Keywords

List of Frequently Asked Questions

  1. 1.

    What is the role of molecular testing in round cell sarcomas?

  2. 2.

    How are undifferentiated round cell sarcomas classified? What are the most common fusions?

  3. 3.

    Is FISH testing for Ewing sarcoma and other undifferentiated round cell sarcomas enough? What is the role of next-generation sequencing (NGS) in Ewing sarcoma and other undifferentiated small round cell sarcomas?

  4. 4.

    What are some of the limitations for using NGS on undifferentiated round cell sarcomas?

  5. 5.

    What is the role of molecular testing in adipocytic tumors?

  6. 6.

    What is the sensitivity of MDM2 FISH for the diagnosis of ALT/WDL? What is the benefit of using FISH testing in problematic lipomatous tumors?

  7. 7.

    When is it appropriate to use MDM2 FISH testing in dedifferentiated liposarcoma? Are there any potential pitfalls?

  8. 8.

    What molecular fusion is seen in myxoid liposarcoma? What other molecular alterations can be seen?

  9. 9.

    What is the role of molecular testing in vascular tumors?

  10. 10.

    Are there any benign vascular tumors that may benefit from ancillary molecular testing?

  11. 11.

    What intermediate or low-grade vascular neoplasms are often confirmed with the use of molecular testing?

  12. 12.

    What is the role of molecular testing in angiosarcoma?

  13. 13.

    What is the role of molecular testing in skeletal muscle tumors?

  14. 14.

    What fusions are seen in alveolar rhabdomyosarcoma? What other alterations can be seen? How sensitive and specific is FISH testing and what are possible reasons for a negative result?

  15. 15.

    What entities are encompassed under the umbrella term “sindle cell/sclerosing rhabdomyosarcoma”? What rearrangements and/or mutations are seen? How does this impact prognosis?

  16. 16.

    What is the role of molecular testing in tumors of uncertain differentiation? What are the common fusions found in these tumors? What testing modalities are commonly employed and are there any limitations?

  17. 17.

    What is the role of molecular testing in fibroblastic/myofibroblastic tumors? What are the common fusions found in these tumors? What testing modalities are commonly employed and are there any limitations?

Frequently Asked Questions

  1. 1.

    What is the role of molecular testing in undifferentiated round cell sarcomas?

    • The differential for undifferentiated round cell sarcomas is broad. In fact, there are so many that the use of mnemonics is often employed just to remember the differential (Table 10.1). The role of molecular is to provide a definitive diagnosis on limited tissue samples to ensure appropriate treatment (e.g., neoadjuvant chemotherapy) and prognostic data when applicable.

    • Molecular testing is often considered an ancillary technique after an initial round of sorting with immunohistochemical stains (lymphoma/leukemia versus Ewing versus Rhabdomyosarcoma); however, many tumors can have overlapping histologic and immunohistochemical features, and in these cases the use of molecular testing is invaluable.

  2. 2.

    How are undifferentiated round cell sarcomas classified? What are the most common fusions?

    • Undifferentiated round cell sarcomas are classified as Ewing sarcoma, round cell sarcoma with EWSR1-non-ETS fusions, CIC-rearranged sarcoma, and sarcoma with BCOR genetic alterations [1] (Table 10.2).

    • Ewing sarcoma (EWS) is the prototypical small round cell sarcoma that involves EWSR1 on 22q12 with members of the ETS (E-26 transformation specific) transcription factors creating EWSR1-ETS fusions.

      • Most commonly involved fusion involves FLI1 [2] on 11q24 in ~85% of cases followed by ERG on 21q22 in ~10% of cases [3].

      • Other less common fusions involved ETV1 (ETS-variant gene 1) on 7p22 [4], ETV4 (ETS-variant gene 4) on 17q12 [5], and FEV (fifth Ewing sarcoma variant) on 2q33 [6].

    • Because EWSR1 is a member of FET family, other members including FUS (Fused in Sarcoma) can rarely substitute for EWSR1 creating FET/ETS fusions.

      • Known fusions include FUS-ERG and FUS-FEV [7, 8].

      • TAF15 other member of FET family could, in theory, substitute for FUS or EWSR1.

    • Round cell sarcoma with EWSR1-non-ETS fusions.

      • EWSR1-NFATC2 and EWSR1-PATZ1 tend to be not respond as well to neoadjuvant chemotherapy [9].

      • DNA methylations studies confirm distinct profiling from EWSR1-ETS and CIC or BCOR rearranged sarcomas [10, 11].

    • CIC-rearranged sarcomas account for the vast majority of “Ewing-like” sarcomas.

      • Most commonly involve CIC-DUX4 but other partners include FOXO4 [12, 13], NUTM1 [14], and NUTM2A [15].

      • Can also show trisomy 8 and MYC amplifications [16].

      • CIC-LEUTX can be seen as a subset of angiosarcomas [17].

      • Not chemosensitive like EWS and have a worse overall survival [18].

        • Tend to affect the deep soft tissue of the trunk and lower extremities of young adults (third to fourth decade).

        • Exception are the CIC-NUTM1 fusion which involves the bones of young children [14].

      • Potential pitfall.

        • A subset of angiosarcomas can show CIC gene abnormalities, most often mutations, but rearrangements have also been reported [17].

    • BCOR-rearranged sarcoma .

      • Most frequent is BCOR-CCNB3; but others include internal tandem duplication (BCOR-ITD), KMT2D-BCOR, BCOR-ZC3H7B, and BCOR-MAML3 [19, 20].

      • Tend to affect the bones of child/adolescents (80% in first two decades) [21, 22].

