Case 5 -

Based on the Immunophenotype, Presence of EWSR1 Rearrangement and Absence of a Defined Lesion on a Subsequent Chest CT, a Diagnosis of Ewing Sarcoma / Primitive Neuroectodermal Tumor (ES/PNET) Was Made Andrew Horvai, Univ of California/SF, San Francisco, CA

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Clinical History A 71 year old man, 60-pack year smoker, sought attention from his primary care physician for cough of several months duration and recent onset of left hip pain. Past medical history was notable for coronary artery disease. A chest X-ray revealed an ill-defined left sided mediastinal or hilar density. A lytic lesion was noted in the intertrochanteric portion of the left femur. He underwent fine needle aspiration biopsy of the left femur lesion. Case 5 - Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Case 5 - Figure 1 The plain AP radiograph of the chest demonstrates an ill-defined opacity in the left hilum or mediastinum. Case 5 - Figure 2 The plain AP radiograph of the left femur shows a destructive, lytic, lesion involving the intertrochanteric region. Cortical erosion is present. Case 5 - Figure 3 A coronal CT of the pelvis shows an ill-defined, lytic, permeative lesion of the femur without definite soft tissue extension. Case 5 - Figure 4 Papanicolau (d) stained smear and H&E stained cell block of fine needle aspiration material shows clusters of dyshesive, small ovoid to round cells with scant cytoplasm, granular chromatin and prominent nucleoli. Case 5 - Figure 5 Papanicolau (d) stained smear and H&E stained cell block of fine needle aspiration material shows clusters of dyshesive, small ovoid to round cells with scant cytoplasm, granular chromatin and prominent nucleoli. Case 5 - Figure 6 The tumor cells are weakly positive for CD99 in a membrane pattern. Case 5 - Figure 7 Weak, focal synaptophysin staining is also observed. Figure legends (post meeting material) Case 5 - Figure 8 Proximal femoral resection specimen. The patient sustained a pathologic fracture prior to surgery. Case 5 - Figure 9 Post-operative plain AP radiograph demonstrating left hip arthroplasty. Case 5 - Figure 10 CD99 immunopositivity in ES/PNET is typically diffuse in a membrane pattern. Case 5 - Figure 11 Five year survival of pediatric-type sarcomas in adults. Rhabdomyosarcoma (RMS) and EWS/PNET show distinctly worse prognosis in adults than children when controlled for stage but the same is not true for desmoplastic small round cell tumor (DSCRT). Case 5 - Figure 12 Gradualism in ES/PNET evolution. Although most adult malignancies require stepwise accumulation of mutations over decades, in many pediatric tumors such as ES/PNET, the initiating genetic change may represent a "shortcut." Additionally, the progenitor cell required for transformation is unique to the pediatric population. Introduction: The category of 'small round blue cell tumors" (SRBCT) encompasses a wide variety of malignant neoplasms of a disparate lineages. Arriving at an accurate diagnosis can be challenging as the morphologic features, almost by definition, are strikingly similar between various entities. Generally, the differential diagnosis must be tailored to the clinical presentation so as to avoid an exceptionally broad, time consuming and costly "shotgun" approach to diagnosis. One of the most important variables to consider when formulating a SRBCT differential diagnosis is the patient age, given the differences in demographics between various tumors in this category. The case presented here focuses on the specific considerations and practical diagnostic implications of a SRBCT in late adulthood as well as the clinicopathologic features of so-called "pediatric" sarcomas in adults. The patient is a 71 year old man with a history of a cough, an ill-defined hilar or mediastinal lesion on chest X-ray (Case5-a) and an aggressive, lytic lesion in the left femur. (Case5-b and Case5-c) The latter was biopsied. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: A fine needle aspiration (Case 5-d) of the left femur and core biopsy (Case 5-e) demonstrated predominantly necrotic tissue at scanning magnification. At higher magnification, a few dyshesive clusters of primitive appearing small cells with scant cytoplasm and coarse, granular chromatin and prominent nucleoli were observed. No obvious differentiation or matrix production (cartilage, bone, or collagen) by tumor cells was observed. The tumor cells were weakly positive for CD99 (Case5-f), synaptophysin (Case5-g) and CD56. They were uniformly negative for chromogranin, keratin cocktail (CAM5.2+AE1/AE3), CK7, CK20, CDX2, LCA and TTF-1. Fluorescence in-situ hybridization (FISH) from the fixed tissue was positive for the EWSR1 rearrangement. Differential Diagnoses: The differential diagnosis included metastatic carcinoma, especially small cell lung carcinoma, or less likely a neuroendocrine carcinoma from another site, primary intraosseous lymphoma, Ewing sarcoma, or other malignancies which can rarely have SRBCT morphology such as melanoma or osteosarcoma. Final Diagnosis: Based on the Immunophenotype, Presence of EWSR1 Rearrangement and Absence of a Defined Lesion on a Subsequent Chest CT, a Diagnosis of Ewing Sarcoma / Primitive Neuroectodermal