Journal of Surgical Oncology 2015;111:520–531

Molecular Diagnostics in Soft Tissue Sarcomas and Gastrointestinal Stromal Tumors STEPHEN M. SMITH, MD,1 JOSHUA COLEMAN, MD,1 JULIA A. BRIDGE, 1 AND O. HANS IWENOFU, MD *

2 MD,

1

Department of Pathology & Laboratory Medicine, Wexner Medical Center at The Ohio State University, Columbus Ohio 2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska

Soft tissue sarcomas are rare malignant heterogenous tumors of mesenchymal origin with over fifty subtypes. The use of hematoxylin and eosin stained sections (and immunohistochemistry) in the morphologic assessment of these tumors has been the bane of clinical diagnosis until recently. The last decade has witnessed considerable progress in the understanding and application of molecular techniques in refining the current understanding of soft tissue sarcomas and gastrointestinal stromal tumors beyond the limits of traditional approaches. Indeed, the identification of reciprocal chromosomal translocations and fusion genes in some subsets of sarcomas with potential implications in the pathogenesis, diagnosis and treatment has been revolutionary. The era of molecular targeted therapy presents a platform that continues to drive biomarker discovery and personalized medicine in soft tissue sarcomas and gastrointestinal stromal tumors. In this review, we highlight how the different molecular techniques have enhanced the diagnosis of these tumors with prognostic and therapeutic implications.

J. Surg. Oncol. 2015;111:520–531. ß 2015 Wiley Periodicals, Inc.

KEY WORDS: molecular diagnostics; treatment; soft tissue sarcomas; GIST

INTRODUCTION Soft tissue sarcomas (STS) are malignant mesenchymal neoplasms that show significant heterogeneity both within and across different histologic subtypes. The advent of immunohistochemistry resulted in some degree of categorization with good diagnostic accuracy though with inherent limitations. The more recent emergence of high throughput technologies like next generation sequencing has enabled the identification of fusion genes like NAB-STAT6 in solitary fibrous tumor, CIC-DUX4 in Ewing-like round undifferentiated sarcomas and EWSR1-FLI1 in Ewing sarcoma which serves not only as important biomarkers but also molecular drivers of oncogenesis. Indeed, it is conceivable that as molecular characterization of sarcomas continue, there would be a gradual paradigm shift from the conventional classification of soft tissue sarcomas from the traditional putative tissue of origin to defining recurrent chromosomal translocations or gene expression profiles that are specific for the diagnostic entities. The recognition of these chromosomal translocations and fusion genes has increasing become the focus of potential targeted therapeutics, prognostication and biologic drivers of sarcomagenesis. Advances in molecular techniques over the past two decades including: karyotype analysis, southern blots, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), gene expression and microRNA profiling and high through-put next-generation sequencing (NGS) have translated to remarkably improved diagnostic capabilities for the modern pathologist. At the present juncture, we can apply the molecular code of soft tissue malignancies to identifying targetable mutations, development of therapeutics, design of clinical trials and overall risk stratification [1]. Herein, we review the utility of how these different technologies in the analysis of STS enhance the precision of histologic diagnosis and also how the modern diagnostician’s role has evolved from diagnosis to guidance of therapy and prognosis in these rare entities.

ADIPOCYTIC TUMORS The diagnostic accuracy of adipocytic tumor classification has dramatically improved over the past several decades, due largely in

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part to the development of fluorescent in situ hybridization (FISH) and cytogenetics [2]. One of the critical clinical questions in adipocytic neoplasms is the reliable distinction between well differentiated adipocyte-like well differentiated liposarcoma and lipomas. Routine Hematoxylin and Eosin stain, whilst the mainstay of diagnosis, does have it limitations. The differentiation of atypical lipomatous tumors (ALT)/well-differentiated liposarcomas (WDLS) from benign lipomatous entities on the molecular level is increasing in importance for diagnosis, prognostication and treatment. Classically, the defining genetic feature of WDLS/ALT is supernumerary “ring” and giant rod chromosomes (Fig. 1A–C), seen commonly in chromosomal karyotypes of lesional cells. Importantly, these rings and giant rods result in amplification of the 12q14–15 region, the same amplicon which contains the HMGA2 gene. The functional protein expressed in 90% of WDLS is MDM2, although this may be found in other sarcomas and is therefore not specific for WDLS [3]. Thus, one major diagnostic utility is to distinguish lipomas from liposarcomas by detection of amplification of MDM2 by FISH. Of note, the amplification of the MDM2 and CDK4 genes are also frequently observed in dedifferentiated liposarcomas and can be useful in the distinction of this entity from other spindle cell sarcomas particularly in the retroperitoneal location [4]. Interestingly, the MDM2 protein is a negative regulator of the tumor suppressor p53 by interaction with a second regulator termed as MdmX [5]. This is of particular interest given that several drugs have been designed, including RG7112

*Correspondence to: O. Hans Iwenofu, MD, Department of Pathology and Laboratory Medicine, Wexner Medical Center at The Ohio State University, 410 West 10th Avenue, Columbus, OH 43210. Fax: (614)293-2779. E-mail: [email protected] Received 10 October 2014; Accepted 11 December 2014 DOI 10.1002/jso.23882 Published online 10 March 2015 in Wiley Online Library (wileyonlinelibrary.com).