      • More common in males and have a similar overall survival compared to EWS [19].

  3. 3.

    Is FISH testing for Ewing sarcoma and other undifferentiated round cell sarcomas enough? What is the role of next-generation sequencing (NGS) in Ewing sarcoma and other undifferentiated small round cell sarcomas?

    • FISH testing, primarily through break -apart assays, is no longer considered enough by most soft tissue pathologists. This is in part due to some overlapping histology and immunohistochemistry with non-ETS fusions and other round cell sarcomas with specific gene rearrangements that can have a different prognosis (see below). The role of NGS is to identify specific gene fusions that may dictate treatment and prognosis.

    • EWSR1 break-apart FISH testing can sometimes create false positives in non-EWS tumors [23].

      • Often with tumors showing concurrent SMARCB1 deletions since genes are located only 5.5 Mb from each other.

    • Around 50% of previously diagnosed undifferentiated round cell sarcomas, most commonly defined as negative for EWSR1 rearrangement by FISH, have disease defining fusions [24,25,26], most commonly CIC and BCOR.

    • Because both EWSR1 and PATZ1 are located on the same chromosome and are ~2 mb away from each other, a FISH break-apart probe may result in a false negative due to the short distance between the inversion of the involved genes.

    • A subset of CIC-rearranged sarcomas may be missed using FISH as opposed to NGS testing [27,28,29].

      • Exact reason for false negatives is unknown, but it may be due to cryptic insertions beyond the resolution of the FISH assay.

  4. 4.

    What are some of the limitations for using NGS on undifferentiated round cell sarcomas?

    • Occasionally, CIC-rearranged sarcomas can be missed on RNA-based sequencing and this may be related to a failure of algorithmic analysis [29].

      • This may be in part due to repetitive sequences that can be seen with DUX4 on chromosomes 4q35.2 and 10q26.3 and are filtered out by algorithms.

      • Use of “Grep” command may help detect fusion when missed by other programs such as FusionMap, FusionFinder, and ChimeraScan programs [30].

    • There is still a small subset of undifferentiated round cell sarcomas that lack an identifiable fusion. These cases may benefit from array-based DNA-methylation profiling to determine if they cluster with a known group (e.g., CIC, BCOR) or if it will change management (i.e., use of neoadjuvant chemotherapy).

  5. 5.

    What is the role of molecular testing in adipocytic tumors?

    • Many adipocytic neoplasms have specific molecular mutations or rearrangements (Table 10.3). The primary role for molecular testing is to confirm or establish the diagnosis in problematic situations (see below).

  6. 6.

    What is the sensitivity of MDM2 FISH for the diagnosis of ALT/WDL? What is the benefit of using FISH testing in problematic lipomatous tumors?

    • Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) is defined by the amplification of MDM2 originating from the region of 12q14-q15, and because of its relative increased sensitivity and specificity compared to other methods such as Q-PCR and immunohistochemistry, FISH is now considered the gold standard for diagnosis [31,32,33].

      • Sensitivity in literature is considered to be greater than 90% [31, 32]; however, many studies were done on non-problematic tumors.

      • Cytogenetic analysis often shows supernumerary ring and giant chromosomes that originate from the 12q14-q15 region [34].

    • In problematic tumors, using the following criteria for MDM2 FISH testing may identify up to 1/3 of cases that would otherwise be classified as lipoma [35, 36]:

      • Recurrent lipomas.

      • Tumors with equivocal cytologic atypia.

      • Retroperitoneal, intra-abdominal, and pelvic tumors.

      • Deep extremity tumors larger than 10 cm in patients over 50.

  7. 7.

    When is it appropriate to use MDM2 FISH testing in dedifferentiated liposarcoma (DDLPS)? Are there any potential pitfalls?

    • Similar to ALT/WDLs, DDLPS are usually straightforward and do not need molecular testing if background WDL is present along with the high-grade component.

    • There are certain situations when testing is helpful, and these include:

      • When the differential includes primary retroperitoneal myxoid liposarcoma.

      • When then dedifferentiated component looks like another tumor such as myxoid liposarcoma or pleomorphic liposarcoma.

    • Potential pitfalls in dedifferentiated liposarcomas; the following situations might be misinterpreted as MDM2 amplification:

      • STAT6, which is associated with solitary fibrous tumors (NAB2-STAT6 fusions) [37], is located in 12q13 and can show amplification by FISH [38] and can also show nuclear immunohistochemical staining for STAT6.

      • DDIT3, which is associated with myxoid liposarcoma (FUS-DDIT3) is located on 12q13.2, and DDLPS can show amplification of DDIT3 in tumors that often have myxoid liposarcoma-like morphology [39].

    • Both ALTs and DDLPS can occasionally demonstrate multiple faint alphoid signals that represent satellite DNA of chromosome 12 [31].

      • This could be misinterpreted as gain of copy number.

  8. 8.

    What molecular fusion is seen in myxoid liposarcoma (MLPS)? What other molecular alterations can be seen?

    • Translocations of FUS-DDIT3 [40] in >95% and EWSR1-DDIT3 [41] in less than 5% are considered pathognomonic for MLPS.

      • Break-apart FISH for DDIT3 is considered sensitive and specific [42].

    • Approximately 50% have TERT promoter mutations [43].

    • ~25% of PI3K/mTOR mutations, most often gain of function mutations [44].

    • Diagnosis of high-grade MLPS when >5% round cell change.

      • High-grade MLPS have higher rate of metastasis and death [45].

      • Presence of necrosis is also associated with adverse prognosis [45].

  9. 9.