Tumor (ES/PNET) Was Made. Discussion The differential diagnosis of SRBCT in adults As mentioned above, the diagnostic approach to SRBCT of bone must be tailored to the clinical setting. If age is not a consideration, the differential diagnosis includes ES/PNET, lymphoma, small cell osteosarcoma, mesenchymal chondrosarcoma or metastatic tumors such as neuroblastoma or poorly differentiated variants of some carcinomas and other sarcomas. However, it must be remembered that in the adult population metastasis is 50 to 100 times more common than all primary bone malignancies combined and thus rises to the top of the differential diagnosis. [1] The tumors most likely to metastasize to bone and display a SRBCT phenotype include metastatic neuroendocrine carcinomas (skin, lung, gastrointestinal tract or thyroid) and melanoma. In the present case, the suspicious lung lesion further supported metastasis, so the discussion below will emphasize small cell carcinoma in the differential. Primary osseous lymphoma (diffuse large B-cell lymphoma and T-cell lymphoblastic most commonly) remain diagnostic possibilities into adulthood. ES/PNET occurs very rarely in older adults but needs to be considered if all other diagnoses can be excluded. [1] How might one distinguish between these possibilities? Radiographic correlation is, of course, essential in establishing any diagnosis of bone, particularly with respect to malignancy. However, since all of the diagnostic considerations produce aggressive, lytic lesions, it may not be possible to discriminate between them on routine studies. Almost by definition, the routine histology will be very similar in all diagnoses within the SRBCT differential. With the exception of osteoid production by tumor cells confirming the diagnosis of osteosarcoma, other clues lack specificity. Clues that might suggest small cell carcinoma include cohesiveness, nuclear molding and stippled chromatin whereas lymphoma might be supported by a background of small, mature lymphocytes, apoptotic cells and marked discohesion. Ewing sarcoma typically shows garland-like geographic necrosis, and occasional Homer-Wright rosettes. Lymphoglandular bodies or crush artifact on cytologic preparations may suggest lymphoma. [2] However, none of the above features are particularly sensitive or specific. Thus, ancillary studies including immunohistochemistry or molecular methods are usually necessary. Table 1 lists the immunohistochemical stains most commonly used when discriminating between ES/PNET, small cell carcinoma and lymphoma. [3, 4, 5, 6, 7, 8, 9, 10] CD99, CD56 and CD45 (LCA) are the most sensitive and specific markers. CD99 positivity can be seen in a variety of SRBCTs, but in ES/PNET it is usually diffusely and strongly positive in a membrane pattern (Case5-j). As shown in Table 1, markers classically associated with small cell carcinoma (keratins, synaptophysin) do not exclude the diagnosis of ES/PNET so should be interpreted with care. MAP2 is a relatively novel marker that appears to be specific for small cell carcinoma over ES/PNET although it is also expressed in neuroblastoma. [6, 7] Table 1. Immunohistochemical profile of SRBCT of bone in adults. Antigen Percent positive ES/ PNET Small cell carcinoma Lymphoma CD99 95 9 92 Fli1 73 7 67 CD56 13 94 27 CD57 22 29 <5 Keratin AE1/AE3 27 81 <5 Synaptophysin 37 49 ? MAP2* <5 92 ? CD45 <5 <5 90 By far the most specific and sensitive test in the differential diagnosis of ES/PNET in adults is demonstration of EWSR1 gene rearrangement. FISH is the most widely available test and will detect the EWSR1 rearrangement regardless of the fusion partner (Table 2). For the same reason, a positive FISH result cannot exclude other non-ES/PNET tumors, such as clear cell sarcoma, desmoplastic small round cell tumor and others, that also harbor EWSR1 translocations. [11, 12] However, of these, only ES/PNET occurs with any frequency in bone. RT-PCR, from fixed, paraffin embedded or frozen tissue, or cytogenetics from fresh tissue, are more specific in that these techniques demonstrate both components of the gene or chromosome fusion, respectively. To date, no EWSR1 translocations have been identified in either small cell carcinoma or lymphoma so these studies have exceptional utility in the setting of the case presented here. Table 2. EWSR1 rearrangements in EWS/PNET . Translocation EWSR1- Incidence (%) t(11;22)(q24:q12) FLI1 85 t(21;22)(q22;q12) ERG 10 t(7;22)(p22;q12) ETV1 <1 t(7;22)(q21;q12) E1AF <1 t(2;22)(q33;q12) FEV <1 Inv(22) ZSG <1 t(4;22)q(31;q12) SMARC A5 <1 Clinical considerations Although most SRBCTs are restricted to the pediatric population, significant differences in epidemiology exist between these tumors. The most common examples are ES/PNET, mesenchymal chondrosarcoma and desmoplastic small round cell tumor (Table 3). Of these, only ES/PNET and mesenchymal chondrosarcoma present in bone. [13, 14] The prognosis of most pediatric-type sarcomas, as a group, is worse in adults than in children. Especially in localized disease, overall survival in adults is only about one third that in children. [15] However, important differences exist between tumor types and prognosis (Case5-k). [15, 16] For example, the majority of adult rhabdomyosarcomas