Molecular Pathology of Sarcoma

Molecular Pathology of S a rc o m a Robin D. LeGallo, MD KEYWORDS  Sarcoma  Soft tissue tumors  Molecular oncology  Diagnosis

ABSTRACT

T

he rapid growth of tissue-based molecular pathology has changed the practice of the surgical pathologist signing out soft tissue tumors. This information is presented in a practical and succinct manner focusing on clinically validated findings that have diagnostic or therapeutic relevance. The approach is morphologically based and focuses on differential diagnoses and clinical scenarios. Molecular techniques can be an invaluable ancillary tool.

LIPOMATOUS TUMORS

Criteria for sending lipomatous tumors for cytogenetic/molecular analysis >15 cm in size Deep location Retroperitoneal tumor Recurrent tumor Myxoid cut surface Unusual location Young age Small biopsies

Histology Lipomas are composed of mature adipocytes with only slight variation in size. Myxoid change may be prominent and the term vascular myxolipoma has been used but likely does not represent a distinct entity.

Cytogenetic and Molecular Pathology Cytogenetic aberrations can be identified in most lipomas. The karyotypes tend to be simple, near diploid and heterogeneous, although recurrent abnormalities have been described. The most common abnormality is a rearrangement of 12q13–15, locus of HMAG2, seen in up to twothirds of the abnormal karyotypes.1 This region may be involved in a translocation, inversion, or deletion with t(3;12)(q27;q15) being most frequent, although more than 80 partner chromosomal bands have been reported.1,2 HMAG2 rearrangements are not unique to lipomas and have been reported in other benign and malignant neoplasms, most importantly well-differentiated liposarcoma, but in this tumor it is also characterized by amplification of this region.3,4 Other common recurrent abnormalities include rearrangement of 6p21–23 involving HMGA1, which appear mutually exclusive to the 12q13–15 rearrangements.5 Deletions at 13q, frequently 13q12–22, are seen in about one-fifth of cases.5 Most of these rearrangements can be detected on routine karyotype, although they infrequently may be cryptic, requiring detection by fluorescence in situ hybridization (FISH).

LIPOMA Lipomas are the most common soft tissue tumor, generally occurring in adults in superficial locations but can be deep seated (Table 1).

LIPOBLASTOMA Clinical Lipoblastoma is a tumor of childhood with 90% of tumors diagnosed in the first decade of life.6

The author has nothing to disclose. Department of Pathology, University of Virginia Health System, 1215 Lee Street, HEP 3064, Charlottesville, VA 22908, USA E-mail address: [email protected] Surgical Pathology 5 (2012) 961–984 http://dx.doi.org/10.1016/j.path.2012.08.009 1875-9181/12/$ – see front matter Ó 2012 Elsevier Inc. All rights reserved.

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Table 1 Lipomatous tumors Lipoblastoma Lipoma

Spindle cell/Pleomorphic lipoma Chondroid lipoma Hibernoma Well-differentiated liposarcoma/ Atypical lipomatous tumor Myxoid liposarcoma Dedifferentiated liposarcoma Pleomorphic liposarcoma

8q11–13 rearrangement 12q13–15 rearrangement 6p21 rearrangement 13q deletion 13q deletion or rearrangement 16q deletion or rearrangement t(11;16)(q13;p13) 11q13 rearrangement with deletion 12q13–15 amplification t(12;16)(q13;p11) t(12;22)(q13;q12) 12q13–15 amplification complex karyotype Complex karyotype

Extremities are the most common site but it has been described in numerous locations.

PLAG1 HMGA2 HMGA1 Unknown Unknown C11orf95-MKL2 MEN, AIP MDM2, CDK4, CPM, SAS HMGA2 FUS-DDIT3 EWS-DDIT3 MDM2, CDK4, HMGA2 Unknown

Cytogenetic and Molecular Pathology

Lipoblastoma is characterized by a lobular architecture with rounded lobules of lipoblasts and mature fat set in a myxoid stroma notably separated by fibrous bands.

The karyotype of spindle cell/pleomorphic lipoma tends to be more complex than other lipoma variants. The most common aberrations are 16q and 13q, either as deletions of rearrangements.8 Spindle cell lipoma tends to be polysomic for MDM2, showing an increase in MDM2 and CEP12 signals by FISH.9

Cytogenetic and Molecular Pathology

CHONDROID LIPOMA

Histology

Lipoblastoma is characterized by a simple karyotype with abnormalities of 8q11–13 involving PLAG1.7 Most of these are translocations but polysomy has also been reported in cases with and without other 8q abnormalities.1 These aberrations can be seen on conventional karyotype or detected with FISH studies using a PLAG1 probe.

SPINDLE CELL/PLEOMORPHIC LIPOMA Clinical Spindle cell/pleomorphic lipoma has a strong predilection for the posterior neck and upper back of men in their third to fourth decade.

Histology Spindle cell lipoma typically shows bland spindle cells and mature adipose tissue, and has characteristic ropey collagen fibers. Large, hyperchromatic multinucleated and floret cells, as well as lipoblasts, are seen in the pleomorphic variant. CD34 is a useful immunohistochemical marker, as it stains the spindle cell component.

Clinical Chondroid lipoma is a rare lipoma variant typically presenting in the proximal extremities of adult women.

Histology Chondroid lipoma shows vacuolated, chondroidlike lipoblasts and mature fat embedded in a myxoid and hyalinized matrix.

Cytogenetic and Molecular Pathology The cases reported in the literature show a recurrent translocation t(11;16)(q13;p13), which was recently shown to result in the fusion gene C11orf95-MKL2.10 It is noted that rearrangements of 11q13 are also seen in hibernomas but appear to have differing underlying genetic alterations.11

HIBERNOMA Clinical Hibernomas most commonly occur in the thigh of young adults, although numerous locations have been reported; it is rare in children.

Molecular Pathology of Sarcoma Histology Hibernoma is typically identified by its characteristic multivacuolated brown fat.12 Myxoid degeneration may cause confusion with other lipomatous lesions.

Cytogenetic and Molecular Pathology The karyotypes tend to be more complex than conventional lipomas, but are characterized by rearrangements of 11q13, although no consistent partners have been identified.1,11 Instead, the break at 11q13 seems to result in the deletion of tumor suppressor genes MEN1 and AIP, which appear to be involved in the pathogenesis of this lesion.13

thigh; it is debatable whether it occurs in the retroperitoneum.17 It is rare in children.