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Fig. 1. Adipocytic Tumors. A–C: Well Differentiated Liposarcoma. A: Well differentiated liposarcoma, adipocyte like. Note the scattered nuclear hyperchromasia present. B: Karyotype analysis reveals characteristic giant ring chromosome. C: Fluorescent In Situ Hybridization (FISH) showing amplification of MDM2 in 12q12–14 diagnostic of well differentiated liposarcomas. D–F: Myxoid Liposarcoma. D: Adipocytic neoplasm composed on round to oval cells with mucin pools and chick-wire vascular patter, prototypic for myxoid liposarcoma. E–F: Gene rearrangement is positive for DDIT3 (CHOP) and FUS, diagnostic of myxoid liposarcoma.

and MI-219, to target the anti-p53 properties of this MDM2 [reviewed in [6]]. Myxoid liposarcomas are histologically and molecularly distinct malignant fat tumors that have a predilection for the young. They are characterized by the presence of reciprocal translocation between the DDIT3 (CHOP) gene on chromosome 12 and the FUS gene on chromosome 16, t(12;16)(q13;p11), in >95% of cases, or less commonly, a translocation between the DDIT3 and EWS genes, t(12;22)(q13; p11) [7]. FISH is commonly used in practice to detect rearrangements of the DDIT3, FUS or EWS genes for diagnosis of this entity, with DDIT3 being the most commonly employed probe given that it is found in all known variants of myxoid liposarcoma (Fig. 1C–E).

FIBROBLASTIC TUMORS Dermatofibrosarcoma Protuberans The molecular pathology of dermatofibrosarcoma protuberans (DFSP) continues to be elucidated. Classically, this CD34-positive Journal of Surgical Oncology

tumor demonstrates a classic translocation of the COLA1 gene on chromosome 17q22 with the platelet derived growth factor beta, PDGFb, on chromosome 22q13. Of note, the loss of CD34 signals fibrosarcomatous change and thus increased propensity for metastasis; in higher grade tumors or transformed DFSP, the diagnostic value of gene rearrangement is significantly increased [8,9]. The mainstay of treatment to date has been surgical resection with wide negative margins. However, detection of the COLA1-PDGFb fusion gene has been shown to correlate to response with imatinib therapy, leading to its approval by the FDA in unresectable, recurrent, and/or metastatic DFSP in adult patients [9–14]. A recent phase II study concluded that imatinib therapy as a neoadjuvant agent in DFSP was both efficacious and welltolerated, supporting its usage in COLA1-PDGFb positive tumors [15]. Thus, it is requisite to detect the COLA1-PDGFb rearrangement via FISH or RT-PCR prior to commencing tyrosine kinase inhibitor therapy. The technical difficulties of these assays has lead, in recent literature, to the simplification of a single assay which utilizes multiplex RT-PCR to rapidly and efficiently detect the rearrangement [16] (Fig. 2).

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Fig. 2. Low Grade Fibromyxoid Sarcoma (Evan’s tumor). A: Hematoxylin and Eosin stain showing short fascicles of banal spindle cell tumor with interstitial collagen. The deceptively bland appearance might suggest an erroneous benign diagnosis. B: Dual Color FUS-CREB3L2 (FUS in green and CREB3L2 in orange) custom designed FISH probe set showing positive (two orange/green fused signals) indicative of 7;16 translocation in Low Grade Fibromyxoid Sarcoma. In a recent article, Hong et al. utilized whole genome sequencing to identify genetic aberrations in imatinib-resistant DFSP [17]. Although no significant copy number alterations were identified during imatinib treatment, eight somatic gene mutations were identified. Among them, a CARD10 gene mutation was identified which has known association with the NF-kB signaling pathway [18,19]. Also identified was a SAFB2 gene mutation which has been shown to affect apoptosis and cell growth [20]. And while targeted therapies for many of these aberrations in development, it is worthwhile to consider that identification and profiling of conventional therapy-resistant tumors may yield valuable prognostic information (Fig. 3).

Fibrosarcomas Cytogenetic analysis has identified t(12;15)(p13;q26), t(7;16)(q34; p11) and t(7;16)(q33;p11) translocations as recurrent findings in infantile fibrosarcoma and low grade fibromyxoid sarcomas respectively [21]. Consistent reciprocal translocations involving the FUS gene have been identified in low-grade fibromyxoid sarcoma (Evans tumor), most commonly involving a CREB3L2 gene fusion t(7:16)(q32–34;p11) [22,23]. This is a useful adjunct in distinguishing Journal of Surgical Oncology

Fig. 3. Ewing/Ewing-family Tumors. A: H&E showing uniform round cell morphology of malignant small round blue cell tumor. B: Diffuse membranous reactivity for CD99. C: Fli-1 reactivity by immunohistochemistry. The EWS-Fli1 fusion gene by RT-PCR (not shown) was negative in spite of the morphologic and immunophenotypic overlap with Ewing sarcoma. This was classified as Ewing-like sarcoma. D: Ewing Sarcoma. Diagnosis by RT-PCR. The histology (not shown) is indistinguishable from A. Lane1 and 5: 100bp ladder. Lane 2: Patient sample, Type 1 EWSR1-Fli1. Lane3: Type 2 EWSR1-Fli1 Positive Control. Lane 4: Negative control, no template.

this entity from other histologic mimics like nodular fasciitis, fibromatosis and low grade myxofibrosarcomas. Reports of ring chromosomes have also been documented in fibromyxoid sarcomas [24]. Infantile fibrosarcoma has been documented as having a t(12;15) (p13q26) translocation which is related to a more favorable outcome [25]. This translocation codes for the fusion oncoprotein ETV6-NTRK3 which is also found in radiation induced thyroid cancer and mammary analogue secretory carcinoma [26–30]. Interestingly, recent data suggests that this fusion protein may be a candidate for therapy with the ALK and MET inihibitor crizotinib [31]. Although not yet completely developed, FISH for rearrangements of the ETV6 locus can be utilized for the diagnosis.