    What is the role of molecular testing in vascular tumors?

    • Many vascular tumors have known mutations or rearrangements; however, molecular testing is really only performed in a handful of vascular tumors (Table 10.4), and these are usually problematic or equivocal, borderline cases where the diagnosis is between benign and malignant and/or it will affect treatment/management.

  10. 10.

    Are there any benign vascular tumors that may benefit from ancillary molecular testing?

    • Epithelioid hemangioma is traditionally considered on spectrum and synonymous with the term angiolymphoid hyperplasia with eosinophilia (ALHE).

      • WHO no longer recommends the use of this terminology.

      • Interestingly, many cases of ALHE lack FOS or FOSB rearrangements [46].

    • Epithelioid hemangioma is characterized by recurrent fusion of FOS or FOSB genes in ~50% of cases [46].

      • FOS partners include LMNA, MBNL1, VIM, and lincRNA [47, 48].

      • FOSB partners include ZFP36, WWTR1, or ACTB [47, 49].

    • Molecular testing may be beneficial in cases that are referred to as atypical epithelioid hemangiomas [50], which can show some features such as solid growth and necrosis that would raise the differential of epithelioid angiosarcoma.

  11. 11.

    What intermediate or low-grade vascular neoplasms are often confirmed with the use of molecular testing?

    • Pseudomyogenic hemangioendothelioma (PMHE) .

      • When originally described, it was called epithelioid sarcoma-like hemangioendothelioma [51].

      • Molecular testing can be helpful because the tumor is often confused for other entities such as carcinoma or an epithelioid sarcoma.

        • Showing diffuse keratin (AE1/AE3) and vascular markers expression (ERG, FLI). About 50% express CD31 [52].

      • Characterized by SERPINE1-FOSB and ACTB-FOSB fusions [49, 53].

    • Epithelioid hemangioendothelioma (EHE) .

      • Malignant vascular tumor that most commonly involves the somatic soft tissue but also visceral organs such as the lung and liver [54].

      • In classic cases, molecular testing is likely not needed; however, if surrogate IHC is not available or if there is a need to exclude epithelioid angiosarcoma, then molecular testing is helpful.

      • Characterized by WWTR1-CAMTA1 fusion in >90% of cases [55] and YAP1-TFE3 in approximately 10% of cases [56].

        • CAMTA1 and TFE3 immunohistochemistry are often used as a surrogate for molecular testing [56, 57].

  12. 12.

    What is the role of molecular testing in radiation-associated angiosarcoma?

    • High-level MYC gene amplifications are characteristic of post-irradiation and chronic lymphedema-associated (Stewart-Treves) angiosarcoma [58].

    • Primary role of FISH testing is rule-in/-out angiosarcoma in difficult cases or when IHC is felt to represent a false positive [59].

      • ~25% of cases can show co-amplification of FLT4 [58].

      • FLT4 amplified lesion lack KDR and PLCG1 mutations that can be seen in both secondary and primary angiosarcomas [17].

    • Potential pitfall is that a small subset of primary angiosarcomas can show both MYC overexpression by IHC and MYC amplification, so clinical context is needed [60].

  13. 13.

    What is the role of molecular testing in skeletal muscle tumors?

    • Molecular testing for sarcomas showing skeletal muscle differentiation (rhabdomyoblastic) is continuing to evolve. The main role of molecular testing is for confirmation of alveolar rhabdomyosarcoma and to exclude other sarcomas with specific rearrangements or in specific situations where there may be a clinical need for mutational status (e.g., MYOD1 status in pediatric spindle cell rhabdomyosarcoma) or prognosis (e.g., congenital spindle cell rhabdomyosarcoma).

  14. 14.

    What fusions are seen in alveolar rhabdomyosarcoma? What other alterations can be seen? How sensitive and specific is FISH testing and what are possible reasons for a negative result?

    • Alveolar rhabdomyosarcoma (ARMS).

      • Second most common type of rhabdomyosarcoma [61].

      • Primitive round cell sarcoma characterized by PAX3-FOXO1, most commonly or PAX7-FOXO1 fusions [62].

        • Amplifications of MYCN and CDK4 can often be seen in PAX3-FOXO1 fusions [63, 64].

        • Amplification of 1p36 which encompasses PAX7 can be seen in PAX7-FOXO1 fusions [65].

        • Break-apart FISH for FOXO1 is generally considered to be 100% specific [66] but still a subset (~15%) of ARMS that are negative.

          1. (a)

            Likely a combination of low-level fusions, cryptic fusions, and true fusion-negative cases [67].

      • ALK copy number gains can be seen but has not played a role in therapy [68, 69].

      • Prognosis worse than fusion-negative rhabdomyosarcoma and ERMS [70].

  15. 15.

    What entities are encompassed under the umbrella term “sindle cell/sclerosing rhabdomyosarcoma”? What rearrangements and/or mutations are seen? How does this impact prognosis?

    • Congenital spindle cell rhabdomyosarcoma .

      • Characterized by VGLL2/NCOA2/CITED2 rearrangements.

        • Fusions involve VGLL2-CITED2, VGLL2-NCOA2, SRF-NCOA2, TEAD1-NCOA2 [71, 72].

      • Tend to present within first year and commonly involve the trunk and have a favorable prognosis.

        • Exceptions are those with MYOD1 mutations that tend to have a poor prognosis [72].

    • Spindle cell rhabdomyosarcoma with FUS-TFCP2 and EWSR1-TFCP2 fusions [11].

      • Can involve both bone and soft tissue, involve both children and adults, tend to be confused for an Ewing-like sarcoma, and behave aggressively.