Histology Myxoid liposarcoma is composed of lipoblasts in various stages of maturation in a prominent myxoid matrix; myxoid pools that may be present are referred to as the “pulmonary edema” pattern. The hallmark of this tumor is the arborizing plexiform capillary network known as the “chicken wire” vasculature. Increased cellularity with rounding up of the nuclei showing which may touch or overlap indicates progression to the round cell variant, which portends a worse prognosis.

Cytogenetics/Molecular Pathology

WELL-DIFFERENTIATED LIPOSARCOMA/ ATYPICAL LIPOMATOUS TUMOR Clinical Well-differentiated liposarcoma/atypical lipomatous tumor (WDLS/ALT) refers to deep and superficial locations respectively of the low-grade adipocytic sarcomas occurring in the retroperitoneum, inguinal area, and extremities of adults. Recurrence is generally the rule in the deep locations because of the inability of a complete surgical resection. WDLS has no metastatic potential but can dedifferentiate into a higher-grade sarcoma.

Histology Most WDLS/ALTS are composed of mature adipose tissue with variation in size of the adipocytes with nuclear atypia, which may be most pronounced within the stromal cells of the fibrous septae. WDLS/ALT can be classified as lipomalike, sclerosing, inflammatory, or spindle cell. Myxoid stroma can be present.

Cytogenetic and Molecular Pathology At the chromosomal level, WDLS/ALT is characterized by giant ring or marker chromosomes and less commonly by double minutes, which have shown by FISH studies to be composed of amplified sequences of MDM2, CDK4, HMAG2, SAS, and CPM.4,14,15 MDM2 and CDK4 immunohistochemistry is available but has neither the sensitivity nor specificity as FISH, especially on small biopsies.16

MYXOID/ROUND CELL LIPOSARCOMA Clinical Myxoid liposarcoma represents a third to a half of liposarcomas and primarily occurs in young to middle-aged adults in the deep tissues of the

Myxoid liposarcoma has many recurrent cytogenetic aberrations but notably is characterized by t(12;16)(q13;p11) in up to 95% if cases, which results in the fusion gene FUS-DDIT3 (formerly CHOP).18 The remaining cases may show the variant t(12;22)(q13;q12); EWSR1-DDIT3, which includes myxoid liposarcoma in the group of tumors with EWSR1 rearrangements (Table 2).19 The translocation can be detected by conventional karyotype, reverse transcriptase–polymerase chain reaction (RT-PCR), or FISH analysis.20 Other recurrent cytogenetic abnormalities include –6q, 18, and t(1;16).18

PLEOMORPHIC LIPOSARCOMA Clinical Pleomorphic liposarcoma is a tumor of older adults and most commonly occurs in the extremities

Table 2 EWSR1 rearranged soft tissue tumors Ewing sarcoma

Desmoplastic small round cell tumor Clear cell sarcoma Extraskeletal myxoid chondrosarcoma Myxoid liposarcoma Angiomatoid fibrous histiocytoma Myoepithelial tumors of soft parts

FLI1 ERG ETV1 ETV4 FEV WT1

Common Rare Rare Rare Rare Common

ATF1 NR4A3 (TEC)

Common Common

DDIT3 (CHOP) CREB1 ATF1 POU5F1 PBX1 ZNF44

Rare Common Rare Common Common Rare

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component, although it can be intermingled. The histology is typically that of a high-grade pleomorphic sarcoma, by definition not lipogenic, but can show other lines of differentiation.

Histology

Cytogenetic/Molecular Pathology

Pleomorphic liposarcoma has a background of high-grade spindle or pleomorphic sarcoma with identifiable lipoblasts with scalloped hyperchromatic nuclei. Epithelioid and myxoid variants have been described.21

The karyotypes are more complex than the WDLS counterpart but the ring or marker chromosome containing amplification of 12q13–15 is retained and a useful tool in confirming this diagnosis.9,18 In addition, amplification of 1p32 and 6q23 has been identified by comparative genomic hybridization, a finding not present in WDLS/ALT.23

Cytogenetic/Molecular Pathology Pleomorphic liposarcomas tend to have complex karyotypes with numerous structural and numerical abnormalities, none of which are specific to this tumor. Ring and marker chromosomes may be present, but amplification of 12q14-1 does not consistently occur.18 A single study found about one-third of pleomorphic liposarcomas to have MDM2 amplification by Southern blot or loss of heterozygosity of p53, which appear to be mutually exclusive.22

DEDIFFERENTIATED LIPOSARCOMA Clinical Dedifferentiated liposarcoma refers to a nonlipomatous sarcoma component arising in a low-grade liposarcoma, usually WDLS of the retroperitoneum. The vast majority are de novo and present with the primary tumor, although dedifferentiation can occur in recurrences.

Histology The dedifferentiated component usually shows an abrupt transition from the well-differentiated

Practical Applications Lipoma versus WDLS/ALT This differential most commonly occurs with relatively large and deep tumors of the extremities or inguinal region. Conventional lipomas are rare in the retroperitoneum but probably do exist.24 The 12q13–15 amplification characteristic of WDLS/ALT can be seen on conventional karyotype and confirmed by FISH or assessed on formalin-fixed paraffin-embedded tissue, most commonly with the MDM2 probe (Fig. 1). The MDM2 probe is coupled with the centromere 12 probe (CEP12) and MDM2:CEP12 signal ratio is scored, which differentiates amplification from chromosomal polysomy; a score of 2 or greater is generally considered amplified.9 Conventional lipomas also have recurrent abnormalities, which can be helpful but in most institutions this would require fresh tissue to be sent to cytogenetics, as the abnormalities are seen in only a subset of cases and FISH is not as routinely used.

Fig. 1. (A) Atypical lipomatous tumor shows variation in adipocyte size and rare enlarged hyperchromatic cells. (B) Vysis MDM2 and CEP12 FISH probes shows homogeneous amplified signals for MDM2 with 2 CEP12 signals.

