Original Articles. Utilization of Fluorescence In Situ Hybridization in the Diagnosis of 230 Mesenchymal Neoplasms. An Institutional Experience

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1 Original Articles Utilization of Fluorescence In Situ Hybridization in the Diagnosis of 230 Mesenchymal Neoplasms An Institutional Experience Munir R. Tanas, MD; Brian P. Rubin, MD, PhD; Raymond R. Tubbs, DO; Steven D. Billings, MD; Erinn Downs-Kelly, DO; John R. Goldblum, MD N Context. Mesenchymal neoplasms harbor characteristic translocations and amplification of gene regions amenable to evaluation by fluorescence in situ hybridization (FISH). Objective. To determine the utility of FISH in the diagnosis of mesenchymal neoplasms. Design. Two hundred thirty soft tissue cases analyzed by FISH were reviewed retrospectively. Results. Morphologic patterns where FISH was used included high-grade round cell sarcomas (n = 67), nonmyogenic spindle cell sarcomas (n = 40), low-grade myxoid neoplasms (n = 34), adipocytic neoplasms (n = 20), and melanocytic neoplasms (n = 19). Fifty cases did not fit into the previously mentioned categories. SYT FISH (96% of monophasic synovial sarcomas were positive; 0% of malignant peripheral nerve sheath tumor were positive) and DDIT3 FISH (100% of myxoid/round cell liposarcomas; no other neoplasm positive) were very sensitive and specific. EWSR1 FISH was very sensitive and specific in the More than any other subspecialty within pathology, with the exception of perhaps hematopathology, molecular studies have become important in the diagnosis of soft tissue neoplasms. Many diagnostic entities have characteristic molecular alterations, and, as such, methods that allow detection of these alterations are useful for diagnostic confirmation (Table 1). Several methods exist to evaluate for these genetic events, including conventional cytogenetics, reverse transcription polymerase chain reaction, and fluorescence in situ hybridization (FISH). The focus of this study was to evaluate the utility of FISH, which for a variety of reasons (discussed later) has become the molecular test of choice at Accepted for publication March 2, From the Department of Pathology, Cleveland Clinic, Cleveland, Ohio. The authors have no relevant financial interest in the products or companies described in this article. Note from authors: This article and the study it describes were referenced in a recently published review article discussing the use of fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms (Adv Anat Pathol. 2009;16[6]: ) prior to this publication. Reprints: John R. Goldblum, MD, Cleveland Clinic, Department of Anatomic Pathology, L25, 9500 Euclid Ave, Cleveland, OH ( goldblj@ccf.org). differential diagnosis of melanocytic neoplasms (88% of clear cell sarcomas were positive; all melanomas were negative). EWSR1 FISH was sensitive among high-grade round cell sarcomas (positive in 100% of desmoplastic small round cell tumors and 96% of Ewing sarcoma/ primitive neuroectodermal tumors) but not specific because clear cell sarcoma, extraskeletal myxoid chondrosarcoma, and a subset of round cell liposarcomas also harbor rearrangements of EWSR1. FUS FISH was very sensitive in detecting low-grade fibromyxoid sarcomas (91% positive) but not specific because most myxoid/ round cell liposarcomas also contain rearrangements of FUS. All atypical lipomatous tumors were positive for amplification of MDM2, whereas all lipomas were negative. FOXO1A FISH was positive in,70% of cases of alveolar rhabdomyosarcoma. Conclusion. FISH is a useful adjunct in the diagnosis of mesenchymal neoplasms. (Arch Pathol Lab Med. 2010;134: ) many institutions. To examine patterns of use of FISH in the diagnosis of soft tissue neoplasms, and to evaluate the utility of the method, we reviewed 230 consecutive soft tissue cases where FISH was performed. MATERIALS AND METHODS FISH studies were performed on interphase nuclei present on formalin-fixed paraffin-embedded tissue sections as previously reported. 1 Five split-apart probe sets are used at our institution to evaluate most translocations including probes to the EWSR1, FUS, DDIT3, SYT, and FOXO1A genes (Abbott Molecular/Vysis, Des Plaines, Illinois). Each set contains a pair of dual-color, splitapart probes, which hybridize to targets that flank the most common breakpoints in a given gene. Spatial separation of the 2 differently colored probes indicates a rearrangement of the gene, which in turn indicates the presence of a translocation involving that gene (Figure 1, a and b). Ten percent or greater of the neoplastic cells must show a rearrangement of the gene region to be interpreted as a positive result. On occasion, 1 signal will be consistently absent in a split-apart FISH test, indicating the presence of a complex rearrangement for which the clinical significance is uncertain (possibilities include a deletion of that gene region or its involvement in a cryptic rearrangement). A dual-color MDM2 FISH probe that hybridizes to the MDM2 gene region (12q13 15) has been developed at the Cleveland Clinic (Cleveland, Ohio) to aid in the diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma. This probe is compared Arch Pathol Lab Med Vol 134, December 2010 Mesenchymal Neoplasms FISH Tanas et al 1797