Malignant Small Round Blue Cell Tumors (MSRBCTs) MSRBCTs are a group of unrelated tumors that characteristically share uniform round cell morphology, high DNA content hence the bluish hue to the cells and diffuse growth pattern. These tumors cover a wide array of different malignancies including: Ewing’s sarcoma/ Primitive Neuroecteodermal Tumors, Rhabdomyosarcomas (especially alveolar and embryonal subtypes), mesenchymal chondrosarcoma, lymphomas, small cell variant of osteosarcomas, malignant melanoma, desmoplastic small round cell tumor, Wilm’s tumor, neuroblastoma, poorly differentiated synovial sarcoma and neuroendocrine tumors. Although histomorphology remains vital in the analysis these cases, the

Molecular Diagnostics in Sarcomas and GIST use of immunohistochemistry is limited due to overlapping immunophentotypic mimicry between some of the tumors. The development of molecular diagnostics has revolutionized the way these entities are now approached. Many RT-PCR and FISH assays have been widely used to distinguish these tumors. Interestingly, the recent next-generation sequencing-based assay designated as ChildSeqRNA, developed to approach childhood sarcomas, includes a significant number of these small round blue cell tumor entities in its database [32]. We will focus our discussion in this section on rhabdomyosarcomas, Ewing’s Sarcoma and Ewing’s family of tumors and desmoplastic small round blue cell tumor (Fig. 4).

Rhabdomyosarcoma The morphology of rhabdomyosarcoma may present as a diagnostic challenge to the pathologist. Often this entity presents as a small round blue cell tumor, creating a histomorphologic differential diagnostic list which may include synovial sarcoma, lymphoma, melanoma, Wilm’s tumor and desmoplastic small round cell tumor as previously mentioned. Although advances in immunohistochemistry antibody panels like TLE-1, Fli-1, WT-1, Sox-9, S-100, Pancytokeratin, Desmin, Myogenin, Myo-D1, Sox-10, SATB2, among others, have eased the diagnostician’s challenge of sorting through these vast differential diagnosis, the discovery of entity-specific fusion genes has allowed for precision diagnosis in these tumors. PAX3-FOXO1 (also termed as FKHR) arising from t(2;13)(q35;q14) translocation is the most commonly identified translocation in alveolar rhabdomyosarcomas, being identified in approximately 55% of cases [33,34]. A second translocation of the PAX7-FOXO1 genes, t(1;13)(p36;q14), was identified in 1994; this translocation is identified in approximately 25% of cases [34]. Interestingly, the estimated 4-year overall survival rate in tumors expressing the PAX7-FOXO1 fusion

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gene was significantly higher than those expressing the PAX3-FOXO1 fusion gene [34]. Both the PAX3-FOXO1 and PAX7-FOXO1 fusion transcription factors regulate cell motility and promotes metastasis by acting on multiple downstream effectors, many of which are still being identified [35–38]. Those alveolar rhabdomyosarcomas which are sans PAX/FOXO1 translocations are known to be genetically heterogeneous, with unusual fusion products identified [39]. Interesting, SNP analysis has shown that a subset of these tumors show predominant rearrangement within the MDM2 and CDK4 regions on chromosome 12q13–15, similar to those seen in adipocytic tumors [40,41]. Specific translocations for the embryonal subtype have not yet been identified; it is well-established in the literature that embryonal rhabdomyosarcomas are negative for the FOXO1 rearrangements found in the alveolar form [42,43]. Attempts to identify genetic alterations in embryonal rhabdomyosarcoma have found a myriad of known oncogenes implicated, including TP53 and RAS [44–46]. However, these alterations are inconsistent and highly variable, precluding their use as a diagnostic tool. Reverse-transcriptase PCR and whole-genome analysis of 77 embryonal rhabdomyosarcoma cases revealed no significant difference in genetic signatures between the alveolar and embryonal rhabdomyosarcoma subtypes, suggesting that histomorphology and immunohistochemistry are still central to the diagnosis [47]. Of note, approximately 20% of all rhabdomyosarcomas are fusion gene-negative [34].

Ewing’s Sarcoma/Family of Tumors The tremendous overlap of the histologic and immunophenotypic profile for Ewing’s sarcoma and other entities in the malignant small round blue cell tumor family including: mesenchymal chondrosarcoma, small cell osteogenic sarcoma, desmoplastic small round cell tumor, rhabdomyosarcoma, lymphomas, Wilm’s tumor, neuroblastomas,

Fig. 4. Synovial Sarcoma. A: Biphasic synovial sarcoma. H&E stain showing the admixture of both spindled and glandular. B: Monophasic synovial sarcoma, fibrous type. H &E stain showing fascicles of spindled cells with nuclear hyperchromasia. C: Diffuse TLE-1 immunohistochemical stain reactivity, often seen in synovial sarcomas. D: Fluorescent In Situ Hybridization (FISH) showing clear hybridization signals with positive SS18 (SYT) gene rearrangements diagnostic of synovial sarcoma. Journal of Surgical Oncology