Molecular Pathology of Sarcoma Lipomatous myxoid tumors Many lipomatous tumors characteristically have a myxoid matrix or can undergo prominent myxoid change. The diagnosis can usually be made based on histologic features combined with the clinical information, such as size, location, and age of the patient. Diagnostic difficulties can arise when the histology or clinical scenario is atypical, however as there can be overlap in both morphologic features and presentation of these lesions (Fig. 2). Small biopsies can also be problematic. In addition, other nonlipomatous myxoid tumors, such as a cellular intramuscular myxoma, lowgrade myxofibrosarcoma, and extraskeletal myxoid chondrosarcoma, may enter the differential. Cytogenetic and molecular techniques can often solve diagnostically challenging cases. The t(12;16) and less common t(12;22) of myxoid liposarcoma can be detected on conventional karyotype or FISH analysis using break-apart probes for DDIT3, FUS, and EWSR1. Myxoid liposarcoma is not amplified for MDM2, although it rarely expresses MDM2 by immunohistochemistry.25 This identification of myxoid liposarcoma from other malignant sarcomas has clinical relevance. Unlike well-differentiated liposarcoma, myxoid liposarcoma has the ability to metastasize, is relatively chemosensitive and radiosensitive, and may be subject to molecular-based targeted therapy.26 Dedifferentiated liposarcoma from other pleomorphic sarcomas Distinguishing dedifferentiated liposarcoma from other high-grade sarcomas can be challenging if the well-differentiated liposarcoma is not identified. Dedifferentiated liposarcomas maintain the MDM2 amplification seen in its well-differentiated counterpart, making MDM2 FISH a valuable tool (Fig. 3). Although MDM2 FISH is a highly sensitive assay, it must be noted that high-grade pleomorphic sarcomas frequently show amplification of MDM2 also, reducing the specificity. However, the MDM2:CEP12 ratio is often greater than 8:1 in dedifferentiated liposarcoma, which does not seem to be a feature of other high-grade sarcomas.9

Diagnostic challenges solved by molecular pathology: lipomatous tumors  Lipoma from WDLS/ALT  Myxoid liposarcoma from other myxoid lesions  Dedifferentiated liposarcoma from other high-grade sarcomas

SPINDLE CELL TUMORS DERMATOFIBROSARCOMA PROTUBERANS Clinical Dermatofibrosarcoma protuberans (DFSP) is a tumor of the dermis and subcutaneous tissues that most commonly presents on the extremity or trunk of middle-age adults (Table 3). It can also occur in children, where it tends to have a distinct histology and is termed giant cell fibroblastoma.

Histology DFSP is the prototype of storiform growth pattern and is also characterized by its infiltrative borders into the adjacent subcutaneous tissues. Progression to fibrosarcoma is noted by a cellular herringbone pattern, pleomorphism, and brisk mitotic activity. Giant cell fibroblastoma tends to be more myxoid and has clefted spaces lined by hyperchromatic or multinucleated giant cells. DFSP shows strong CD34 immunoreactivity that is often lost in fibrosarcomatous transformation, which may show staining with p53.

Cytogenetics and Molecular Pathology DFSP is associated with a translocation t(17;22) (q22;q13); COL1A1-PDGF-B. These can be seen on karyotype as unbalanced translocations or more commonly as a supernumerary ring chromosome containing amplified sequences of 17q22 and 22q10–13.27 The translocation can be detected by RT-PCR, but all possible introns of COLA1 have been implicated, resulting in the need for multiplexing primers. FISH using dual-color fusion may have a higher sensitivity.28 The detection of this translocation may be clinically relevant, as DFSP may be sensitive to imatinib mesylate.

DESMOID TUMOR Clinical Desmoid tumors are typically seen in adolescents and young adults, presenting as deep-seated masses favoring the limb girdle, thigh, chest wall, abdominal wall (especially in pregnant and postpartum women) and mesentery, where they are the most common location in patients with Gardner syndrome. The biologic behavior is unpredictable ranging from regression to highly aggressive local disease; they do not metastasize.

Histology Desmoid tumors are composed of uniform bland spindle-shaped fibroblasts with abundant collagen growing in fascicles with characteristic infiltrative borders.

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Fig. 2. Differential diagnosis of fatty myxoid lesions. (A) Lipoblastoma with (B) der and ring chromosome 8. (C) Confirmed by FISH consistent with PLAG1 abnormality. (D) Conventional lipoma with myxoid change, (E) showing t(3;12) on karyotype and (F) lacking MDM2 amplification. (G) Hibernoma with myxoid features, (H) showing an 11q23 rearrangement on karyotype and (I) an intact FUS gene by FISH. (J) Myxoid liposarcoma, (K) confirmed by a t(12;16) on karyotype and (L) rearranged FUS gene by FISH. (M) Well-differentiated liposarcoma of the retroperitoneum with prominent myxoid stroma, (N) showing large marker ring chromosome on karyotype and (O) MDM2 amplification on FISH.

Molecular Pathology of Sarcoma

Fig. 3. (A) Dedifferentiated liposarcoma, (B) showing MDM2 amplification by FISH. (C) High-grade pleomorphic sarcoma, NOS, (D) showing polysomy for the MDM2 gene but no evidence of amplification (multiple copies of both MDM2 and CEP12).

Cytogenetics and Molecular Pathology Recurrent cytogenetic abnormalities include trisomy 8 and 20 and loss of 5q, although none of these are specific to desmoid tumors.29 The vast majority of sporadic desmoid tumors exhibit

activating mutations in exon 3 of CTNNB1, which encodes for beta catenin.30 This is most commonly assessed by immunohistochemisty; desmoids show nuclear localization as the mutation blocks the phosphorylation needed for degradation of

Table 3 Genetic alterations in selected spindle cell tumors Dermatofibrosarcoma protuberans Desmoid tumor Low-grade fibromyxoid sarcoma Low-grade myxofibrosarcoma Inflammatory myofibroblastic tumor Malignant peripheral nerve sheath tumor Nodular fasciitis Solitary fibrous tumor Smooth muscle tumors Synovial sarcoma

t(17;22)(q22;q13); COL1A1-PDGF-B Beta-catenin mutation t(7;16)(q33;p11); FUS-CREB3L2 t(11;16)(p11;p11); FUS-CREB3L1 Complex karyotype 2p23 rearrangements, ALK Complex karyotype t(17;22)(p13;q13.1); MYH9-USP6 9q22 aberrations complex, 1 p deletions t(X;18)(p11.2;q11.2); SYT-SSX1 or SYT-SSX2

Common Common Common Rare Occasional Common Common ? Common Common

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is typically seen as balanced on karyotype but occasionally as a supernumerary ring chromosome.39,40 It can be detected by RT-PCR or by FISH assays.41,42

LOW-GRADE MYXOFIBROSARCOMA INFLAMMATORY MYOFIBROBLASTIC TUMOR

Clinical

Clinical Inflammatory myofibroblastic tumor (IMT) is a tumor of children and young adults that can occur in any location but favors omentum, mesentery, soft tissues, and lungs, the latter being a more common site in older patients.

