 mg of viable snap-frozen tissue as the second priority for workup (note A). Tumor-defining translocations for Ewing sarcoma family of tumors may also be performed by RT-PCR and fluorescence in situ hybridization (FISH) on formalin-fixed tissue scrolls or tissue sections, respectively. Due to increased sensitivity of detection, snap-frozen tumor tissue is the preferred specimen type, and every effort should be made to procure it.This protocol is based on the experience of the Children's Oncology Group.For more information, contact The Children's Oncology Group Biopathology Center; telephone: (614) 722-2890 or (800) 347-2486.A minimum of 100 mg of viable tumor should be snap-frozen for potential molecular studies.4 When the amount of tissue is limited, the pathologist can keep the frozen tissue aliquot used for frozen section (usually done to determine sample adequacy and viability) in a frozen state (−70°C is preferable) for potential molecular studies. Translocations may be detected using RT-PCR on frozen or fixed paraffin-embedded tissue, or FISH on touch preparations made from fresh tissue. Fluorescence in situ hybridization can be performed on tissue sections obtained from formalin-fixed, paraffin-embedded tumor blocks as well, although sensitivity is reduced compared with tough preparations for FISH made from fresh or frozen tissue.In cases where histologic diagnosis of PNET/ES is difficult, immunostaining with monoclonal antibodies against the cell surface glycoprotein CD99, also known as MIC-2, is suggested. Nearly all PNET/ES tumors are positive for this antigen.5 This glycoprotein is diffusely expressed in the vast majority of cases in a membranous pattern. The results of staining using monoclonal antibodies O13, HBA71, and 12E7 are similar, but individual tumors may exhibit better staining with 1 or another antibody.Lymphoblastic lymphomas/leukemias, rhabdomyosarcomas, desmoplastic small round cell tumor, synovial sarcomas, solitary fibrous tumor, extrarenal malignant rhabdoid tumor, neuroendocrine tumors, and mesenchymal chondrosarcoma may demonstrate immunoreactivity to MIC-2.6 In some of these tumors, CD99 immunostaining is often weakly granular and intracytoplasmic; in others (desmoplastic tumor, lymphoblastic lymphoma/leukemia), distinct plasma membrane staining is present, as seen in PNET/ES. The MIC-2 immunostain should always be done in a panel, which usually includes muscle markers (desmin, muscle-specific actin, myoD1, myogenin), neural markers (protein gene product 9.5, S100 protein, neuron-specific enolase, human natural killer–cell antigen [CD57], synaptophysin, neurofilament protein), epithelial markers (epithelial membrane antigen, cytokeratin), and lymphoid markers (CD45, CD30, Tdt, T-cell and/or B-cell markers). The value of other immunohistochemical markers for diagnosis, such as Ki-67, p53, and c-Kit (CD117), has not been established at this time. Primitive neuroectodermal tumor and Ewing sarcoma are consistently vimentin immunopositive.The prevalence of the EWS-FLI1 fusion gene in PNET/ ES (90%–95%) has been shown to be useful diagnostically. In this regard, immunohistochemistry against the carboxy-terminus of the FLI1 has been shown to be sensitive in the diagnosis of PNET/ES7 (see "Chromosomal Translocations"), although the FLI1 antibody will stain other tumor types,89 including vascular tumors and lymphoblastic lymphoma.In the past, PNET/ES has been a diagnosis of exclusion (of other primary and metastatic tumor types) without specific confirmatory markers. Immunostains (CD99, FLI1), although supportive of the diagnosis in the proper context, remain relatively nonspecific. Detection of the specific fusion gene (EWS-FLI1 or other variants) provides valuable molecular confirmation of the diagnosis unavailable by other technologies (see "Chromosomal Translocations").It is now generally accepted that Ewing sarcoma and PNET form a single group of bone and soft tissue tumors. The characteristic translocations involve the EWS gene at 22q12 and either the FLI1 gene at 11q24 or the ERG gene at 21q22. The presence of t(11;22) (EWS-FLI1) and t(21;22) (EWS-ERG) is strongly correlated with PNET/ES. The most common gene fusion is the EWS-FLI1 (90%–95% of patients). Recent investigations10 