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melanomas and lymphomas —underscores the importance of molecular analysis to confirm this diagnosis. The most common cytogenetic finding in this tumor is the classic t(11;22)(q24;q12) translocation, resulting in a fusion of the EWSR1 transcript to an transcription factor, FLI1, which occurs in over 90% of Ewing’s sarcomas. The EWSR1FLI1 oncoprotein modulates the activation of more than 1000 abnormal genes as well as repressing multiple tumor-suppressor genes [48, reviewed in [49]]. Rearrangements between EWSR1 and ERG (on chromosome 21) are the next most common finding, representing 3–5% of tumoral cells. PCR is commonly used to detect these fusion genes. The rare EWSR1/FUS which has been documented can be detected using FISH, although this translocation is not specific for Ewing’s sarcoma [50]. Within the past 1–2 years, molecular pathology has identified a subset of small round cell sarcomas which morphologically resemble Ewing’s sarcoma, but are classified based on the distinct chromosomal aberrations not seen in traditional Ewing’s sarcoma. Mariño-Enríquez and Fletcher, in their recent review of these “Ewing-like sarcomas”, noted that “the application of cytogenetics and molecular genetic techniques allows for the identification of an increasing number of genetically defined subgroups within this category, which may represent distinct biologic entities.” [49]. For example, poorlydifferentiated Ewing’s-like sarcomas, such as those containing the CIC-DUX4 fusion gene, t(4;19)(q35;q13), may not be as sensitive to Ewing sarcoma-type chemotherapeutic regimens [49]. Interestingly, this cytogenetic translocation might be found in up to two-thirds of EWSR1-negative Ewing’s sarcomas [51].

Desmoplastic Small Round Cell Tumor Desmoplastic small round cell tumor (DSRCT) may be a challenging given its morphologic and immunonophenotypic overlap to other lesions particularly Wilm’s tumor. DSRCT is uniquely associated with an EWSR1-WT1 translocation, t(11;22)(p13;q12), creating a fusion product of EWS gene transcribed from chromosome 22 fused to WT1 gene from chromosome 11. Immunohistochemical expression, however, may lead to misdiagnosis dependent upon which protein terminus the antibody is directed against [52,53]. To that end, RT-PCR to detect DSRCT has been an essential tool in the distinction between the two entities and confirmation of diagnosis [54,55].

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from clinical trials are still preliminary [62]. Among these is the consideration of cediranib, a VEGFR inhibitor, which has demonstrated disease control in patients with metastatic, unresectable ASPS [63,64]. To that end, microarray analysis and quantitative real-time polymerase chain reaction may thusly become routine in profiling these tumors for therapeutic guidance. Recently, Kubota et al. utilized 2D-DIGE electrophoresis with subsequent Western blotting to pool and identify unique proteins from ASPS. Among these, the authors identified an oncogene product, suppressor of variegation, enhancer of zeste, and trithorax (ZET), as being overexpressed in ASPS and further demonstrated its inhibitory effects on phosphatase 2 A (PP2A), a tumor-suppressor protein. These findings are promising in treatment of tumors showing this overexpression, which may consequently be treated by PP2A activators such as FTY720 or forskolin; the authors of this study demonstrated antitumoral effects in vitro [65].

Synovial Sarcoma Perhaps the model of modern molecular diagnostics and confirmatory testing is the diagnosis of synovial sarcoma via FISH and RT-PCR analysis [66,67]. Classically, this sarcoma demonstrates the t(X; 18)(p11.2;q11.2) translocation, which results in the fusion of the SS18 (also known as SYT) and SSX genes. A number of product variants of fusion genes have been described, however the most common of these is the SS18/SSX1 product (occurring in approximately 65% of cases), followed by the SS18/SSX2 product (occurring in approximately 35% of cases) [68]. The SS18/SSX fusion product functions to inhibit Mcl1 which ultimately results in apoptosis suppression [69]. RT-PCR, although the most commonly used tool to detect the fusion genes, may not necessarily identify the rare SS18/SSX4 [55]. To that end, FISH is preferred if a negative SS18/SSX is identified via RT- PCR, but morphologically and immunophenotypically, the tumor is strongly suspicious for synovial sarcoma. Gene profiling studies identified the protein, transducer-like enhancer of split 1 (TLE-1), as a marker for synovial sarcoma, with high specificity and sensitivity for this diagnosis over other soft tissue sarcomas [70]. Chaung et al. recently restudied TLE1 expression and molecular profiles of synovial sarcomas, confirming an earlier finding of a positive correlation between TLE-1 staining and SS18-SSX translocation as detected by conventional PCR [71,72].