Low-grade myxofibrosarcoma is a tumor of older adults and elderly and tends to present as a slow-growing painless superficial lesion. Myxofibrosarcoma can be of any histologic grade and low-grade myxoid fibrosarcoma frequently recurs as a higher-grade tumor.43

Histology

Histology

IMT has a variable histologic appearance, ranging from a haphazard arrangement of myofibroblasts in an inflamed edematous and myxoid stroma to organized fascicle and even heavily collagenized scarlike tissue.

Low-grade myxofibrosarcoma is characterized by abundant myxoid matrix with scattered spindled or stellate hyperchromatic tumor cells and frequently pseudolipoblasts are present. Curvilinear vessels are thicker than what is seen in myxoid liposarcoma.

Cytogenetics and Molecular Pathology Balanced translocations involving the ALK locus at 2p23 are seen in one-third to one-half of IMTs of children and young adults. The fusion partner is variable and includes TPM3(1q21), TPM4(19p13), CLTC(17q11), CARS(11p15), ATIC (2q35), SEC31A(4q21), and RANBP2(2q13).33 The tumor cells may be immunoreactive for ALK or the rearrangement can be detected by FISH using an ALK break-apart probe.

Cytogenetics and Molecular Pathology Most low-grade myxofibrosarcomas have abnormal karyotypes that tend to be complex, but no defining features have been identified.44,45

MALIGNANT PERIPHERAL NERVE SHEATH TUMOR Clinical

Low-grade fibromyxoid sarcoma (LGFMS) typically presents in young adults, but the age range is broad. The proximal extremities and trunk are favored sites. The recognition of LGFMS is important, as it characteristically has late recurrence, averaging 15 years after diagnosis.34

Malignant peripheral nerve sheath tumors (MPNSTs) are a relatively rare sarcoma and about half arise in the setting of neurofibromatosis type 1 (NF1). Sporadic MPNST usually arises in middleaged adults; there is a younger mean age, male predominance, and increased mortality in patients with NF1. Both tend to present as large deepseated tumors in association with a major nerve, often in the proximal extremities, buttocks, and paraspinal region.

Histology

Histology

The tumor shows a biphasic pattern of myxoid nodules alternating with short, whorling collagenized fascicles. When hyalinized nodules cuffed with tumor cells are present, the tumor is referred to as hyalinizing spindle cell tumor with giant rosettes. Immunohistochemistry has not typically been helpful in this tumor, but cytoplasmic immunoreactivity toward MUC4 has been reported to help differentiate LGFMS from histologic mimics.35

Conventional MPNST is a cellular spindled cell lesion with a fascicular growth pattern and alternating areas of hypercellularity and hypocellularity. Rare tumors may be purely epithelioid, show rhabdoid features, or have heterologous skeletal muscle differentiation (Triton tumor).

LOW-GRADE FIBROMYXOID SARCOMA Clinical

Cytogenetics and Molecular Pathology The defining translocation of LGFMS is t(7;16) (q33;p11); FUS-CREB3L2.36,37 A variant fusion FUSCREB3L1 has been described.38 The translocation

Cytogenetics and Molecular Pathology The karyotype of MPNST tends to be complex and often shows a near triploid complement of chromosomes with frequent gains and losses. Aberrations of 9 p, 17q, and 17 p, the loci of CDKN2A, NF1, and p53 are common but not uniformly seen.46–48 Hence, there are no recurrent

Molecular Pathology of Sarcoma diagnostic or prognostic useful alterations, although recent array data suggest gains of regions in 12 p and 12q, including the locus for CDNK1, may have portend a worse prognosis.49

NODULAR FASCIITIS Clinical Nodular fasciitis can present in any site and any age but most commonly is a tumor of young adults, presenting as a rapidly growing lesion in the deep dermal or subcutaneous tissues. It is usually smaller than 3 cm.

Histology The histologic features of nodular fasciitis are variable depending on its phase. Early lesions have the loose “tissue culture” appearance with a myxoid background containing scattered inflammatory cells and extravasated red blood cells. Older lesions tend to be more collagenized and organized looking. The immunophenotype is myofibroblastic.

Cytogenetics and Molecular Pathology Although nodular fasciitis has historically been considered a reactive pseudoneoplastic proliferation, recent data confirm clonal rearrangement in most lesions. Nodular fasciitis is characterized by t(17;22)(p13;q13.1); MYH9-USP6.50 Interestingly, USP6 rearrangements also characterize aneurysmal bone cysts.51 The translocation of nodular fasciitis is cryptic on karyotype owing to similarity in banding patterns but can be detected by FISH or RT-PCR.

aberrations at 9q22 have been reported in 3 cases of pleural-based SFT.53

SMOOTH MUSCLE TUMORS Clinical Leiomyoma and leiomyosarcoma are typically tumors of adults and can be cutaneous or in the deep soft tissues although the later is suggestive of malignancy.

Histology Smooth muscle tumors (SMTs) show short interlacing fascicles of spindled cells with blunt nuclei; malignancy is characterized by increased cellularity, pleomorphism, and mitotic activity. Leiomyosarcoma may have an epithelioid or myxoid appearance. Smooth muscle differentiation is evidenced by variable immunoreactivity toward smooth muscle actin, desmin, and h-caldesmon.

Cytogenetics and Molecular Pathology There are no genetic features that are consistently clinically useful in the diagnosis of SMTs. The karyotype of leiomyosarcoma tends to be more complex than its benign counterpart but deletions of 1 p are seen in both.54,55 Uterine leiomyomas may involve rearrangements of HMGA2 at 12q15, a gene frequently rearranged in conventional lipomas.56 Germline mutations or deletions of fumarate hydratase at 1q42 characterize the familial cancer syndrome hereditary leiomyomatosis and renal cell carcinoma.57

SYNOVIAL SARCOMA SOLITARY FIBROUS TUMOR Clinical Solitary fibrous tumor (SFT) was first described as a pleural-based tumor but has now been described in just about all locations of the body, including skin and deep soft tissues.

Histology The histology is variable but classic cases show a hemangiopericytoid vascular pattern with bland ovoid haphazardly arranged between the vascular channels. Some cases are heavily collagenized, whereas others show high cellularity, pleomorphism, mitotic activity, and necrosis, features portending aggressive biologic behavior.52 The tumor cells are immunoreactive toward CD34, CD99, and BCL2.