suggest that different types of EWS-FLI1 fusions (type 1 vs type 2) may have prognostic implications. Patients with type 1 fusions (in which EWS exons 1–7 link with FLI1 exons 6–9) fare better than patients with type 2 fusions (involving other sites within the relevant genes). This relationship remains under active investigation.There are several tumor-defining translocations that are detected in a small percentage (<5%) of PNET/ES. These characteristic translocations include t(7;22)(p22;q12) EWS-ETV1, t(17;22)(q12;q12) EWS-E1AF, t(2;22)(q33;q12) EWS-FEV, and t(1;22)(p36;12) EWS-ZSG. Although these translocations are relatively rare with PNET/ES, the practicing surgical pathologist should be aware of these in the event that EWS-FLI1 and EWS-ERG translocations are not detected by cytogenetics, RT-PCR, or FISH. It is definitely possible to render a diagnosis of PNET/ES in the absence of a tumor-defining translocation, and the detection of PNET/ES-associated translocations is not mandatory to make such a diagnosis.Ultrastructural studies are valuable despite the putative diagnostic power of immunohistochemistry and molecular studies.11 These tumors usually have limited cytoplasmic organelles. Some cytoplasmic regions may contain an increased amount of polyparticulate glycogen. The latter corresponds to the classic "dot-positivity" noted with the periodic acid–Schiff stain. Furthermore, one may also find intermediate filaments corresponding to vimentin and cytokeratin. In those tumors with neuroendocrine differentiation, neurosecretory granules may occur, but they are pleomorphic and larger than the 100-nm-diameter spherical granules of neuroblastoma. Intermediate-type junctions are often present, but true desmosomes are not usually seen.Cytologic material is usually sufficient to diagnose PNET/ES (with supportive immunostains) (note A). However, rhabdomyosarcomas may not be readily distinguished from PNET/ES in soft tissue lesions. An important limitation of fine-needle aspiration biopsy is the limited amount of tissue for additional molecular diagnostic studies3 and banking (note A). Fluorescence in situ hybridization studies for pertinent translocations may be performed on fine-needle aspiration biopsy material, although it is still hampered by sampling error. In spite of these limitations, fine-needle aspiration biopsy can provide a specific diagnosis of a suspected PNET/ES in most cases.12If cytologic material includes fluid, such as pleural effusions or fluid from a liquefactive tumor, the fluid should be centrifuged and the resulting pellet fixed with formalin prior to making a paraffin block. The resulting cell block allows for histopathologic examination and immunocytochemical, RT-PCR, and FISH analyses.Relevant historical factors include any previous therapy and family history of malignancy. If preoperative therapy has been given, assessment may be limited to the estimate of viable and necrotic tumor.The typical case of PNET/ES shows a lobular growth pattern of tumor cells that are distinctly monotonous in their nuclear uniformity. Nuclei measure 10 to 15 μm in diameter with distinct nuclear membranes, finely granular chromatin, and 1 to 2 inconspicuous nucleoli. Cytoplasm is poorly defined, scant, and pale staining and may be vacuolated due to irregular glycogen deposition. Atypical variants may show increased nuclear size or more pronounced atypia. Multinucleate giant cells are not seen. Large areas of perivascular tumor necrosis with "ghost cells" (filigree pattern) may be striking. Areas of neuroectodermal differentiation (Homer Wright rosettes; rarely Flexner-Wintersteiner rosettes or primitive neuroepithelium) may be evident in some tumors.Currently, extraosseous PNET/ES is treated in the same manner as intraosseous Ewing sarcoma. There are no histologic subtypes of established prognostic importance.6 However, a neural pattern purportedly suggests a better prognosis, and a filigree pattern may confer a worse outcome. Unusual patterns such as alveolar/rosette formations may be seen in treated tumors, but no prognostic implication for these patterns is known.Core needle biopsies can obtain sufficient material for special studies and morphologic diagnosis, but sampling problems may limit