consists of the pleomorphic subtype which carries a dismal prognosis compared to the more common alveolar and embryonal subtypes seen in children. In contrast, at least for ES/PNET, the primary site and stage of presentation do not appear to account for the effect of age on prognosis. [16] Rather, the discrepancy is most likely related to the ability to tolerate treatment regimens. It must be remembered that most SRBCT are with systemic, cytotoxic chemotherapy regimens regardless of stage at presentation. Often, older patients are not able to tolerate the morbidity of the treatments and in many cases, optimal regimens in adults have not been established. [17] The implementation of targeted therapies, especially for translocation driven sarcomas, will hopefully have profound impact on adult patients with SRBCTs. Table 3. Incidence of "pediatric" SRBCT in adults Tumor Mean age (yrs) Age > 30 (% of cases) ES/PNET 15 10 Mesenchymal chondrosarcoma 27 40 Desmoplastic small round cell tumor 22 25 Alveolar rhabdomyosarcoma 8 15 Embryonal rhabdomyosarcoma 7.5 <1 Wilms tumor 3.5 3 Neuroblastoma <1 <1% Tumor evolution What accounts for the different epidemiology of so-called "pediatric" sarcomas compared to "adult" sarcomas? Although a complete discussion of this topic is beyond the scope of this presentation, two key aspects: genetic shortcuts and cellular environment highlight fundamental mechanisms of tumorigenesis and are discussed below (Case5-L). Tumorigenesis depends on the stepwise transformation of a normal cell, through a series of intermediate stages, to a final population of genetically unstable cells that have the capacity to invade and metastasize. [18] Well characterized models of carcinogenesis (e.g. colon) illustrate that mutations in multiple growth promoting, cell adhesion and tumor suppressing genes are required to produce malignancy; a process that may require 25 to 30 years to unfold with specific, morphologically recognizable, intermediate lesions (e.g. dysplasia). [19] In contrast, the process in the majority of sarcomas, especially ES/PNET and other translocation-associated tumors, is far less well understood since neither the cell of origin nor the intermediate lesions are morphologically recognizable. Nevertheless, an intriguing hypothesis proposes that the product of a fusion gene, alone, confers sufficient transforming function to supplant multiple, independent mutations. Consequently, sarcomas that use genetic "shortcuts" require less time to evolve malignant potential and present earlier in life. Further supporting this hypothesis is the observation that many sarcomas common to adults are characterized by complex genetic changes. [20] While other mutations (p16 INK4A, p53) likely do contribute to ES/PNET evolution, the EWS-FLI1 fusion serves as a useful model to test the "shortcut" hypothesis. ES-FLI1 is a disordered protein with unstable 3-dimensional structure that makes it quite adept at multiple interactions with cell cycle machinery, transcriptional co-activators, RNA splicing and telomerase. [21, 22] Therefore, a simple initiating genetic event such as EWS-FLI1 fusion substitutes for the multiple mutations that, over time, might individually disrupt each of the above signal transduction and regulatory pathways. Of course, somatic gene fusions are not unique to pediatric malignancies, nor to sarcomas, so the mechanism is likely more complex than outlined here. [23] An alternative mechanism suggests that the cellular background required to give rise to a pediatric tumor is somehow restricted to that population. Hence, a fusion protein, even if acquired in later adulthood when the correct stem cells are no longer present, may not manifest in malignancy. Although epidemiologic evidence does support the concept, several problems exist with this explanation. A mesenchymal progenitor cell likely does give rise to ES/PNET, [24, 25] but studies suggest that these cells are present in adults. More importantly, indirect evidence demonstrates that the transforming capacity of the EWS fusion gene is not specific to mesenchymal stem cells. EWS-ERG fusions are able to transform lymphoid precursors and committed lymphoid cells to produce lymphomas in mice. [26] Although similar results have not been obtained for other EWS fusions, possibly owing to lethal effects in stem cells, these data suggest that a specific cell type is not uniformly required for fusion genes to function. Thus, the discrepancy in age of incidence is also unlikely to be due to simply the host cell type. It is possible, however, that functional or structural changes may take place in host cells that change the accessibility of the fusion genes for rearrangement in older patients. Conclusions Sarcomas typical of pediatric population do, albeit rarely, occur in adults and should be considered in the differential diagnosis when supported by histology Difference in outcome of pediatric sarcomas in adults is likely reflects chemotherapy tolerance / response Pediatric sarcomas use genetic “shortcuts” to tumorigenesis, the host cell may not be critical References Wold LE, Adler C-P, Sim FH, Unni KK, eds. Atlas of Orthopedic Pathology. Philadelphia: Saunders; 2008. 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