Cytogenetics and Molecular Pathology No recurrent diagnostic abnormalities have been reported in solitary fibrous tumors, although

Clinical Synovial sarcoma can occur across the ages but is usually found in adolescents and young adults. The classic location is in the vicinity of the large joints, although not in the joint space itself; however, it has been described in numerous locations. It usually presents as a palpable, often painful, deep-seated mass and a prolonged history is common.

Histology The histology is variable and biphasic synovial sarcoma can easily be identified by its spindle-cell and epithelial component, which may make slitlike spaces or overt glandular formation. Monophasic synovial sarcoma is typically a cellular lesion with a herringbone growth pattern and frequently has a hemangiopericytomatous vascular pattern. Cytokeratin and epithelial membrane antigen (EMA) react with the epithelial and focally within the spindle cell component, whereas the latter is

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Cytogenetics and Molecular Pathology The hallmark of synovial sarcoma is the t(X;18) (p11.2;q11.2), resulting in the fusion product SYT-SSX1 (65%), SYT-SSX2 (35%), and, rarely, SYT-SSX4. The translocation can typically be seen on conventional karyotype but rare cryptic variants have been reported. Break-apart FISH assays are highly sensitive and can detect all fusions but not distinguish between partners; RT-PCR is also highly sensitive and specific and can determine partners but may miss the rare variant translocations.60 Early literature suggested that the SYT-SSX1 fusion had a worse prognosis but larger studies did not confirm this finding.61

Practical Applications Low-grade spindle-cell neoplasms with myxoid features The differential diagnosis of this group of tumors depends on morphology as well as the clinical presentation of the tumors. Highly myxoid lowgrade spindle cell lesions may include myxoma, low-grade myxofibrosarcoma, and myxoid liposarcoma. Superficial fibrous and myxoid lesions include nodular fasciitis, DFSP, and LGFMS, whereas deeper tumors, often intra-abdominal and mesentery, include desmoid tumor, LGFMS, IMT, SFT, and gastrointestinal stromal tumors among others. The underlying genetic alterations implicated in these lesions can be exploited for diagnostic purposes using immunohistochemistry, cytogenetics, FISH, and molecular assays (Fig. 4). Notably, FUS FISH is useful in LGFMS and myxoid liposarcoma. The mutations ckit, beta-catenin, and ALK aberrations of gastrointestinal stromal tumor, desmoid tumor, and IMT, respectively, can be assessed using immunohistochemistry, FISH assays, or RT-PCR. High-grade spindle-cell lesions The differential diagnosis of a cellular spindle-cell lesion with a herringbone pattern inevitably raises the differential diagnosis of synovial sarcoma and MPNST; tumors that can have tremendous morphologic and immunohistochemical overlap.62 The detection of the t(X;18) characteristic of synovial sarcoma can be done by FISH using an SYT break-apart probe or by RT-PCR using fresh or formalin-fixed tissues (Fig. 5).

Diagnostic challenges solved by molecular pathology: spindle cell tumors  Nodular fasciitis versus smooth muscle tumor  Low-grade fibromyxoid sarcoma versus desmoid tumor  Inflammatory myofibroblastic sarcoma versus gastrointestinal stromal tumor  Dermatofibrosarcoma fibrous histiocytoma

protuberans

versus

 Myxoid liposarcoma versus myxoma  Monophasic synovial sarcoma versus malignant peripheral nerve sheath tumor

EPITHELIOID NEOPLASMS OF SOFT TISSUE ALVEOLAR SOFT PART SARCOMA Clinical Alveolar soft part sarcoma (ASPS) is a rare soft tissue malignancy of young to middle-age adults with a female predominance (Table 4). The extremities followed by mesentery and omentum are the most common locations; head and neck is a less common site.

Histology ASPS has an organoid growth pattern with tumor nests separated by vascular fibrous septae. The cells are polygonal with uniform nuclei, often with a central nucleolus and characteristic voluminous clear to eosinophilic granular cytoplasm with distinctive periodic acid-Schiff–positive crystals. Immunoreactivity for TFE3 is a helpful finding.63

Cytogenetics and Molecular Pathology ASPS is characterized by a nonbalanced der(17)(X;17)(p11;p15), resulting in an ASPSCR1TFE3 fusion.64 The karyotype otherwise tends to be simple with no recurrent diagnostic abnormalities. Type 1 and type 2 fusions have been described in equal proportions and vary in the breakpoint in TFE3. The translocation can be seen on karyotype as der17 but RT-PCR and FISH studies are commonly used.65,66 It is noted that that the translocation in Xp11.2 translocation carcinomas in almost identical but the t(X;17) (p11;q25) is balanced in this tumor.67

ANGIOMATOID FIBROUS HISTIOCYTOMA Clinical Angiomatoid fibrous histiocytoma (AFH) most commonly arises in the superficial soft tissues of

Molecular Pathology of Sarcoma

Fig. 4. (A) Cellular and myxoid intra-abdominal desmoid tumor, (B) showing strong nuclear beta catenin immunoreactivity and (C) 120 on karyotype. (D) DFSP with (E) ring chromosome on karyotype consistent with (17;22), (F) supported by FISH. (G) Gastrointestinal stromal tumor, (H) showing strong immunoreactivity for ckit. (I) Inflammatory myofibroblastic tumor, (J) showing an ALK rearrangement by FISH. (K) LGFMS, (L) showing a FUS rearrangement by FISH.

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Fig. 5. (A) Monophasic synovial sarcoma, (B) confirmed by an SYT rearrangement by break-apart FISH. (C) Malignant peripheral nerve sheath tumor, (D) with intact SYT gene, which shows 3 copies consistent with trisomy.