    • Adult spindle cell/sclerosing rhabdomyosarcoma .

      • A subset shows MYOD1 mutations [73, 74].

        • Usually homozygous mutation in exon 1 (pL122R) but can also have heterozygous mutations.

        • More commonly seen in sclerosing subtype and can also harbor coexistent PIK3CA mutations [74].

    • Histiocyte-rich rhabdomyoblastic tumor .

      • Provisional entity most often confused with spindle cell rhabdomyosarcoma [75].

      • Does not have MYOD1 mutations, is usually encapsulated with surrounding lymphoid aggregates and a prominent histiocytic inflammatory infiltrate.

      • Good prognosis with no recurrences or metastasis [76].

      • Mentioned because angiomatoid fibrous histiocytoma would be on the histologic differential and molecular testing may be needed to exclude.

  16. 16.

    What is the role of molecular testing in tumor of uncertain differentiation? What are the common fusions found in these tumors? What testing modalities are commonly employed and are there any limitations?

    • Many soft tissue tumors of uncertain differentiation have unique molecular rearrangements that routinely primarily to confirm a diagnosis. See below for specifics on the individual tumors that are commonly tested.

    • Angiomatoid fibrous histiocytoma (AFH) .

      • Primarily occurs in children and young adults (<40 years) primarily in the dermis/subcutis of the extremities, although can occur anywhere where normal lymph nodes are found [77].

      • Most frequent fusion is EWSR1-CREB1 seen in >90% of cases [78].

        • Less commonly EWSR1-ATF1 and less commonly FUS-ATF1.

        • FISH break-apart probes are generally sensitive for detecting either the EWSR1 or the FUS rearrangement in AFH but up to 25% cases can be missed [79].

          1. (a)

            May possibly represent cryptic rearrangements not detectable through FISH probes or represent other fusion(s).

        • Immunohistochemistry.

          1. (a)

            Most show co-expression of EMA and desmin [77].

          2. (b)

            Recently, many have been shown to variably express ALK although this does not correlate with molecular alteration [80].

    • Synovial sarcoma (SS) .

      • Primarily occurs in the deep soft tissue of the extremities of adolescents or young adults and vast majority occur before age 50 [81].

      • Monophasic subtype more common than biphasic.

      • Because synovial sarcomas express EMA and cyokeratins [82], molecular testing can help exclude sarcomatoid carcinoma, in cases with treatment effect where the original material is not available for review and in more poorly differentiated cases.

      • Most common fusion is SS18-SSX1 between exon 10 of SS18 and exon 6 of SSX and less commonly involves SSX2, SSX4 or SS18L-SSX1 [83, 84].

        • Many centers employ the use of break-apart FISH which in some studies shows sensitivity of around 85% versus 95% when compared to PCR [83].

    • Alveolar soft part sarcoma .

      • Mainly affects young adults, most commonly involves the deep soft tissue of the extremities followed by the trunk [85].

        • More commonly affects head and neck in children.

      • Characterized by ASPSCR1-TFE3 fusion [86].

      • Molecular testing either by FISH or NGS only necessary in difficult or selected cases.

        • Strong and diffuse TFE3 by IHC and classic morphology is considered sufficient for the diagnosis [87].

    • Clear cell sarcoma .

      • Mainly affects young adults, most commonly as deep-seated locations of the distal extremities, with the majority arising near the foot/ankle [88].

      • Because of the expression of melanocytic markers [89], immunohistochemical distinction from melanoma not possible and molecular has become the mainstay for definitive distinction.

      • Most common fusion involves EWSR1-ATF1 most commonly between exon 8 of EWSR1 and exon 4 of ATF1 [90].

        • Other variant translocations involve fusion of EWSR1-CREB1 [89].

        • Rare cases can also show concurrent BRAF mutations [91], further blurring the differential with melanoma.

    • Extraskeletal myxoid chondrosarcoma .

      • Most commonly affects adults (median age 50 years) in the deep soft tissue of the proximal extremities and limb girdles [92].

      • Most commonly involves rearrangements of NR4A3 with either EWSR1 or TAF15 [93].

        • Rare fusions involving TCF12-NR4A3 and TFG-NR4A3 have also been identified [94, 95].

        • More recently, a HSPA8-NR4A3 fusion has been identified [96].

      • No other sarcoma has been found to have NR4A3 fusions, so its detection is considered pathognomonic.

    • Desmoplastic small round cell tumor (DSRCT) .

      • Most commonly affects children and young adults in the abdomen/peritoneal cavity [97].

        • Striking male predominance.

      • Because of its histologic overlap with other small round cell tumors (e.g., Ewing, alveolar rhabdomyosarcoma), the use of ancillary immunohistochemical stains, and molecular testing is often employed.

        • Most cases show expression for keratins, desmin, and WT1 (C-terminus) [97].

        • On molecular level, characterized by recurrent fusion most commonly involving first seven exons of EWSR1 and exons 8–10 of WT1 [98].

    • Intimal sarcoma .

      • Malignant sarcoma involving the great vessels of the heart and is now considered the most common [99].

      • FISH for MDM2 amplification is often necessary as these tumors can have non-distinctive histology.

      • The use of array-CGH can often also show amplification of KIT and PDGFRA, gain of EGFR, and loss of CDKN2A [99].

  17. 17.

    What is the role of molecular testing in fibroblastic/myofibroblastic tumors? What are the common molecular alterations found in these tumors? What testing modalities are commonly employed and are there any limitations?