2 Table 1. An Overview of Molecular Alterations in Mesenchymal Neoplasms Diagnostic Entity Cytogenetic Alteration Genes Involved Ewing sarcoma/peripheral neuroectodermal tumor t(11;22)(q24;q12) FLI1-EWS t(21;22)(q22;q12) ERG-EWS Alveolar rhabdomyosarcoma t(2;13)(q35;q14) PAX3-FKHR t(1;13)(p36;q14) PAX7-FKHR Myxoid/round cell liposarcoma t(12;16)(q13;p11) CHOP-FUS t(12;22)(q13;q11 12) CHOP-EWS Desmoplastic small round cell tumor t(11;22)(p13;q12) WT1-EWS Synovial sarcoma t(x;18)(p11.2;q11.2) SSX1-SYT SSX2-SYT Clear cell sarcoma t(12;22)(q13;q12) ATF1-EWS Extraskeletal myxoid chondrosarcoma t(9;22)(q22;q12) NOR1-EWS Low-grade fibromyxoid sarcoma t(7;16)(q34;p11) CREB3L2-FUS t(11;16)(p11;p11) CREB3L1-FUS Atypical lipomatous tumor/well-differentiated liposarcoma 12q rings and giant markers MDM2 (amplified) Reprinted with permission by Lippincott, Williams & Wilkins from Tanas MR, Goldblum JR. Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review. Adv Anat Pathol. 2009;16(6): with the number of signals present for a centromeric reference probe for chromosome 12 (CEP 12). A ratio of the average number of MDM2 and CEP 12 signals counted in the neoplastic cells is calculated. A ratio of 2.0 or more indicates amplification of the MDM2 gene region, which is considered a sensitive and specific molecular signature of atypical lipomatous tumor/welldifferentiated liposarcoma. 2 The 230 soft tissue cases evaluated in this study were identified via a search through our Department of Anatomic Pathology database from December 2005 to June The cases were composed predominantly of consultation cases (80%) but also included cases from routine surgical pathology practice at the Cleveland Clinic (20%). RESULTS There were 5 major morphologic patterns that emerged in which FISH was used including high-grade round cell sarcomas, spindle cell sarcomas, low-grade myxoid neoplasms, adipocytic neoplasms, and melanocytic neoplasms without an epidermal component. FISH, not surprisingly, was most commonly used in the evaluation of high-grade round cell sarcomas (n 5 67). This diagnostic category contains many of the sarcomas that fall under the description of small round blue cell tumors, characterized by primitive-appearing round cells with little to no distinguishing features by light microscopic evaluation (Table 2; Figure 2, a and b). Diagnostic entities included Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), poorly differentiated synovial sarcoma (SS), alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. Sarcomas in this group that did not have evidence of a translocation by FISH included rhabdomyosarcoma, not otherwise specified, and high-grade round cell sarcomas, not otherwise specified (Table 2). Immunohistochemistry could be used to help distinguish between many of these entities (eg, CD99 for ES/PNET, desmin/myogenin for alveolar rhabdomyosarcoma). However, because these markers are not absolutely specific, molecular studies were sought Figure 1. a, Fluorescence in situ hybridization probes for FOXO1A (13q14). The red rhodamine-labeled probe and fluorescein isothiocyanate green-labeled probes when adjoined (FOXO1A intact) give a yellow signal. The red and green probes when spatially separated indicate rearrangement of FOXO1A. Note that only 1 copy of FOXO1A is rearranged. b, A normal cell with both copies of FOXO1A intact. Reprinted with permission by Lippincott, Williams & Wilkins from Tanas MR, Goldblum JR. Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review. Adv Anat Pathol. 2009;16(6): Arch Pathol Lab Med Vol 134, December 2010 Mesenchymal Neoplasms FISH Tanas et al