Table 4 Genetic alterations in soft tissue tumors with epithelioid features Alveolar soft part sarcoma Angiomatoid fibrous histiocytoma

Clear cell sarcoma Epithelioid angiosarcoma Epithelioid hemangioendothelioma Epithelioid sarcoma Extraskeletal myxoid chondrosarcoma

Myoepithelial tumors of soft tissue

der(17)(X;17)(p11;p15); ASPSCR1-TFE3 t(2;22)q(33;q12); EWSR1-CREB1 t(12;22)(q13;q12); EWSR1-ATF t(12;16)(q13;p11); FUS-ATF t(12;22)(q13;q12); EWSR1-ATF1 t(12;22)(q34;q12); EWSR1-CREB1 Complex karyotype t(1;3)(p36.3;q25); WWTR1-CAMTA1 22q11-12 abnormalities; INI1 (SMARCB1) t(9;22)(q22;q12); EWSR1-NR4A3 t(9;17)(q22;q11); TAF15-NR4A3 t(9;15)(q22;q21); TCF12-NR4A3 t(3;9)(q12;q22); TFG-NR4A3 t(6;22)(p21;q12); EWSR1-POU5F1 t(1;22)(q23;q12); EWSR1-PBX1 t(19;22)(q13;q12); EWSR1-ZNF44

Common Common Rare Rare Common Rare Common Common Common Rare Rare Rare Occasional Occasional Rare

Molecular Pathology of Sarcoma the extremities of young patients. It may be associated with systemic symptoms.

remembered that both these fusions have been described in angiomatoid fibrous histiocytoma.

Histology

EPITHELIOID ANGIOSARCOMA

AFH has a hemorrhagic cystic or nodular appearance containing bland histiocytoid cells that may have epithelioid or spindled nuclei. AFH is characterized by its thick fibrous capsule with abundant lymphoid tissue.

Clinical

Cytogenetics and Molecular Pathology AFH harbors a characteristic translocation, the most common being t(2;22)(q33;q12); EWSR1CREB1 followed by t(12;22)(q13;q12); EWSR1ATF and t(12;16)(q13;p11); FUS-ATF.68,69 The translocation can usually be seen on karyotype but FISH studies using break-apart probes for EWSR1 and FUS are likely the most common modes of detection.70,71

Epithelioid angiosarcoma is a rare sarcoma that has a predilection for the skin, superficial soft tissue, breast, bone, liver, and spleen. It may arise in the setting of postirradiation, chronic lymphedema, or arteriovenous fistulas in patients who have had a renal transplantation.

Histology

CLEAR CELL SARCOMA

Epithelioid angiosarcoma is composed of nests and sheets of plump endothelial cells with hyperchromatic nuclei with eosinophilic cytoplasm. Vasoformative spaces may be absent, leaving carcinoma and other epithelioid neoplasms in the differential diagnosis. They typically express vascular markers and commonly coexpress low molecular weight keratin.

Clinical

Cytogenetics and Molecular Pathology

Clear cell sarcoma also referred to as a melanoma of soft parts is a rare but aggressive malignancy that typically occurs in the distal extremities of young adults in association with tendons or aponeuroses.

The karyotype of angiosarcoma tends to be complex and recurrent abnormalities differ between primary and secondary cases. Recurrent abnormalities in primary angiosarcoma include gains of 5 p, 8 p, and 20 p and losses of 4 p, 7 p, and Y; abnormalities of 22q are also seen.76 Mutations in p53 as well as amplification of MDM2 have been described.77 Secondary angiosarcoma seems to be characterized by amplifications at 8q24, 10p12, and 5q35, the former being correlated with amplification of CMYC.78,79

Histology Clear cell sarcoma is characterized by sheets, nests, or fascicles of epithelioid round to fusiform cells with vesicular chromatin and a single prominent nucleolus. A delicate fibrovascular network surrounding the nests is present. Giant cells are common and melanin pigment can be present. The cells are immunoreactive toward S100 and variable toward other melanocytic markers.

Cytogenetics and Molecular Pathology The diagnostic translocation of clear cell sarcoma is t(12;22)(q13;q12); EWSR1-ATF1. Four variant fusions have been described by involving different exons but all fuse the transcriptional activating domain of EWSR1 to the bZIP DNA binding domain of ATF1.72 An alternative t(12;22)(q34;q12); EWSR1CREB1 fusion has been identified, particularly in tumors involving the gastrointestinal tract.73 The translocation can typically be seen on karyotype, which also tends to be complex, although 18 seems to be the only recurrent abnormality. Break-apart EWSR1 FISH assays are useful but cannot distinguish clear cell sarcoma from other EWSR1 rearranged tumors.74 RT-PCR is highly specific but may be difficult to perform on formalin-fixed paraffin-embedded tissues.74,75 It also must be

EPITHELIOID HEMANGIOENDOTHELIOMA Clinical Epithelioid hemangioendothelioma is a low-grade variant of endothelial malignancy that can occur in any age group but is rare in children. It usually involves the dermal or subcutaneous tissues, may be multicentric, and can have bone or visceral involvement.

Histology Epithelioid hemangioendothelioma grows as a cord or nest of polygonal cells with eosinophilic cytoplasm and oval nuclei. Intracellular cytoplasmic lumina are a helpful feature. The background is typically myxoid or hyalinized and the differential diagnosis may include other myxoid neoplasms.

Cytogenetic and Molecular Pathology There is little cytogenetic information on epithelioid hemangioendothelioma but a recurrent

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LeGallo t(1;3)(p36.3;q25) has been reported in the literature, which has recently been shown to result in a WWTR1-CAMTA1 gene fusion.80

EPITHELIOID SARCOMA

rent secondary abnormalities include 11q, 17, 18, 112, and 119. Although NR4A3 translocation can often be seen on conventional karyotype, it may be cryptic but detected using FISH probes EWSR1 and NR4A3.86

Clinical Epithelioid sarcoma is a high-grade sarcoma of young adults and is the most common sarcoma of the distal extremities. A “proximal type” tends to occur in the deep soft tissues of the pelvis, perineum, and buttocks.

Histology Epithelioid sarcoma is an infiltrative lesion composed of a nodule of tumor cells having a bland monomorphic epithelioid, spindled, or rhabdoid appearance. Central necrosis is common and some tumor may be extensively hyalinized. The proximal type tends to have more sheetlike growth with larger epithelioid or rhabdoid cells. Expression of cytokeratins, CD34 and loss of INI1 immunoreactivity are diagnostically helpful features.

Cytogenetics and Molecular Pathology There is little information on the cytogenetics of epithelioid sarcoma, but they appear to be variable and complex with frequent structural aberrations of chromosome 8, 12 p, 18, and 22q11–12.81 Up to 90% of epithelioid sarcomas show loss of protein expression of INI1 by immunohistochemistry, which may correlate to the 22q11–12 abnormalities, as INI1 (SMARCB1) is located on 22q11.2.82 However, analysis of this locus shows genomic abnormalities to be infrequent, suggesting other mechanisms for INI1 inactivation.83,84

EXTRASKELETAL MYXOID CHONDROSARCOMA

MYOEPITHELIAL TUMORS OF SOFT TISSUE Clinical Myoepithelial (ME) tumors of soft tissue appear to be a distinct entity to the salivary counterpart. They tend to occur in the deep tissues of the extremities, head and neck, and mediastinum (including lung) in children and young adults.