    • Many fibroblastic/myofibroblastic tumors have distinct molecular mutations or fusions that are characteristics, but because ancillary testing methods such as immunohistochemistry are so cheap and have a faster turn-around-time, molecular testing is generally not needed. In addition, some of these tumors are benign and the cost of performing the molecular testing cannot be generally justified (Table 10.5). See below for specifics on the individual tumors for potential scenarios where ancillary molecular techniques may be employed.

    • Nodular fasciitis

      • Self-limited mesenchymal neoplasm that most commonly affects young adults of the subcutis of the upper extremities, trunk, head, and neck [100].

      • Most cases show classic histology and show myofibroblastic differentiation with actin positivity [101] in a “tram-track” pattern, so there is no need for molecular confirmation.

      • Difficult or unusual cases may benefit from molecular testing in order to exclude a more worrisome lesion.

        • Most often characterized by rearrangements between USP6 and MYH9 [102].

        • An exceptional case associated with multiple recurrences and metastatic disease has been associated with PPP6R3-USP6 [103].

      • A subset of cellular fibromas of tendon sheath have been found to have USP6 rearrangements [104].

    • EWSR1-SMAD3-positive fibroblastic tumor

      • Benign fibroblastic tumor that often involves the dermis/subcutis of acral sites [105].

      • Typically are positive for ERG but negative for SMA, CD34, CD31, and S100 [105].

        • It is unclear if these tumors represent a spectrum of similar pediatric fibroblastic neoplasms, and indeed, some cases classified initially as one entity are sometimes re-classified as another based on molecular testing [106].

    • Soft tissue angiofibroma

      • Benign fibroblastic neoplasm affecting middle-aged adults in the extremities, most commonly the leg [107].

      • Characterized by NCOA2 rearrangements most often to AHRR [108].

        • Other fusion partners include GTF2I-NCOA2 and GAB1-ABL1 [109, 110].

      • In most cases, molecular testing is not needed to confirm the rearrangements.

        • The main role of molecular testing is to exclude lesions that can have some histologic overlap and can behave more aggressively or have metastatic potential such as solitary fibrous tumor, low-grade fibromyxoid sarcoma, and a low-grade myxofibrosarcoma.

    • Desmoid-type fibromatosis

      • Locally aggressive fibroblastic neoplasm with a propensity for local recurrence that primarily affects young adults and most commonly involves the extremities, trunk, and abdominal cavity [111].

      • Point mutations involved two codon of exon 3 of CTNNB1 are found in the majority of sporadic tumors [112].

        • Immunostain for beta-catenin often used as a surrogate marker.

      • Smaller percentage is associated with Gardner syndrome and show germline mutations in APC gene but can also show sporadic mutations [113].

    • Giant cell fibroblastoma /dermatofibrosarcoma protuberans

      • Although listed separately in WHO, they are thought to be spectrums of the same neoplasm with the former arising primarily in children.

      • Lesions defined by COL1A1-PDGFB fusion.

        • ~2% of cases may be cryptic, and another small percentage may show alternate fusions involving PDGFB including COL6A3-PDGFD and EMILIN2-PDGFD [114].

      • Reasons to perform break-apart FISH or NGS.

        • Extensively myxoid lesion where classic architecture is not present.

        • Metastatic/recurrent disease where original material is not available.

        • Small samples where only “herringbone” pattern is present and the differential would include synovial sarcoma, MPNST, and fibrosarcomatous DFSP.

    • Solitary fibrous tumor

      • Commonly affects adults and can occur at any site but more common in deep soft tissue and extrapleural locations [115].

      • NAB2-STAT6 fusion is pathognomonic [37].

      • Molecular testing not generally needed as STAT6 IHC is generally considered sensitive and specific [38].

        • Dedifferentiated liposarcomas can occasionally show STAT6 expression, so this may be an instance where molecular testing is indicated.

    • Infantile fibrosarcoma

      • Commonly affects children less than 2 years and involves the extremities, trunk, head and neck.

      • Most cases harbor an ETV6-NTRK3 fusion [116].

        • Other cases have NTRK1, NTRK2, BRAF, and MET fusions [117,118,119].

      • Cases with NTRK rearrangements often show pan-TRK IHC expression [120].

    • Low-grade fibromyxoid sarcoma (LGFMS)

      • Malignant fibroblastic neoplasm that most commonly affects young to middle-aged adults of the deep extremities and trunk [121].

      • Characterized by fusions most commonly involving FUS-CREB3L2 or FUS CREB3L1 [122].

        • Less commonly can involve EWSR1 [123].

      • MUC4 IHC is generally considered sensitive and specific for the diagnosis, so routine molecular testing is not needed [124].

        • Selected cases tested when MUC4 IHC is negative or not available.

    • Sclerosing epithelioid fibrosarcoma

      • Malignant fibroblastic neoplasm with subset that is related to LGFMS both histologically and molecularly and can have a similar age and site distribution [125].

      • Most common fusion is EWSR1-CREB3L1 [126].

        • Other fusions show FUS or PAX5 with CREB3L2, CREB3L3, or CREM [127, 128].

      • Similarly to LGFMS, MUC4 IHC is considered sensitive and specific and is present in ~90% of cases [129], so routine molecular testing is not often needed.

Table 10.1 Differential of small round cell tumors
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Table 10.2 Undifferentiated round cell sarcomas
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Table 10.3 Alterations in lipomatous neoplasms
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Table 10.4 Commonly assessed molecular alterations in vascular lesions
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Table 10.5 Fibroblastic and myofibroblastic tumors with characteristic molecular findings that are not routinely tested
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Case 1

Case History

30-year-old previously healthy male presents with progressively worsening double vision for 1 month with associated vertigo and balance difficulties . This has been compounded by neuropathic back pain that radiates to the abdomen. An imaging CT shows a 9 cm chest wall mass that encases the eighth and ninth rib with a mild periosteal reaction without frank osseous invasion. The radiologic differential would be a malignant solitary fibrous tumor versus lymphoma versus, less likely metastatic disease.

