One-third of all sarcomas are characterized by specific recurrent chromosomal translocations, resulting in highly specific gene fusions, usually encoding aberrant chimeric transcription factors [68
]. The other two-thirds lack a recurrent genetic signature and are characterized by numerous aberrations, including chromosomal losses and gains [68
]. The first group offers the best opportunity for molecular evaluation because these translocations are often the only cytogenetic abnormality and are most likely pathogenetically important.
The most common mechanism involves the EWS gene rearrangement, a specific translocation that juxtaposes the functional domain EWS gene with the DNA-binding domain FLI1, ERG, ATF1, DDIT3, WT1 genes [82
]. Ninety-eight percent of small blue round cells will have the EWS gene rearrangement and are prone to misdiagnosis. These tumors have remarkable clinical diversity and often pose a diagnostic problem because they can be difficult to differentiate by light microscopy and sometimes as a result of nonspecific immunoresults. As an example, O13 (CD99) reactivity, initially believed to represent a reliable marker for Ewing’s sarcoma/PNET diagnosis, has been described also in alveolar rhabdomyosarcoma, synovial sarcoma, desmoplastic round cell tumor (DRCT), and so on. Common immunohistochemical similarities among small blue round cell tumors are described (Table ). The fusion transcripts created by these translocations serve as specific tumor markers that can now be detected by RT-PCR. Among round cell tumors, a distinction that should be made is between DRCT and Ewing’s sarcoma/PNET. With DRCT, prognosis is very poor with 35% overall progression-free survival at 5 years and nonmetastatic Ewing’s has a better prognosis. Desmoplastic small round cell tumor has a characteristic translocation, the EWS gene on chromosome 22 is fused with the WT1 gene (Wilms tumor suppressor gene) on chromosome 11 that clearly distinguishes it from Ewing’s sarcoma [57
]. Another distinction that should be made is between rhabdomyosarcoma and Ewing’s sarcoma/PNET. Rhabdomyosarcoma and Ewing’s sarcoma/PNET share two immunohistochemical markers, CD99 and MyoD, but can be distinguished through molecular translocations (Table ) [18
]. Even desmin positivity, once believed to represent a marker for rhabdomyosarcoma, is present in DRCT and in rare cases of Ewing’s sarcoma [33
]. Another important distinction is between poorly differentiated embryonal rhabdomyosarcoma (E-RMS) and solid-alveolar rhabdomyosarcoma (A-RMS) based on the PAX3/FKHR fusion [33
]. A-RMS occurs predominantly in the extremities and the trunk, whereas E-ARMS occurs predominantly in the head and neck region, the genitourinary tract, and the retroperitoneum. Prognosis substantially differs for patients with A-RMS having a poorer survival than those with E-RMS. A-RMS is characterized by two pathognomonic translocations, t(2;13)(q35;q14) and t(1;13)(p36;q14), found in 80% and 15% of the cases, respectively, whereas E-RMS is not associated with recurrent structural chromosome rearrangement [81
]. Further molecular identification by RT-PCR of the EWSR1-ATF1 translocation can also distinguish the two [110
A practical algorithm for diagnostic evaluation of common musculoskeletal tumors
The second mechanism is a non-EWS gene-based functional domain translocation such as FUS and TLS resulting in chimeric fusion transcription factor overexpression. Based on the FUS-DDIT3 transcript, myxoid liposarcoma can be distinguished from other forms of liposarcoma (LS). Antonescu et al. [6
] reported the TLS-CHOP fusion is highly sensitive and specific for myxoid/round cell LS. Other types of liposarcoma, even with a predominant myxoid component, lack the TLS-CHOP rearrangement, confirming they represent a genetically distinct group of LS. Approximately 5% of myxoid liposarcoma/round cell LS have cytogenetically, but not molecularly, indistinguishable 12;22 translocation that also has been identified as a characteristic aberration in clear cell sarcoma of the tendons and aponeuroses. However, histologic differentiation is sufficient despite molecular identification by RT-PCR of the EWSR1-ATF1 translocation also being able to distinguish the two. Low-grade fibromyxoid sarcoma (LGFMS) is an indolent, late-metastasizing malignant soft tissue tumor that is often mistaken for either more benign or more malignant tumor types. This can now be identified with a recurrent balanced translocation t(7;16)(q32-34;p11) (FUS/CREBL32 fusion gene) [77
]. Some well-differentiated lipomas with minimal atypia reportedly show gain of 12q15-q24 sequences rather than rings and markers or balanced translocations of 12q13-15 (typical feature of benign, ordinary lipomas) [66
]. Hence, it is important to make this distinction between benign and malignant lipomas. Distinction between angiomatoid fibrous histiocytoma and malignant fibrous histiocytoma can be made by detection of FUS-ATF1 fusion [104
]. Prognostic criteria have changed substantially because the fibrohistiocytic tumor now is in a separate category, intermediate malignant (rarely metastasizing), occurring mainly in children and adolescents. This tumor was formerly considered a subtype of the broad category of malignant fibrous histiocytoma.