Histology ME tumors have heterogeneous morphology with cells having epithelioid, clear, rhabdoid, or spindled cytology. They may be arranged in nests, cords, or sheets, and the background may be chondromyxoid or hyalinized. The tumor cells are immunoreactive toward cytokeratin, EMA, and S100. Up to one-half of the pediatric cases show loss of INI1 protein by IHC.87

Cytogenetics and Molecular Pathology EWSR1 rearrangements can be identified in almost half of ME tumors of the soft tissues. Translocations include t(6;22)(p21;q12); EWSR1POU5F1, which seems to correlate with a clear cell nested pattern; t(1;22)(q23;q12); EWSR1PBX1, which is associated with a spindled or sclerosing lesion; and, rarely, t(19;22)(q13;q12); EWSR1-ZNF44. In about half the cases, the EWSR1 partner is not identified. Cases without EWSR1 translocations tend to be more superficial and are more likely to appear benign.88

Clinical

Practical Applications

Extraskeletal myxoid chondrosarcoma (EMC) presents in the deep soft tissue of the extremities in middle-age to older adults.

Differentiating clear cell sarcoma from metastatic malignant melanoma Clear cell sarcoma can usually be differentiated from other epithelioid mesenchymal neoplasms based on its immunophenotype with variable immunoreactivity with melanocytic markers. However, the morphologic and immunophenotype of malignant melanoma can be identical. When clear cell sarcoma presents in unusual locations or age groups, the distinction is nearly impossible without the detection of the EWS-ATF, diagnostic of clear cell sarcoma (Fig. 6).

Histology Classic features of EMC include a lobular arrangement of cords of ovoid to spindled tumor cells in an abundant chondromyxoid matrix.

Cytogenetics and Molecular Pathology The hallmark of EMC is rearrangements of 9q22, locus of NR4A3, (TEC). In approximately 75% of cases it is involved in a t(9;22)(q22;q12); EWSR1NR4A3, but variant translocations include t(9;17) (q22;q11); TAF15-NR4A3, t(9;15)(q22;q21); TCF12NR4A3, and t(3;9)(q12;q22); TFG-NR4A3.85 Recur-

Diagnosing angiomatoid fibrous histiocytoma When AFH presents in the superficial soft tissue of a young person with the classic histologic features, the diagnosis can be made on morphology

Molecular Pathology of Sarcoma

Fig. 6. (A) Clear cell sarcoma, (B) showing EWSR1 rearrangement by FISH. (C) Metastatic melanoma, (D) showing intact EWSR1 gene.

and immunohistochemistry. However, AFH is increasingly being described in unusual locations and with unusual morphologies, including marked cellular atypia.69,71 In these instances, detection of the EWSR1 and, less commonly, FUS translocations can help confirm a diagnosis (Fig. 7). Diagnosing myoepithelial tumors of soft tissues ME tumors are increasingly being recognized in soft tissues and the differential diagnosis varies based on the histologic features, patient age, and anatomic location. Myoepithelial tumors can mimic chordoma, both have S100, EMA immunoreactivity, and can show loss of INI1 staining, although brachyury immunoreactivity in chordoma may be useful. Extraskeletal myxoid chondrosarcoma also has histologic overlap with an epithelioid predominant ME tumor and it must be remembered that EWSR1 break-apart FISH will not distinguish these tumors (Fig. 8). The spindled or sclerosing variant of an ME tumor can mimic many other low-grade spindled cell lesions (Fig. 9).

Diagnostic challenges solved by molecular pathology: epithelioid soft tissue tumors  Clear cell melanoma

sarcoma

versus

metastatic

 Angiomatoid fibrous histiocytoma versus epithelioid sarcoma  Extraskeletal myxoid chondrosarcoma versus myoepithelial tumor  Myoepithelial tumor versus chordoma

SMALL ROUND BLUE CELL TUMORS ALVEOLAR RHABDOMYOSARCOMA Clinical Alveolar rhabdomyosarcoma (ARMS) is the most aggressive variant of rhabdomyosarcoma presenting in older children and young adults, typically in the extremities or proximal deep soft tissues (Table 5).

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LeGallo

Fig. 7. (A) Angiomatoid fibrous histiocytoma of the hand with marked cytologic atypia, (B) showing a rearranged EWSR1 gene by FISH. (C) Epithelioid sarcoma of the hand. (D) Lack of protein expression of INI1.

Histology Conventional ARMS shows the “alveolar” pattern of monomorphic small round cells with high N/C ratios clinging to fibrovascular septa. The solid variant consists of a nested or sheetlike growth

pattern with a variable amount of desmoplastic stroma. Giant cells may be present and muscle differentiation is seen as plump rhabdomyoblasts. Muscle markers are expressed and the early transcription factors, such as myogenin and myoD1, are particularly useful.

Fig. 8. (A) Extraskeletal myxoid chondrosarcoma, (B) showing EWSR1 rearrangement by FISH.

Molecular Pathology of Sarcoma

Fig. 9. (A) Myoepithelial carcinoma with spindle-cell features, (B) showing EWSR1 rearrangement by FISH.

Cytogenetics and Molecular Pathology ARMS is characterized by t(2;13)(q35;q14) or t(1;13)(p36;q14), which juxtaposes the DNA binding domain of either PAX3 or PAX7, the transcriptional activating domain of FOX01A (FKHR).89 This fusion can be detected in up to 85% of cases by either RT-PCR or FISH but its absence does exclude the diagnosis of ARMS, which is still based on morphology.90,91 Fusion amplification is common in t(1;13) so multiple broken signals may be seen. ARMS is an independent predictor of poor prognosis compared with other forms of rhabdomyosarcoma. Although the PAX3 and PAX7 tumors show similar prognosis for localized disease, the PAX3 tumors have a dismal prognosis when metastatic with an overall survival of less than 10%.92

DESMOPLASTIC SMALL ROUND CELL TUMOR Clinical Desmoplastic small round cell tumor (DSRCT) was first described in the abdomen of adolescent

males and young men, where it predominates but has not been described in both genders in numerous extra-abdominal sites. It has a predilection to grow along mesothelial surfaces, making complete surgical excision impossible.