Histologic Features

A CT-guided biopsy demonstrates a basaloid population of epithelioid cells with crush artifact and scattered admixed spindle cells (Fig. 10.1). Immunostains are positive for AE1/AE3 (focal), EMA (rare), and the spindled cells are positive for SOX10. The cells are negative for CD3, CD20, CK7, CK20, TTF1, desmin, chromogranin A, and SALL4.

Fig. 10.1
figure 1

Axial CT shows a posterior chest wall mass (a) that is composed of small round blue cells admixed with spindle cells (b) that are positive for SOX10 (c) that can of be seen in EWSR1-PATZ1 fusion. Interestingly, however, NGS revealed an EWSR1-FLI1 fusion (d)

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Choice of Molecular Testing

Given the presence of admixed spindled cells that are SOX10 positive, the preliminary differential was a round cell sarcoma with an EWSR1-PATZ1 fusion (EWSR1-non-ETS fusion). As this particular fusion is not responsive to traditional chemotherapy, a decision was made to perform RNA sequencing to confirm or rule out the fusion as opposed to simply using an EWSR1 break-apart FISH probe which has a faster turn-around time.

Molecular Study

An Archer® NGS fusion study revealed an ESWR1 (Exon 7)-FLI1 (Exon 5) fusion. (The NGS fusion study was performed using 26 gene FusionPlex Sarcoma panel, ArcherDx, Boulder, CO; validated in the molecular diagnostic laboratory for clinical testing.)

Final Diagnosis

Ewing Sarcoma with EWSR1-FLI1 fusion .

Case Discussion

This case demonstrates that although break-apart FISH would have detected the EWSR1 rearrangement, RNA sequencing provided the correct information for the diagnosis and also provided the treating clinicians with the correct clinical information to begin EWS chemotherapy regimen that would not have been started if this case truly demonstrated a PATZ1 fusion.

Case 2

Case History

A 13-year-old female noticed a lump in her groin for 2 weeks. She had a history of a neoplasm removed from her right knee 5 years ago. Ultrasound showed a 4 cm lymph node concerning for malignancy. Upon further investigation, the patient had been diagnosed with an angiomatoid fibrous histiocytoma. Given that lesion was removed years ago without recurrence, the current lesion is suspicious for metastasis.

Histologic Features

Sections show a tumor composed of spindled to epithelioid cells arranged in a syncytial pattern that is involving a lymph node (Fig. 10.2). Immunostains are positive for EMA and desmin.

Fig. 10.2
figure 2

Low power shows a metastatic deposit in a lymph node (a). Higher power shows uniform histiocytoid cells (b) that are positive for both EMA (c) and desmin (d). NGS reveal an EWSR1-ATF1 fusion (e)

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Molecular Study

An Archer® NGS fusion study found an EWSR1-ATF1 gene fusion.

Final Diagnosis

Metastatic angiomatoid fibrous histiocytoma .

Case Discussion

While angiomatoid fibrous histiocytomas can have surrounding lymphoid aggregates, the current case illustrates a rare metastasis to the locoregional lymph nodes that happens in <5% of cases. EWSR1-CREB1 fusions are the most common fusions in angiomatoid fibrous histiocytoma, but ATF1 fusions are more frequently associated with extrasomatic soft tissue cases. In the current case, FISH break-apart would have been an option. However, one potential limitation is that FISH testing cannot always discriminate between a simple terminal deletion of the 3’ EWSR1 and translocation involving the remaining 5′ portion of EWSR1 with another gene. In addition, rare fusions involving FUS can happen and would be missed by that assay.

Case 3

Case History

A 25-year-old male presented with several week history of pleuritic chest pain . He was treated by his PCP with a trial of steroids and Z-Pak which failed to improve his symptoms. On initial examination, a right upper quadrant ultrasound demonstrated two hepatic masses. A follow-up MRI showed innumerable masses along the liver that appear external to liver parenchyma. There was also a large confluent mass anteriorly between the liver and the diaphragm that measured 10 cm.

Histologic Features

Core biopsy shows a malignant small round cell tumor arranged in nest and sheets with intervening fibrous stroma (Fig. 10.3). Immunostains are positive for AE1/AE3, Desmin, and CD99 (patchy) and are negative for WT1, CK5/6, MYOD1, CD45, Melan-A, and TTF1.

Fig. 10.3
figure 3

Axial MRI shows multiple peritoneal surface masses throughout the abdomen (a). Biopsy shows a small round cell tumor growing in sheets with intervening fibrous stroma (b) that is strongly positive for AE1/AE3 (c) and desmin (d). NGS revealed an EWSR1-WT1 fusion (e)

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Molecular Study

An Archer® NGS fusion study revealed an EWSR1-WT1 fusion.

Final Diagnosis

Desmoplastic small round cell tumor .

Case Discussion

The most common transcript for DSRCT involves the first 7 exons of EWSR1 fusing to exons 8–10 of WT1; however variant translocations exist. This patient underwent 8 cycles of chemotherapy and repeat imaging revealed persistent abdominal lesions with persistent mediastinal lymphadenopathy. The patient is currently scheduled for resection of the mediastinal lesions with a plan for subsequent abdominal exploration and cytoreductive surgery. Despite multimodality therapy, the 5-year overall survival rate is low.