Alveolar Soft Part Sarcoma The classically described aberration associated with alveolar soft part sarcoma (ASPS) is der(17) t(X;17)(p11;q25), which involves a nonreciprocal translocation of the ASPSCR1 (ASPL) and TFE3 genes [56]. The ASPSCR1-TFE3 aberrant transcriptional factor upregulates MET transcription enabling cell survival. Interestingly, the ASPSCR1 gene may be joined in frame upstream of either the third or fourth exon of TFE3, yielding 2 fusion variants, ASPSCR1-TFE3 type 1 and type 2, respectively [56]. RT-PCR for this translocation is highly sensitive and specific for the diagnosis of ASPS, which is helpful when immunohistochemistry for TFE3 is inconclusive [57,58]. To this end, it has been recommended that a biomarker strategy be utilized for diagnosis of ASPS [59]. Recently FISH for rearrangements of ASPSCR1, TFE3 or a customized dual signals single-fusion FISH probe set for the ASPSCR1-TFE3 fusion gene has been developed and validated experimentally [60]. Classically, these tumors are resistant to conventional chemotherapy which, although often indolent in nature, has lead to a lack of definitive treatment options for advanced/metastatic disease [61]. Stacchiotti et al. have recently reviewed potential MET and angiogenic inhibitors which are showing promise in treating advanced disease, however the data Journal of Surgical Oncology

Clear Cell Sarcoma of Soft Tissue Clear cell sarcoma (also know as malignant melanoma of soft parts) is deep seated malignant neoplasm of soft tissue exhibiting immunophenotypic mimicry with malignant melanoma and characterized by a t(12;22)(q13;q12) rearrangement resulting in a EWSR1-ATF1 fusion gene product [73]. The pattern of S-100 and HMB-45 positivity in this tumor strikingly overlaps with malignant melanoma of cutaneous origin could lead to an erroneous diagnosis. To that end, the diagnosis is enabled by the demonstration of either t(12;22)(q13;q12) and/or EWSR1 by conventional cytogenetic analysis and FISH respectively for confirmation [74]. Interestingly, the EWSR1-CREB1 translocation (t(2;22)(q34;q12)), is also often present [73]. This fusion gene is found to be coexpressed with the EWSR1-ATF1 genes in several other entities, including angiomatoid fibrous histiocytoma, clear cell sarcoma-like tumor of the gastrointestinal tract, primary pulmonary myxoid sarcoma and hyalinizing clear cell carcinoma [75]. Of yet, how expression of these fusion genes affects the diverse biologic behaviors of these entities is still poorly understood.

Molecular Diagnostics in Sarcomas and GIST INI1 Loss Tumor Family INI1 (hSNF5/SMARCB1), located on chromosome 22q11.2, directly represses cyclin D1 and activates p16 and p21 cyclin-dependent kinases, resulting in cell-cycle arrest [76,77]. INI1 loss in malignant rhabdoid tumors had been well-documented in the literature [78,79], and in 2009, Hornick et al. documented a loss of INI1 in epithelioid sarcomas by immunohistochemistry [80]. Other sarcomas, including synovial sarcoma, extra-skeletal myxoid chondrosarcoma, GIST and chordoma have also been shown to demonstrate loss or rearrangement of INI1 as well, complicating the diagnosis [81–85]. Although the diagnosis of these tumors is generally made via morphology and immunohistochemistry, FISH probes have been developed with high sensitivity for INI1 deletion [86]. Interestingly, the identification of INI1 loss in sarcomas has identified multiple candidate chemotherapeutic agents. Flavopiridol and tamoxifen are presently being tested considering their effect on cyclin D1 [87]. Other cyclindependent kinase inhibitors are also being tested in patients INI1-loss tumors [88,89]. Thus, although data is early, the loss INI1 in epithelioid sarcomas and malignant rhabdoid tumors may suggest prognostic and therapeutic considerations.

VASCULAR TUMORS Association of angiosarcoma with Maffucci syndrome, retinoblastoma, xeroderma pigmentosum and Klippel–Trénaunay– Weber syndrome, among others, suggest a possible genetic linkage to angiosarcoma development [reviewed in [90]]. Mutations in the angiopoietin-TIE pathway have been identified, leading to the development of angiopoeitin inhibitors, such as trebananib, or AMG 386 [91]. Currently, trebananib is being evaluated for patients with advanced non-resectable angiosarcoma [92]. At present, the use of FISH in demonstrating MYC amplification has been useful in distinguishing post radiation angiosarcomas from atypical vascular lesions which can be otherwise extremely difficult on histopathologic bases alone [93–95]. The understanding of the cytogenetics of epithelioid hemangioendothelioma (EHE) has developed over the past decade significantly. In 2001, Mendlick et al. described a t(1;3)(p36.3;q25) translocation within a subset of EHE [96]. Ten years later, this chromosomal abnormality was further clarified to involve the WW domain-containing transcription regulator 1 (WWTR1) and calmodulin-binding transcription activator 1 (CAMTA1) genes—the former a chimeric transcription factor expressed in endothelial cells and the latter a protein normally expressed in brain tissue only [97,98]. In challenging cases, FISH and RT-PCR exploiting this translocation may be utilized for diagnosis [97,99]. Interestingly, a subset of EHE in young patients was also recently found to demonstrate a YAP1-TFE3 fusion gene which was not morphologically distinct from other EHE [100]. At present, the NCCN guidelines do not utilize these fusion proteins in diagnosis, prognosis or treatment of EHE.