diagnostic accuracy.13 Open incisional biopsy is generally the preferred and most widely used technique because it consistently provides a larger sample of tissue and maximizes the opportunity for a specific pathologic diagnosis.3 Excisional biopsy may not include an adequate margin of normal tissue, even with an operative impression of total gross removal.3Resection specimens may be intralesional, marginal, wide, or radical in extent.14 Intralesional resections extend through tumor planes, with gross or microscopic residual tumor identifiable at surgical margins. A marginal resection involves a margin formed by inflammatory tissue surrounding the tumor. A wide radical resection has surgical margins that extend through normal tissue, usually external to the anatomic compartment containing the tumor. For all types of resections, marking (tattoo with ink followed by use of a mordant) and orientation of the specimen (prior to cutting) are highly recommended for accurate pathologic evaluation.3 Full representative mapping of the specimen is also recommended,3 as discussed later.A full sagittal section of the resection specimen,15 as illustrated in the Figure, allows for mapping of the entire central face of the tumor and adjacent marginal tissue. Freezing of the specimen and cutting with a bone saw (with intraosseous specimens) may best achieve this result. This face of the specimen can be documented by a black-and-white photograph or photocopy of the specimen when vacuum-sealed in a plastic bag. As shown in Figure 1, this central full face of the specimen and lesion can be mapped and blocked postfixation (and decalcification as necessary) for complete microscopic examination, including estimate of percentage of tumor necrosis.The extent of resection (ie, gross residual disease vs complete resection) has the strongest influence on local control of malignancy.1617 The definition of what constitutes a sufficiently "wide" margin of normal tissue in the management of PNET/ES has evolved. In the current Children's Oncology Group study of PNET/ES, the following margins are considered adequate.If the response to chemotherapy is poor, wider margins may be required. If margins are deemed inadequate by these criteria, postoperative radiotherapy often is indicated.A summary of the prognostic factors is detailed below.18 Of the various prognostic markers listed, age at onset, size, site, and stage bear the most significant relationship with outcome.Histologic response to chemotherapy is an excellent predictor of outcome in osteosarcomas and may also be of value in PNET/ES. This feature may be graded by the Huvos classification, as detailed below.1920 Details for evaluating tissue necrosis versus viability can be found elsewhere.20In osteosarcomas, grades III and IV are considered favorable. Grades I, IIA, and IIB are considered to be failure of chemotherapy and will prompt a chemotherapy regimen change. In the literature,20 some may consider any degree of necrosis greater than 90% to be favorable.A recent Childhood Cancer Group/Pediatric Oncology Group study of resected PNET/ES evaluated the response to preoperative chemotherapy using the following grading.21Because the Huvos and Childhood Cancer Group/Pediatric Oncology Group grading schemes use similar numbering but significantly different necrosis levels, it is important for the report to include the actual estimated percentage necrosis rather than necrosis grade. This allows the oncologist and surgeon to interpret and translate the percentage necrosis into the necrosis scheme used at their specific hospital(s).The AJCC/UICC TNM staging system for bone tumors is as follows.2223Primitive neuroectodermal tumor/Ewing sarcoma (either intraosseous or extraosseous) is classified as high grade, and hence stages IA and IB below are excluded for PNET/ES.The AJCC/ UICC TNM staging system2223 for soft tissues is as follows.* Superficial tumor located exclusively above superficial fascia. Deep tumor is located exclusively beneath superficial fascia or extends superficially into or through the fascia. Retroperitoneal, mediastinal, and pelvic sarcomas are classified as deep.Primitive neuroectodermal tumor/Ewing sarcoma (either intraosseous or extraosseous) is classified as high grade.