3 Table 2. Diagnosis Summary of Fluorescence In Situ Hybridization (FISH) Results for Major Morphologic Categories EWSR1, No./ SYT, No./ to confirm these diagnoses. Almost all examples of ES/ PNET and poorly differentiated SS harbored rearrangements of EWSR1 or SYT, respectively, whereas alveolar rhabdomyosarcoma was characterized by rearrangement of FOXO1A (13q14) in only 66% of cases. The frequency of translocations in these sarcomas approximate what has been reported in the literature The next most common use of FISH was in the morphologic category of spindle cell sarcomas (n 5 40). The main differential diagnosis was nonmyogenic spindle cell sarcomas, in which the principal diagnostic considerations were monophasic SS versus malignant peripheral nerve sheath tumor (MPNST), entities that have a similar histologic appearance (Figure 2, c and d). 13 The difficulty in distinguishing between these 2 entities is exacerbated by the lack of a consistently robust marker that can be used to distinguish them; SS typically contains only focal immunoreactivity for epithelial markers (cytokeratins and FISH Probes FOXO1A, No./ DDIT3, No./ FUS, No./ MDM2, No./ High-grade round cell sarcomas (n 5 67) Ewing sarcoma/pnet 22/23 (96) a Poorly differentiated synovial sarcoma 5/5 (100) Alveolar rhabdomyosarcoma 4/6 (66) Rhabdomyosarcoma, NOS 0/5 (0) Round cell liposarcoma 3/3 (100) Desmoplastic small round cell tumor 2/2 (100) High-grade round cell sarcoma, NOS 1/17 (6) 0/5 (0) 0/1 (0) 0/3 (0) 0/1 (0) Spindle cell sarcomas (n 5 40) Monophasic synovial sarcoma 26/27 (96) Malignant peripheral nerve sheath tumor 0/7 (0) Spindle cell sarcoma, NOS 0/6 (0) Low-grade myxoid neoplasms (n 5 34) Low-grade fibromyxoid sarcoma 10/11 (91) Myxoma 0/4 (0) Myxofibrosarcoma 0/2 (0) 0/3 (0) Myxoid liposarcoma 5/5 (100) 2/2 (100) Low-grade myxoid neoplasm, NOS 0/1 (0) 0/12 (0) Adipocytic neoplasms (n 5 20) Lipoma 0/2 (0) 0/3 (0) 0/7 (0) Atypical lipomatous tumor 0/1 (0) 3/3 (100) Dedifferentiated liposarcoma 4/4 (100) Pleomorphic liposarcoma 0/1 (0) Malignant melanocytic neoplasms (n 5 19) Malignant melanoma 0/11 (0) Clear cell sarcoma 7/8 (88) Abbreviations: NOS, not otherwise specified; PNET, peripheral neuroectodermal tumor. a Numbers represent the proportion/percentage of cases demonstrating rearrangement (or amplification for MDM2) of that particular gene region by FISH split-apart probes. Totaling numbers from each individual entity within a morphologic category may not equal the total number of cases within each category because multiple FISH tests were used on the same case in some instances. Reprinted with permission by Lippincott, Williams & Wilkins from Tanas MR, Goldblum JR. Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review. Adv Anat Pathol. 2009;16(6): epithelial membrane antigen), whereas MPNST usually exhibits focal positivity for S100 protein, which can also be seen in SS. The main FISH test used to distinguish SS from MPNST was FISH for rearrangement of the SYT gene region (18q11). This test was sensitive and specific for SS; 96% were characterized by rearrangement of the SYT gene, whereas none (0%) of the MPNSTs harbored a rearrangement of the SYT gene, mirroring what has been reported in the literature (Table 2). 3,14 A minority of sarcomas had SYT FISH performed on them, which was negative, but did not have sufficient light microscopic or immunohistochemical features allowing for diagnosis of MPNST; these sarcomas were in the end classified as spindle cell sarcoma, not otherwise specified. FISH was also useful in the diagnosis of low-grade myxoid neoplasms (n 5 34). The differential diagnosis in this morphologic category include entities such as lowgrade fibromyxoid sarcoma, myxoma, myxofibrosarcoma, Arch Pathol Lab Med Vol 134, December 2010 Mesenchymal Neoplasms FISH Tanas et al 1799

4 Figure 2. Examples of morphologic categories evaluated by fluorescence in situ hybridization. a and b, High-grade round cell sarcomas: Ewing sarcoma/primitive neuroectodermal tumor (a) and alveolar rhabdomyosarcoma (b) (hematoxylin-eosin, original magnifications 3200). c and d, Spindle cell sarcomas: monophasic synovial sarcoma and malignant peripheral nerve sheath tumor (hematoxylin-eosin, original magnifications 3200). e and f, Low-grade myxoid neoplasms: low-grade fibromyxoid sarcoma (e) and intramuscular myxoma (f) (hematoxylin-eosin, original magnifications 3100). and myxoid liposarcoma (Table 2; Figure 2, e and f), which all have myxoid stroma, lack of a characteristic immunohistochemical profile, and (with the exception of examples of myxofibrosarcoma) minimal/mild to no 1800 Arch Pathol Lab Med Vol 134, December 2010 cytologic atypia. Thus, molecular studies were important in distinguishing between many of these entities. The most commonly used FISH probe set for this morphology was for the FUS gene region (16p11), which is split apart in Mesenchymal Neoplasms FISH Tanas et al