LEIOMYOSARCOMA These are sarcomas of smooth muscle origin with complex karyotypes [101]. Comparative genomic hybridization analysis of leiomyosarcomas (LMS) has detected losses in numerous loci, including the PTEN suppressor gene (10q) in 20 of 29 cases examined and Rb suppressor gene (13q) in 17 of 29 cases [101]. Oncogenic gain was identified in 16 of 29 cases on locus 17p [102,103], which interestingly has been implicated in malignant triton tumors [104]. To this end, the role of molecular pathology as a diagnostic tool has been limited [105]. In 2010, a gene expression profiling study was undertaken to identify and further characterize LMS [106]. Fifty-one samples of LMS samples Journal of Surgical Oncology

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were analyzed using 44 K spotted cDNA microarrays, unsupervised hierarchical clustering and principal component analysis, revealing three reproducible molecular subtypes based on analysis of 3038 genes: one with enriched muscle contraction and the actin cytoskeleton genes (e.g., CALD1, DMD, ACTG2, CASQ2); one with enriched genes related to protein metabolism, regulation of cell proliferation, and organ development (e.g., TCF4, FBN1, SNAI2); and one predominantly expressing genes related to metal binding, extracellular proteins, proteins involved in the wound response, and ribosomal proteins involved in protein synthesis (e.g., MRPS12, EEF1D, GPX1). The same study used array CGH of 20 LMS samples to identify clusters of genes commonly lost or gained to each subtype. Losses of the 1p36.32 region was common amongst one subtype, but not found in every case. Amplification of the MET oncogene (7q31) was also identified in the same group. This finding is of particular interest in therapeutic development, given the recent development and ongoing trials of MET/ HGF pathway inhibitors [107]. The differing molecular signatures inherent to these subtypes are hypothesized to underlie the heterogeneity in drug responses observed in LMS patients [106]. Recently, Edris et al. correlated these expression profile subtypes with a reference collection of known gene expression profiles from human cell lines, identifying regions with strong yield as therapeutic targets [108]. Among the 11 candidate molecules identified—two, Cantharidin and MG-132—targeted all three subtypes of LMS. One of many mechanisms underlying the genomic instability in LMS is PTEN dysfunction (reviewed in [109]). Point mutations of the PTEN locus on chromosome 10q in LMS have been detected using PCR. PTEN has been widely studied in multiple tumors, and shown to be the balance to the PI3K cellular proliferation cascade which works in conjunction with the mTOR pathway. To that end, the identification of the PTEN mutation in these tumors may signal the potential for mTOR inhibitors as a therapeutic option in LMS patients [110].

GASTROINTESTINAL STROMAL TUMORS A number of GIST molecular-based subgroups have been described. Together KIT and PDGFRA mutations are identified in approximately 85% of GIST. The most common mutation is in the KIT gene on chromosome 4q12–13 which perpetually activates a coded KIT receptor tyrosine kinase. Sequencing of the KIT gene in these entities has identified mutations in exons 8, 9, 11, 13 and 17, with the first two of these being found predominantly in small bowel GIST and the latter three being found in all sites [111]. The second most common mutation is in the platelet-derived growth factor receptor A (PDGFRA), found in approximately 10% of GIST [112]. Like KIT, PDGFRA is a tyrosine kinase which central to GIST pathogenesis and the target of imatinib therapy. Unlike KIT, however, PDGFRA-mutant GIST has been found to show preferential location to the stomach [113]. Interestingly, GIST mutations secondary to chemotherapy have been described as developing in the KIT and PDGFRA tumors [114,115]. Examination of exons 13 and 14 of the KIT gene in imatinib-resistant tumors revealed mutations preventing imatinib binding. In these patients, sunitinib has been shown to have greater benefit [116]. Identification of these mutations is central to predicting response to tyrosine kinase inhibitors [113]. Per the current NCCN guidelines, “approximately 50% of patients will benefit from imatinib if their tumors harbor a KIT exon 9 mutation” [117]. Interestingly, while PDGFRA gene mutations are generally associated with a response to imatinib therapy, the D842V resistance mutation in exon 18 has predicted a poor response to tyrosine kinase inhibitor treatment [118]. Other mutations have also been discovered of late and are prognostically significant. Succinate dehydrogenase (SDH) mutations have been identified in wild-type GISTs [119]. SDH-deficient GISTs demonstrate an energy metabolism defect as an oncogenic mechanism; in cases where SDH mutations are not identified, an epigenetic loss of

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SDH is thought to underlie or significantly contribute to pathogenesis. The loss of the SDHB subunit is now widely regarded as diagnostic for SDH-deficient GIST, while the loss of other subunits supports the diagnosis in conjunction with immunohistochemistry and morphology [120]. Using array CGH, Lee et al. have recently documented that an increase in glutathione S-transferase theta 1 (GSST1) copy number in GISTs were correlated with poor response to imatinib therapy, comparable with similar findings in chronic myelogenous leukemia documented by Koh et al. [121,122]. In both SDH and GSTT1 mutations, anti-VEGFR agents such as sunitinib and regorafenib have shown promise [123].

Emerging Role of MicroRNA’s in Sarcomas and GIST MicroRNA (miRNA) are single stranded non-coding RNAs of 18–25 nucleotides involved in multiple biological processes making their detection desirable for causal, diagnostic, prognostic and therapeutic potentials. In a study of 27 sarcomas, Subramanian et al. determined identified distinct miRNA profiles among different histological subtypes [124]. In this study, GIST demonstrated a 9.4 fold change of overexpression of miR-140* compared to other tumors. The miR-140 family has been implicated in prognostic factors associated with chordoma, carcinoma of the breast and non-small cell lung carcinoma [125–127]. Similar work by Renner et al. in 2012 demonstrated upregulation of the miR-200 family in synovial sarcomas, as well as the tumor-associated miR-9 and miR-9* in myxoid liposarcomas compared to adipose tissue [128]. The same study also identified coexpression of 63 miRNAs within the 14q32.2 region, “separating primary sarcoma samples and sarcoma cell lines into two molecular subgroups” [128]. A possible causal role involving loss of decoy function of miR-29 on HuR in the regulation of A20 resulting in NF-kB constitutive activation has been proposed to be an important circuitry in sarcomagenesis [129]. Further evidence of the roles of miRNA has been discovered by studying miRNA in vitro. For example, miR-494 has been shown to directly inhibit KIT in GIST when overexpressed, suggesting its role as a potential therapeutic target [130].