The third common mechanism involves the fusion of a catalytic domain of a tyrosine kinase receptor with a ubiquitously expressed protein providing a dimerization domain resulting in a constitutively activated, ligand-independent, chimeric tyrosine kinase. This latter mechanism is involved in the pathogenesis of inflammatory myofibroblastic tumor as a result of ALK rearrangements (TPM-ALK and so on) and congenital fibrosarcoma/cellular mesoblastic nephroma resulting from ETV6-NTRK fusion. Inflammatory myofibroblastic tumor of the urinary bladder is an unusual spindle cell neoplasm that displays cytologic atypia, infiltrative growth, and mitotic activity mimicking malignant tumors such as leiomyosarcoma, rhabdomyosarcoma, and sarcomatoid carcinoma. In inflammatory myofibroblastic tumor of the urinary bladder, positivity for ALK-1 by immunohistochemistry ranges from 33% to 89%, whereas ALK-1 protein expression in leiomyosarcoma and sarcomatoid urothelial carcinoma has not been reported, suggesting ALK-1 immunohistochemical studies may be useful in the differentiation of inflammatory myofibroblastic tumor from other spindle cell lesions in the urinary bladder [100
]. In a similar mechanism, deregulation of the platelet-derived growth factor B-chain gene through fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant cell fibroblastoma results in a chimeric autocrine growth factor that is diagnostic [92
]. A histopathologic diagnosis is sufficient for dermatofibrosarcoma protuberans; however, the identification of this molecular event has important consequences in treatment, which is described later.
Lastly, a less common mechanism involves recurrent events within complex karyotypic changes. Very few neoplasms have been described in this category, including malignant peripheral nerve sheath tumor and dedifferentiated liposarcoma. Immunohistochemistry or FISH for detecting MDM2 or CDK4 alterations, two genes commonly amplified on 12q13-15 in atypical lipomatous tumor/well-differentiated liposarcoma [93
], might be useful in the setting of a difficult differential diagnosis.
Most musculoskeletal tumors present with complex karyotypes lacking consistently identifiable specific genetic changes or expression profile signatures. These include most benign tumors and 60% of sarcomas [68
]. Furthermore, these sarcomas tend to occur in older patients and exhibit high-grade pleomorphic cytology and p53 dysfunction. These include leiomyosarcoma, fibrosarcoma, myofibroblastic sarcoma, osteosarcoma, chondrosarcoma, pleomorphic rhabdomyosarcoma, and malignant fibrohistiocytic histiocytoma. Ongoing research could yet identify defining molecular events in these neoplasms.
Despite major advances in the cytogenetic characterization of benign and intermediate tumors of the bone, the incorporation of these alterations as molecular diagnostic tests has been less successful than in malignant tumors. This is because, in general, benign bone tumors are adequately treated by either an intralesional procedure (curettage and burr drilling, cryosurgery) or by marginal excision, depending on relevant anatomy. Furthermore, the recurrence rates of the chromosomal abnormality are not sufficient to achieve clinical molecular diagnostic specificity. Commercial FISH probes are still scarce and unavailable for routine molecular diagnosis.
Benign tumors fall broadly into one of the two of the previously mentioned mechanisms: (1) recurrent events within complex karyotypic changes; and (2) complex karyotypic changes without defined consistent events. Among benign tumors with recurrent events, FISH may be useful in distinguishing between the giant cell tumors of bone and aneurysmal bone cysts [91
]. Telomeric associations are the most frequent chromosomal aberrations in giant cell tumor of bone, most commonly 19q, 11p, 16q, 17p, 18p, 20q, and 21p. All aneurysmal bone cysts exhibit involvement of chromosome segments 17p11-13 and/or 16q22 [91
]. When confronted with a rearrangement, especially concerning 16q22 or 17p3, an associated aneurysmal bone cyst should be excluded. Chondroid lipoma is a rare tumor occurring in the subcutis or muscle of adults; it may be confused with liposarcoma and chondrosarcoma and shows microscopic features of both lipoma and hibernoma. It can be differentiated by identifying a recurrent translocation of t(11;16)(q13;p13) [11
]. Upregulation of PTHrP and Bcl-2 expression characterizes the progression of osteochondroma toward peripheral chondrosarcoma and is a late event in central chondrosarcoma [15
]. In this case, immunohistochemistry can be a useful tool in predicting prognosis if not in clarifying the diagnosis of a patient. Adamantinoma display Ewing-like histologic features described as “adamantinoma-like” Ewing’s sarcoma [39
]. Recently, using RT-PCR on archival tissue, t(11;22) or t(21;22) was not found in any of 12 informative adamantinomas [39
]. Lipomas represent the most cytogenetically diverse benign tumors of fat tissue. Although 98% of lipomas have normal karyotypes, specific genetic abnormalities have been described in sporadic lipomas (12q13-15, t[3;12], 6p, 13q) [63
], lipoblastoma (8q11-13) [31
], hibernoma (11q13) [32
], spindle cell/pleomorphic lipoma (13q12, 16q13) [20
], and atypical lipomatous tumors (rings and giant markers secondary to 12q13-15 amplifications) [19
]. Fibrous lesions, desmoplastic fibromas, desmoid tumors, and other miscellaneous tumors have cytogenetic abnormalities [80
]; however, further distinguishing subtypes does not affect treatment criteria.
Molecular diagnosis should also be used in difficult distinctions between a benign and malignant diagnosis when the consequences of an incorrect interpretation are substantial. In this category, RT-PCR for detection of FUS-CREB3L2 fusion can be useful to distinguish a low-grade fibromyxoid sarcoma from other benign fibrous or neural proliferations when the immunohistochemical or ultrastructural findings are inconclusive [64
]. A similar example includes the differential diagnosis between myxoid liposarcoma in children versus lipoblastoma, a diagnostic dilemma that can be settled by identifying the FUS-CHOP fusion (PLAG1 protein) by RT-PCR or the presence of an 8q abnormality by FISH [31
]. A summary of common clinical scenarios described in this section in which molecular testing would be helpful is provided (Table ).