5 either a t(7;16) or t(11;16) in low-grade fibromyxoid sarcoma 15,16 or in the t(12;16) in myxoid liposarcoma. 11 The second most commonly used FISH test was for DDIT3 gene region (12q13), which is split apart in t(12;16) and t(12;22) seen in myxoid liposarcoma. 11,12 As with highgrade round cell sarcomas and spindle cell sarcomas, a number of these neoplasms, which did not contain a translocation identifiable by FISH, were classified as lowgrade myxoid neoplasm, not otherwise specified. MDM2 FISH has been used only very recently at our institution due to the recent development of a MDM2 gene region probe (n 5 20) (Table 2). The main utility of this probe is in distinguishing atypical lipomatous tumors (well-differentiated liposarcoma), which are characterized by amplification of the MDM2 gene region (12q13 15), from lipomas, which do not contain amplification of MDM2. 2,17 19 The differential diagnosis of atypical lipomatous tumor and lipoma exists due to the lipoma-like variant of atypical lipomatous tumor, which can have only scattered atypical cells. Most of the examples of atypical lipomatous tumor seen in consultation are the lipoma-like variant, which can be diagnostically challenging due to the focality of the cytologic atypia. Although immunohistochemistry for MDM2 is available, it is not as sensitive or specific as MDM2 FISH for the diagnosis of atypical lipomatous tumor. 19 All atypical lipomatous tumors harbored amplification of the MDM2 gene region (100%), whereas all variants of lipomas (including spindle cell and pleomorphic lipomas) were negative for amplification of MDM2. The lipomas evaluated by FISH were typically deep seated (eg, intramuscular lipomas) or contained prominent fibrous septa (so-called fibrolipomas) or myxoid stroma. Some spindle cell/pleomorphic lipomas not presenting in the typical clinical setting (middle-aged to elderly man, upper back/posterior neck) were also evaluated by MDM2 FISH. Although less specific in this setting, MDM2 FISH was also used to provide support for the diagnosis of dedifferentiated liposarcoma when the well-differentiated component was not easily identifiable; this was possible because dedifferentiated liposarcomas maintain the molecular signature of MDM2 amplification found in the well-differentiated liposarcomas from which they arise. 20 Importantly, use of MDM2 FISH in this setting is complicated by the fact that up to 40% of pleomorphic sarcomas show amplification of the MDM2 gene region, 2 thereby significantly decreasing the specificity of the test in this differential diagnosis. However, due to its high degree of sensitivity, it is useful when it is negative, arguing strongly against the diagnosis of dedifferentiated liposarcoma. In one case, this test was used to favor the diagnosis of pleomorphic liposarcoma over the diagnosis of dedifferentiated liposarcoma, when it was determined that MDM2 was not amplified (Table 2). The last major class of neoplasms for which FISH was used (n 5 19) was melanocytic neoplasms (Table 2) in which the differential diagnosis was malignant melanoma versus clear cell sarcoma (malignant melanoma of soft parts). Malignant melanoma and clear cell sarcoma are notable for their similar histologic appearances including a nested/packeted growth pattern, spindle cells with vesicular nuclei and prominent nucleoli, and an identical immunohistochemical profile including immunoreactivity for S100 protein as well as melanocyte-specific markers including HMB-45, Melan-A (Mart1), tyrosinase, and microphthalmia transcription factor. In a patient with a classic clinical history, a young patient with a deep-seated lesion involving tendons/aponeuroses of the distal extremities, clear cell sarcoma is favored, whereas melanoma is favored when there is a greater degree of pleomorphism. However, because of the aforementioned overlap in their features, molecular studies can play an important role in differentiating occasional cases with clinical or morphologic overlap. EWSR1 FISH was used to confirm the diagnosis of clear cell sarcoma, which harbors a t(12;22), resulting in rearrangement of the EWSR1 gene region. 