NEXT-GENERATION SEQUENCING Next-generation sequencing (NGS), also referred to as highthroughput or massively-parallel sequencing, is a term coined in reference to well-established ’first-generation’ technologies such as Sanger and pyrosequencing. It represents a group of related technologies that have in common the ability to sequence millions of template molecules comprising millions to billions of bases. As with conventional sequencing technologies, some of these rely on light emission for detection of base incorporation during the sequencing reaction [131]. Novel technologies have also found their place in the NGS boom, however, such as the use of pH change [132], or the change in electrical/ion current through a pore or channel [133], among others. Sequencing of genomic DNA in the clinical realm takes three forms, primarily. The most grandiose of these is whole-genome sequencing (WGS), which attempts to capture data at each position of an individual’s (or tumor) genome. A more limited (approximately 2% of the whole genome) and less costly approach to large-scale genomic analysis is whole-exome sequencing (WES). Finally, the least costly and most commonly used approach in oncology at present entails targeted sequencing of one to several hundreds of clinicallyinformative genes or highly-mutated regions therein (i.e., “hotspots”). In addition to economy of time and resources, this approach can afford enhanced sensitivity relative to WES and WGS. Each base can be sequenced many more times over with redundant or overlapping fragments, leading to enhanced “depth of coverage,” and affording more opportunities to detect low-abundance variants. This is Journal of Surgical Oncology

Fig. 5. This figure depicts a simplified schema for next-generation sequencing on several commonly used platforms. A: Genomic DNA is extracted from tumor tissue. For whole genome sequencing (WGS, see right side), the DNA must be sheared into fragments and purified for the optimal fragment size range. For whole exome sequencing (WES) and some more limited multigene panels, a further step may be added to select the DNA fragments corresponding to regions of interest by hybridizing to oligonucleotide baits (not shown). Alternately, amplification based methods (left side) may be used to generate templates from the regions of interest (arrows represent PCR primers). B: For WGS, WES, and multigene panels, DNA fragments are ligated to platform-specific adapter oligonucleotides (orange bars), which contain sequences required for recognition and further processing by the various sequencing technologies. C: For the IonTorrentTM platform (Life Technologies, Grand Island, NY; see left side), sequencing template fragments are captured on beads (IonSphere ParticlesTM, or ISPs) via interactions with oligonucleotides complementary to the adapter sequences (purple bars). Emulsion PCR replicates the sequence across the surface of the bead, effectively amplifying the signal given off by base incorporations during sequencing. For IlluminaTM platforms (Illumina, San Diego, CA; see right side), each template is bound to the surface of a fluidic chamber by its adapter, subsequently undergoing local amplification across neighboring oligonucleotide anchors (purple bars), in a process termed “bridging amplification.” D: For IonTorrent sequencing, the ISPs are distributed into individual wells (not shown). Nucleotides are flooded across the openings of the wells, one base at a time, in a fashion analogous to pyrosequencing. If a base is incorporated, the hydrogen ions released during phosphate bond formation are detected by a pH-sensitive layer at the bottom of the well and transduced into electronic signals. In the Illumina system, all four bases are introduced during each cycle. After incorporation, the unique fluorophores that identify each base are captured by a high-resolution camera for each clonal template cluster. E: The raw data from each successive cycle is interpreted by the instrument, and base calls are assembled into sequences that represent each of the original library fragments. These short sequences (horizontal blue bars) are mapped to the human reference genome by specialized bioinformatic software. Where mismatches with the reference sequence occur (in the figure, the green boxes representing adenine are discordant with the red-labeled, wild-type thymidine of the above reference), a variant may be observed. Further information is necessary to distinguish bone fide mutations from common polymorphisms. F: “Actionable” mutations are principally those that may predict response (or resistance) to a particular therapy. Other mutations may also yield diagnostically or prognostically useful information, however.

Journal of Surgical Oncology EWS1-FLI1, EWS1-ERG, FUS-ERG EWSR1-WT1 PAX3-FOXO1, PAX7-FOXO1, PAX3-AFX SS18-SSX1, SS18-SSX2, SS18-SSX4 CIC-DUX4 INI1 INI1 MYC WWTR1, CAMTA1, YAP1-TFE3 loss of PTEN and Rb ETV6-NTRK3 COLA1-PDGFb FUS-CREB3L2, FUS-CREB3L1 EWS-TEC, TAF2N-TEC, TCF12-TEC ASPL-TFE3 EWSR1-ATF1; EWSR1-CREB1 TGFBR3-MGEA5, VGLL3 amplification EWSR1-CREB1, EWSR1-ATF1 ALK-TPM3, ALK-CLTC, ALK- TPM, ALK-CARS, ALK-RANBP2