Case 4

Case History

A 50-year-old male had a history of essential thrombocytosis diagnosed 20 years ago. His most recent blood cell counts revealed the following: WBC 3.3 K/μL, Hb 9.3 g/dL, Hct 38%, Plt 331 K/μL. He had had worsening anemia of the past year with increasing splenomegaly. A CT of the abdomen and pelvis revealed a 19.5 cm spleen as well as numerous soft tissue masses encompassing the right and left kidney but clinically the patient has been asymptomatic.

Histologic Features

Core biopsy shows a variably cellular lesion composed of small, bland spindled cells arranged in vague fascicles set in a myxo-collagenous stroma with scattered hyperchromatic pleomorphic cells with smudgy chromatin (Fig. 10.4). An immunostain is positive for CD61 and negative for MDM2.

Fig. 10.4
figure 4

Coronal CT shows multiple soft tissue masses bilaterally included around the kidneys (a). Biopsy of retroperitoneum at low power shows a fibrous spindle cell lesion with scattered enlarged and hyperchromatic cells (b). Higher power reveals the pleomorphic cells show smudgy chromatin and multinucleate forms (c) that are positive for CD61 (d)

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FISH Results

Negative for MDM2 amplification.

Final Diagnosis

Sclerosing extramedullary hematopoietic tumor .

Case Discussion

Sclerosing extramedullary hematopoietic tumor is an extramedullary complication associated with myeloproliferative neoplasms. The presence of atypical megakaryocytes in the retroperitoneum raises the differential of a well-differentiated liposarcoma; however, they stain appropriately with CD61 and FISH for MDM2 is negative to help exclude a liposarcoma. In this case, the diagnosis would have been extremely difficult if the clinical history was not available.

Case 5

Case History

A 35-year-old female with no significant past medical history presented with a back mass that was clinically felt to be a lipoma or a cyst. Nothing was done at that time on initial presentation, and she returned to clinic a couple months later because the mass increased in size and it was excised by a surgeon.

Histologic Features

Sections show a cellular lesion composed of spindled and epithelioid cells arranged in sheets and nests with areas of peritheliomatous growth with associated necrosis, brisk mitotic activity and areas of clear cell and myxoid change. In addition, there are nodular areas of fascicular growth with intervening dense fibrous septa (Fig. 10.5). Immunostains are positive for CD99, FLI1 and WT1 and are negative for CD45, AE1/AE3, Cam5.2, PAX8, desmin, hmb-45, SOX10, and BCOR.

Fig. 10.5
figure 5

Low power shows nodular growth pattern with intervening fibrous septa (a) and areas of clear cell change with zones of necrosis (b). Medium power shows strong, membranous CD99 (c) and nuclear WT1 (d). NGS revealed a fusion transcript of CIC (exon 20) and FOXO4 (exon 2) (e)

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Molecular Study

An Archer® NGS fusion study revealed a fusion transcript of CIC (exon 20) and FOXO4 (exon 2).

Final Diagnosis

CIC-rearranged sarcoma .

Case Discussion

CIC-rearranged sarcoma is an undifferentiated round cell sarcoma that most commonly involves a CIC-DUX4 fusion. The nodular growth pattern in the current case along with the zone of necrosis and strong nuclear expression of WT1 is suggestive of a CIC-rearranged sarcoma. Interestingly the presence of clear cell change can sometimes be associated with EWSR1-FEV; however, because response to chemotherapy is dependent on the specific fusion transcript detected NGS testing is becoming the gold standard in the diagnosis of round cell sarcomas.

Case 6

Case History

An 80-year-old female with a left chest wall mass that was felt to be recurrent myxoid liposarcoma . She was originally diagnosed at an outside facility with a retroperitoneal primary myxoid liposarcoma 5 years ago and is status post two resections. The current lesion measures up to 16 cm. No history of molecular/FISH testing is found.

Histologic Features

Sections show a predominant low-grade myxoid adipocytic lesion with a plexiform vasculature and signet-type lipoblasts . A few sections demonstrate more increased cellularity and atypia with spindled cells arranged in fascicles with scattered pleomorphic forms and conspicuous mitotic activity. Within the cellular areas, focal osteoid formation is present (Fig. 10.6). An immunostain for MDM2 is focally positive.

Fig. 10.6
figure 6

Low power shows a nodular myxoid lesion (a) with plexiform vasculature and scattered signet-type lipoblasts (b). Higher power demonstrates spindled fascicular growth with conspicuous mitoses with pleomorphic forms (c) and osteoid formation (d). FISH testing demonstrates amplification of MDM2 (e)

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FISH Results

Positive for MDM2 amplification with a ratio of MDM2 fluorescent signal to chromosome 12 centromere signal of 15.

Final Diagnosis

Dedifferentiated liposarcoma.

Case Discussion

The patient’s history of a retroperitoneal primary myxoid liposarcoma is noted. Although primary retroperitoneal myxoid liposarcomas do exist, they are rare and a retroperitoneal dedifferentiated liposarcoma with areas reminiscent of myxoid liposarcoma would be much more likely. As the current specimen showed a high-grade spindle cell component which would be unusual for a high-grade myxoid liposarcoma, FISH testing was employed and is amplified.

Case 7

Case History

A 25-year-old female presented with a complaint of swelling and a slowly enlarging mass of the lower lip. Clinical exam demonstrated a mobile and well-circumscribed lesion suggestive of a benign process.