21,22 We found that 88% of clear cell sarcomas were positive for EWSR1 FISH, whereas all melanomas were negative, making it a highly sensitive and specific test for clear cell sarcoma. There were 50 cases in which various FISH studies were performed that did not fit into the previously mentioned categories. These cases were not numerous enough to allow generalization into additional morphologic categories. Examples included a case of perineurioma where FUS FISH was performed to exclude the possibility of lowgrade fibromyxoid sarcoma. A similar example involved a case of fibromatosis in which FUS FISH was performed also to exclude the possibility of low-grade fibromyxoid sarcoma. COMMENT We have found FISH to be a very useful adjunct to the diagnosis of soft tissue neoplasms, exploiting the presence of nonrandom translocations and amplification of gene regions that characterize many soft tissue neoplasms. This may be especially true in more diagnostically challenging cases (approximately 80% of the cases that used FISH at our institution were consults). Approximately 9% of our soft tissue consults (at the time of this study) used FISH. The most common morphologic categories in which it was used include high-grade round cell sarcomas, nonmyogenic spindle cell sarcomas, low-grade myxoid neoplasms, adipocytic neoplasms, and melanocytic neoplasms, in descending order of frequency. In general, FISH was used to distinguish between entities with similar histologic appearance and sometimes overlapping immunohistochemical profiles. For most entities, FISH had a high degree of sensitivity; entities such as ES/PNET and monophasic SS had rearrangements by FISH documented in 96% of the cases. For some genes, it was very specific as well; for example, none of the examples of MPNST had a rearrangement of SYT. Importantly, because roughly 20% of alveolar rhabdomyosarcomas do not harbor rearrangements of FOXO1A 5 8 (30% in our study), a negative FOXO1A FISH study does not exclude the diagnosis of alveolar rhabdomyosarcoma; in these cases, the diagnosis rests upon recognition of characteristic histologic features and a supportive immunohistochemical profile. FISH can be used to make distinctions of clinical import. For example, alveolar rhabdomyosarcoma has been shown to portend a poorer prognosis than embryonal rhabdomyosarcoma, and different therapeutic approaches for these 2 entities have been suggested. 23 FISH for FOXO1A can be helpful in establishing the diagnosis of alveolar rhabdomyosarcoma in cases that have overlapping features with embryonal rhabdomyosarcoma (embryonal rhabdomyosarcoma does not have a characteristic translocation). Determination of whether a well-differen- Arch Pathol Lab Med Vol 134, December 2010 Mesenchymal Neoplasms FISH Tanas et al 1801

6 tiated lipomatous neoplasm of the extremity is an atypical lipomatous tumor or a lipoma by MDM2 FISH is helpful in determining whether a reexcision and indefinite clinical follow-up is necessary (for atypical lipomatous tumor). FISH offers several advantages over conventional cytogenetics. The main advantage of FISH is that nondividing (interphase) nuclei can be evaluated, making it unnecessary to evaluate the neoplastic cells in culture. This, in turn, allows retrospective analysis of formalinfixed paraffin-embedded tissue, the most commonly archived tissue format. The technique confers an advantage in small biopsies, where it may not be possible to submit tissue for cytogenetics, but FISH can still be performed, sometimes on as few as 40 to 50 cells. FISH also offers several benefits over reverse transcription polymerase chain reaction. For a given translocation with multiple breakpoints, multiple polymerase chain reaction primers are necessary to evaluate for all the possible fusion transcripts. 1 Even then, it is still possible that a subset of translocations with variant breakpoints might not be detectable. This stands in contrast with FISH, where most commercially available FISH probes span all known translocation breakpoints. Therefore, one set of FISH probes (instead of the multiple polymerase chain reaction primers and polymerase chain reactions) can identify essentially all known breakpoints of a given translocation, resulting in increased sensitivity. In addition to advantages in evaluating multiple breakpoints, FISH is also helpful in situations in which one translocation partner is largely conserved (present in nearly all the possible fusion transcripts) but the second translocation partner varies. Examples of this include ES/ PNET (with the various translocation partners adjoined to EWSR1) and myxoid/round cell liposarcoma. Regarding the latter example, probes for DDIT3 can detect both the t(12;16) and the t(12;22) translocations in myxoid/round cell liposarcoma. Again, this avoids the situation in which multiple primer sets are necessary to evaluate all of the possible fusion products derived from the variant translocation partner or partners. The same gene is often involved in translocations found in different soft tissue neoplasms. Therefore, the same set of probes can also be used for a gene rearranged in multiple diagnostic