MYC amplification on chromosome 8 t(1;3)(p36.3;q25), t(11;X)(q13;p11) 10q loss; 13q loss t(12;15)(p13;q25) t(17;22)(q22;q13) t(7;16)(q34;p11), t(7;16)(q33;p11) t(9;22)(q22;q12) der(17) t(X;17)(p11;q25) t(12;22)(q13;q12), t(2;22)(q34;q12) t(1;10)(p22:q24), marker/ring chromosome 3 t(2;22)(q33;q12), t(12;22)(q13;q12) t(1;2)(q25;p23), t(2;2)(p23;q13), t(2;11)(p23;p15), t(2;17)(p23;q23), and t(2;19)(p23;p13.1)

LHFP C11orf95-MKL2

HMGA2-LPP fusion; LHFP mutations

FUS-DDIT3, EWSR1-DDIT3 MDM2 amplification

Genes

t(11;22)(q24;q12), t(21;22)(q22q12), t(16;21)(p11;q22) t(11;22)(p13;q12) t(2;13)(q35;q14), t(1;13)(p36;q14), t(X;2)(q13;q35) t(X;18)(p11;q11) t(4;19)(q35;q13.1) 22q11.2 loss 22q11.2 loss

t(12;16)(q13;p11), t(12;22)(q13;q12) Supernumerary ring and giant marker chromosomes, 12q14–15 amplification 12q13–15, 6p21–23 and 13q mutations; t(3;12)(q27–28;q13–15) monosomy 13 or 13q partial deletion t(11;16)(q13;p13)

Cytogenetic Abnormalities

* Note: These are benign entities that have been included for comparison. **These entities are not discussed in the review.

*Spindle Cell lipoma *Chondroid lipoma Small blue round cell tumors Ewing’s sarcoma Desmoplastic small round cell tumor Alveolar rhabdomyosarcoma Synovial Sarcoma Poorly-differentiated “Ewing-like” sarcomas Epithelioid sarcoma, proximal-type Malignant rhabdoid tumors Vascular Tumors Angiosarcoma (post-radiation) Epithelioid hemangioendothelioma Leiomyosarcoma Fibroblastic sarcoma Infantile fibrosarcoma Dermatofibrosarcoma protuberans (DFSP) Low grade fibromyxoid sarcoma (Evan’s Tumor) **Extraskeletal Myxoid Chondrosarcoma Tumors of uncertain histiogenesis Alveolar soft part sarcoma Clear cell sarcoma of soft tissue **Myxoinflammatory fibroblastic sarcoma **Angiomatoid Fibrous Histiocytoma **Inflammatory Myofibroblastic Tumor

Adipocytic tumors Myxoid cell liposarcoma Atypical lipomatous tumor/well-differentiated liposarcoma, dedifferentiated liposarcoma *Lipoma

Tumor

TABLE 1. List of Common Aberrant Cytogenetic Abnormalities in Human Sarcomas

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particularly necessary when assaying for subclonal mutations in a heterogeneous neoplasm. The reader is referred to Figure 5 for a brief depiction of several sequencing methods that are currently in routine clinical use for oncology, as well as several reviews on the topic [134–136]. The initial clinical applications of NGS in oncology have mostly concerned the detection of somatic point mutations, small insertions and small deletions, in a manner analogous to traditional sequencing methods. Increasingly, however, specialized techniques are being employed for assessment of larger-scale copy number changes and structural rearrangements [137–139]. The latter are of particular interest for sarcoma oncology, and a growing number of papers report success with these technologies in the sensitive and specific detection of tumordefining translocations [140,141]. In contrast to conventional assays, for example, RT-PCR and FISH, NGS methods may also be designed to detect novel rearrangements [139,142]. It goes without saying that as more of these relatively rare neoplasms are sequenced, new mutations are expected to surface with relevance to diagnosis, prognosis, and perhaps most importantly, therapy prediction (Table 1). Indeed, genomic analysis is already opening the doors to personalized therapy for sarcomas. Targeted therapy in routine clinical use has largely been limited to gastrointestinal stromal tumors, treatable by tyrosine kinase inhibitors such as imatinib when activating mutations of C-KIT or PDGFRA are present. Since these mutations can be found in a number of different exons, however, conventional sequencing is time consuming and costly. More expedient screening approaches, for example, exon-wide scanning by highresolution melting curve analysis, suffer from their own set of technical limitations. NGS methodologies offer the benefits of sensitive and specific mutational screening for GISTs in a single assay, detecting not only activating mutations but also those associated with resistance [1]. Additionally, NGS has brought the promise of personalized medicine to many other types of sarcoma. In one report, for example, 60% of tumors analyzed (15 of 25, including pleomorphic high-grade spindle cell sarcoma, leiomyosarcoma, pleomorphic liposarcoma, intimal sarcoma, clear cell sarcoma, and monophasic synovial sarcoma) disclosed targetable mutations for which clinical trials were available [1]. In another report, PIK3CA was recurrently mutated in myxoid/round cell liposarcomas, suggesting the potential for a novel therapeutic strategy [143]. One particular challenge is presented by sarcomas that defy clear histopathologic classification, but genomewide analysis can nonetheless disclose targe mutations in some patients, providing the hope of therapeutic response even when the diagnosis is not firm [144]. In conclusion, we present the diverse array of how molecular techniques have enhanced routine methods of evaluation and emerged as powerful tools in the diagnostics, oncogenesis, prognostication and therapeutics of soft tissue sarcomas and GIST. As data from the different molecular platforms continue to emerge, we believe these will become increasingly definitional and critical determinants of the different tumor subsets.

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