Histologic Features

Sections show a relatively well-circumscribed submucosal spindle cell lesion. The spindle cells show fascicular growth and are monomorphic with relatively bland elongated to wavy nuclei with finely granular chromatin, inconspicuous nucleoli, and minimal cytoplasm (Fig. 10.7). Immunostains are positive for EMA (patchy) and are negative for CD34, desmin, S100 protein, and STAT6.

Fig. 10.7
figure 7

Low power shows a well-circumscribed submucosal mass (a) that is composed of loose fibrous spindle cell areas (b) admixed with more cellular basophilic areas (c). Higher power shows the monotonous spindle cell population with overlapping nuclei (d). NGS revealed a SS18-SSX2 fusion (e)

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Molecular Study

An Archer® NGS fusion study revealed a SS18-SSX2 fusion transcript.

Final Diagnosis

Monophasic synovial sarcoma .

Case Discussion

The most common fusion in synovial sarcoma is SS18-SSX1, but a majority of SS18-SSX2 are the monophasic subtype and they are more common in females. Less than 10% of synovial sarcomas involve the head and neck region and a well-circumscribed growth can give a false impression of a benign process. Although NGS was performed, break-apart FISH for SS18 would have been a reasonable ancillary testing method choice.

Case 8

Case History

A 40-year-old female presented to her dermatologist with a left lower leg nodule . The clinical differential was broad and included lymphoma, a granulomatous process, a deep fungal infection versus a metastasis of unknown primary.

Histologic Features

A punch biopsy shows a cellular dermal-based mesenchymal neoplasm composed of basaloid cells growing in nests and sheets. The cells have scant cytoplasm and mitotic activity is conspicuous (Fig. 10.8). Immunostains are positive for desmin, myogenin, and myoD1 and are negative for AE1/AE3, S100, chromogranin, CK20, TTF1, TdT, and CD45.

Fig. 10.8
figure 8

Low power shows a cellular dermal basaloid neoplasm (a). Higher power shows a small round cell tumor growing in sheets and nests (b). The tumor cells are positive for desmin (c) and myo-D1 (d). NGS revealed a PAX3-FOXO1 fusion (e)

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Molecular Study

An Archer® NGS fusion study revealed a fusion transcript of PAX3 (exon 7) and FOXO4 (exon 2).

Final Diagnosis

Alveolar rhabdomyosarcoma .

Case Discussion

The patient underwent chemotherapy, and at the time of resection, the tumor was approximately 10% viable with negative margins and one lymph node positive for metastatic disease. Approximately 8 months later, she had a recurrence at the original site that was resection and most recent imaging studies have been negative.

Case 9

Case History

A 60-year-old female with a history of clear cell sarcoma that was diagnosed at an outside facility with break-apart FISH for EWSR1. She had lung metastases but had been on 4 cycles of pembrolizumab but developed a possible recurrence in the groin.

Histologic Features

Biopsy showed a subcutaneous clear cell neoplasm composed of relatively uniform cells arranged in pseudo-alveolar nests with surrounding fibrous septa (Fig. 10.9). Immunostains were positive for Melan-A and SOX10 and negative for AE1/AE3.

Fig. 10.9
figure 9

Low power shows a subcutaneous clear cell neoplasm with intervening fibrous septa (a). Higher power shows a relatively monotonous population of epithelioid cells arranged in pseudo-alveolar nests (b). FISH testing demonstrates an EWSR1 rearrangement (c)

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Molecular Study

An Archer® NGS fusion study revealed no database fusions.

Reflective FISH Results

Positive for EWSR1 gene rearrangement in 33% of cells.

Final Diagnosis

Recurrent clear cell sarcoma.

Case Discussion

This is a challenging case that was discussed at length at the multidisciplinary tumor boards. It was suggested that perhaps there is potentially a DNA only fusion without an RNA transcript secondary to the immunotherapy that may account for the NGS testing being negative. Conversely, it is possible that the EWSR1 FISH is a false positive. The FISH probe used in the most recent cytogenetic test is actually proximal to the 5′ portion of EWSR1 and not a part of the gene itself. So, it is possible that the break is next to but not within the actual gene. That being said, the mutational burden was also low and other mutations commonly seen in melanoma (the main histologic differential) were not seen. Taken together, it was felt clinically that the lesion most likely represents clear cell sarcoma and the patient is currently continuing immunotherapy treatment.

Case 10

Case History

A 75-year-old male presented with a right posterior thigh mass. A CT exam showed a solid enhancing intramuscular mass that measured 5.2 cm. A CT biopsy was performed for diagnosis given the concern for a soft tissue sarcoma.

Histologic Features

A biopsy shows a myxoid mesenchymal neoplasm composed of monotonous spindled cells with admixed more epithelioid cells growing in reticular cords (Fig. 10.10). Immunostains show patchy EMA and are negative for AE1/AE3, S100 and synaptophysin.

Fig. 10.10
figure 10

Sections show a myxoid spindle lesion with eosinophilic cytoplasm growing in cords (a) with a transition to more cellular areas (b) with the cells becoming more epithelioid (c). NGS revealed an EWSR1-NR4A3 fusion (d)

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Molecular Study

An Archer® NGS fusion study revealed an EWSR1-NR4A3 fusion.

Final Diagnosis

Extraskeletal myxoid chondrosarcoma .

Case Discussion

The growth pattern of the cells is highly suggestive of an extraskeletal myxoid chondrosarcoma and the absence keratin and myoepithelial markers would make myoepithelioma less likely. However, because the immunophenotype of EMC is often non-distinct, routine molecular testing is performed when this diagnosis is entertained and the finding of a NR4A3 rearrangement is pathognomonic.