entities (see Figure 1). For example, EWSR1 is rearranged in ES/PNET, desmoplastic round cell tumor, extraskeletal myxoid chondrosarcoma, some myxoid/round cell sarcomas, and clear cell sarcoma. Use of FISH requires some speculation on the part of the ordering pathologist about what type of underlying genetic abnormalities may be present, and thus a fusion can obviously only be detected if it is considered as a possibility. Conventional cytogenetics, on the other hand, allows for an unbiased determination of the entire karyotype with essentially no assumptions made a priori. Furthermore, some rearrangements, including insertions, may be difficult to detect by FISH. In this setting, confirmatory testing with reverse transcription polymerase chain reaction may be helpful. Finally, FISH studies cannot detect smaller genetic alterations such as point mutations, which can be detected by polymerase chain reaction analysis or direct sequencing. One issue contemplated during this study is the definition of terms used such as sensitivity and specificity. Several sarcomas, exemplified by ES/PNET and monophasic SS, essentially all harbored rearrangements of genes that were evaluated by FISH (EWSR1 and SYT, respectively). The argument could be made that, because rearrangements of the genes involved in these sarcomas are so prevalent, they become the gold standard for diagnosis of these entities; a sarcoma is defined entirely by the presence or absence of these genetic alterations. Assuming this is true, SYT FISH would be positive in 100% of SSs. Thus, there would be no false-negative results. Interpreted this way, if an SYT FISH test is negative, the neoplasm simply is not an SS. In this scenario, concern about retrospective bias when interpreting the histology/immunohistochemistry of the case after receiving a negative FISH result is a valid one. However, we made several observations to the contrary. There was one example each of ES/PNET and monophasic SS (Table 2) that did not harbor a rearrangement of their respective genes, meaning that there were rare cases where we felt that the histologic and immunohistochemical features of the neoplasm were characteristic enough to make the diagnosis despite negative FISH results. Thus, a low false-negative rate is present and is due to several factors. First, there are occasional false-negative results due to inherent technical difficulties associated with FISH described previously: A neoplasm may harbor the molecular alteration you are testing for; however, it just may not be detectable by FISH (eg, cryptic translocations, insertions). In these cases, a secondary laboratory method, such as reverse transcription polymerase chain reaction or conventional cytogenetics could be considered the gold standard. Another factor contributing to a false-negative rate is that our understanding of the molecular alterations present in these neoplasms is currently incomplete/ nearing completion. We are increasingly recognizing the presence of rare variant translocations in ES/PNET (translocations involving FUS rather than EWSR1) 24 and SS (a t(x;20) involving SS18L1 rather than SYT). 25 In other entities such as alveolar rhabdomyosarcoma, as mentioned previously, as many as 20% do not harbor 1 of the 2 described chromosomal translocations. Thus, FISH for 1 gene may not encompass all of the genetic alterations for many entities. If sufficient morphologic and immunohistochemical evidence is present to suggest the diagnosis, a negative FISH test does not necessarily exclude the diagnosis in the previous examples, and additional ancillary studies may be necessary. Perhaps in the future, we will reach the point where we will feel, with a reasonable degree of certainty, that essentially all recurrent genetic events in soft tissue neoplasms have been described and can be assayed for. Until then, the histologic appearance and immunohistochemical profile of these entities, in the hands of experienced pathologists, will probably be considered the gold standard for the near future. Acknowledging that FISH cannot be used as a standalone technique for all soft tissue neoplasms (but instead as an important adjunct to these previously mentioned techniques) we have nonetheless found FISH to be useful in evaluating most soft tissue neoplasms in which molecular analysis is desired. References 1. Downs-Kelly E, Goldblum JR, Patel RM, et al. The utility of fluorescence in situ hybridization (FISH) in the diagnosis of myxoid soft tissue neoplasms. Am J Surg Pathol. 2008;32(1): Weaver J, Downs-Kelly E, Goldblum JR, et al. 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