Histology DSRCT shows a nested or organoid growth of uniform hyperchromatic small round blue cells with indistinct cytoplasm embedded in abundant thermoplastic stromal. Rarely, overt glandular formation is present. The immunophenotype is polyphenotypic with expression of epithelial, muscle, and neural markers, although myogenin of negative.

Cytogenetics and Molecular Pathology The cytogenetics of DSRCT are often complex but the balanced t(11;22)(p13;q12); EWS-WT1 fusion is found in the vast majority of cases and can be detected on routine karyotype.93 Variant translocations include t(2;21;22)(p23;q22;q13) and t(11;17)(p13; q11.2). The breakpoint is most common in intron 7 of EWS and intron 7 of WT1 but variant breakpoints

Table 5 Genetic alterations in small round blue cell tumors Alveolar rhabdomyosarcoma Desmoplastic small round cell tumor

Ewing sarcoma

t(2;13)(q35;q14); PAX3-FOX01A t(1;13)(p36;q14); PAX7-FOX01A t(11;22)(p13;q12); EWSR1-WT1 t(2;21;22)(p23;q22;q13) t(11;17)(p13;q11.2) t(11;22)(q24;q12); EWSR1-FLI1 t(21;22)(q12;q12); EWSR1-ERG t(7;22)(p22;q12); EWSR1-ETV1 t(2;22)(q33;q12); EWSR1-E1AF t(17;22)(q12;q12); EWSR1-FEV t(6;21)(p11;q22); FUS-ERG

Common Occasional Common Rare Rare Common Rare Rare Rare Rare Rare

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LeGallo in intron 8 and 9 in EWS have been associated with atypical presentations.94,95 RT-PCR is highly sensitive and specific. FISH for EWS break-apart probe is commonly used but cannot differentiate DSRCT from other EWS-positive tumors.

EWING SARCOMA

Histology Poorly differentiated synovial sarcoma is characterized by its small-cell, high-grade spindled, or large-cell epithelioid features. The immunophenotype is similar to its conventional counterpart but less frequently expresses keratin.

Clinical

Cytogenetics and Molecular Pathology

Ewing sarcoma occurs most frequently in the second decade of life but may occur in both young children and adults. For extraosseus sites, chest wall, trunk, extremities, abdomen/pelvis, and head and neck are common sites, although Ewing sarcoma has been described just about everywhere.

Poorly differentiated synovial sarcoma shows the characteristic t(X;18)(p11.2;q11.2) but has been shown to be genetically distinct from its conventional counterpart by gene expression profiling showing upregulation of genes specific to chromosomal locus 8q21.11.103

Histology

Practical Applications

Ewing sarcoma is characterized by a monomorphic population of round cells with finely dispersed chromatin and inconspicuous nucleoli. Cytoplasm is scant and may be slightly eosinophilic, amphophilic, or cleared out.

Subtyping rhabdomyosarcoma Although the diagnosis of rhabdomyosarcoma can usually be made on morphology and immunophenotype, as myogenin and myoD1 are specific for skeletal muscle differentiation, the detection of the t(2;13) or t(1;13) may help confirm the alveolar subtype, especially on small biopsies or in the solid variant (Fig. 10). Molecular technique can also be applied to cytologic samples that may not be amenable to routine immunohistochemistry.

Cytogenetics and Molecular Pathology The hallmark of Ewing sarcoma is t(11;22) (q24;q12); EWSR1-FLI1, this plus variant translocations characterize more than 98% of EFTs (Table 5).96,97 Ewing sarcoma otherwise usually shows a relatively simple karyotype with few structural and numerical abnormalities. Among numerical chromosomal changes, trisomy 8 and 12 are most frequently observed.98 The most common secondary abnormality in EFTs is an unbalanced translocation der(16)t(1:16) usually resulting in gain of 1q and partial monosomy 16.99 This aberration is not unique to EWS and can also be seen in retinoblastomas, Wilms, and RMS, among others. Although EWS-FLI1 can usually be detected by routine karyotype, the variant translocations involving EWS are often complex or involve interstitial chromosomal rearrangements and are difficult to detect. FISH is highly sensitive but break-apart technology cannot distinguish Ewing sarcoma from other EWS rearranged tumors. FISH also cannot identify an EWS-FLI1 type 1 variant, which may have prognostic significance. RT-PCR using fresh or frozen tissue also is highly sensitive and specific but numerous primers must be used to detect variant breakpoints or partners.100,101,102

POORLY DIFFERENTIATED SYNOVIAL SARCOMA

Diagnosing DSRCT The diagnosis of DSRCT in classic locations can be made on is polyphenotypic immunoprofile but it is increasingly being described in unusual locations. Small biopsies can also be difficult, as the immunoreactivity toward desmin and cytokeratin may be patchy. Although EWSR1 break-apart FISH is a common diagnostic tool, it must be remembered that many other soft tissue neoplasms harbor EWSR1 translocations. It is the author’s experience the DSRCT often has duplication of the rearranged chromosome, which is less commonly seen in other EWSR1-rearranged tumors (Fig. 11). Differentiating Ewing sarcoma from poorly differentiated synovial sarcoma The distinction between Ewing sarcoma and poorly differentiated synovial sarcoma can be one of the most challenging, as both can express

Diagnostic challenges solved by molecular pathology: small round blue cell tumors  Subtyping of rhabdomyosarcoma

Clinical

 Desmoplastic small round cell tumor versus small cell carcinoma

Poorly differentiated synovial sarcoma presents in the same clinical context as other subtypes, but has a more aggressive clinical course.

 Ewing sarcoma versus poorly differentiated synovial sarcoma

Molecular Pathology of Sarcoma

Fig. 10. (A) Solid alveolar rhabdomyosarcoma, (B) with PAX7-FOXO1 fusion detected by RT-PCR and (C) FOXO1 rearrangement by FISH.

Fig. 11. (A) DSRCT, (B) showing multiple copies of EWSR1 rearrangement by FISH.

Fig. 12. (A, B) Ewing sarcoma, (C) showing EWSR1 rearrangement by FISH. (D) Poorly differentiated synovial sarcoma, (E) showing SYT rearrangement by FISH.

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LeGallo CD99, S100, and epithelial markers to varying degrees. FISH assays for EWSR1 and SYT can easily answer this question and can be done on formalin-fixed paraffin-embedded tissues or cytologic preps (Fig. 12).

13.

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