Skip Navigation LinksHome > July 2013 - Volume 20 - Issue 4 > Sarcoma Diagnosis in the Age of Molecular Pathology
Advances in Anatomic Pathology:
doi: 10.1097/PAP.0b013e31829c2c7b
Review Articles

Sarcoma Diagnosis in the Age of Molecular Pathology

Demicco, Elizabeth G. MD, PhD

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Author Information

Department of Pathology, Mount Sinai Medical Center, New York, NY

The author has no funding or conflicts of interest to disclose.

Reprints: Elizabeth G. Demicco, MD, PhD, Department of Pathology, Mount Sinai Medical Center, 1 Gustave L Levy Place, New York, NY 10029 (e-mail: elizabeth.demicco@mountsinai.org).

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Abstract

Mesenchymal neoplasia presents numerous challenges to pathologic classification. Histologic features can be deceiving, and traditional immunohistochemical markers of differentiation may be of little use in narrowing the diagnosis. Fortunately, great strides have been made in unraveling the genetic and genomic alterations associated with both sarcomagenesis and benign neoplasia. In turn, these advances have led to an expansion of the available diagnostic toolkit for sarcoma pathology. In order to assist the practicing pathologist in integrating these tools into their repertoire, this article will discuss some of the latest advances in sarcoma diagnosis, including an update on translocation-associated sarcomas, and will review a number of sarcoma-specific immunohistochemical studies developed over the past decade. Some of the potential uses and pitfalls of commonly used tests will be addressed. Finally, the discussion will briefly touch upon the impact that advances in molecular technologies, particularly targeted gene expression analysis, may have on altering the face of diagnostic pathology.

Mesenchymal neoplasms pose diagnostic challenges to even the most experienced pathologists. The difficulty is two-fold; sarcomas are rare, accounting for <1% of solid tumors of adults,1,2 and extremely diverse, with well over 50 recognized sarcoma subtypes,3 and many more benign entities. Benign and malignant entities often share similar histologic features, as do malignancies with widely disparate clinical courses. The complexity of sarcoma pathology is reflected in the high discrepancy rates of up to 27% for tumor diagnoses between primary institutions and specialized referral centers.4

In part, these discrepancies result from the difficulty in distinguishing between rare entities with which one is unfamiliar and may encounter only with great infrequency. Histomorphology is often insufficient for tumor diagnosis, although basic cellular morphology—spindle cell, small cell, pleomorphic, or epithelioid characteristics—can narrow the differential. Mesenchymal neoplasia can be further classified by histologic line of differentiation or presence of underlying molecular alterations. Histologic lineage is helpful for tumors that correspond to recognizable normal tissues, including fat, muscle, cartilage, or bone. This terminology is less useful for the myriad of tumors with primitive, vaguely fibroblastic differentiation, or for translocation-associated sarcomas, which may have aberrant features not particular to any one specific lineage. Because of these issues, and as more molecular alterations are identified in different tumor types, the role of molecular diagnostics has grown ever more important.

Advances in sarcoma molecular diagnostics have resulted both from the ongoing identification of previously unrecognized recurrent genetic alterations within the sarcoma genome, and from improved understanding of subtype-specific intracellular signaling events which, in turn, have led to the identification of novel immunohistochemical diagnostic markers.

An understanding of the utility and pitfalls of both molecular studies and newer immunohistochemical markers is required in order to successfully integrate these tools into clinical practice and interpret their results. This review will provide an overview of the current state of molecular diagnostics as they relate to sarcomas, including updates on the molecular classification of sarcomas, and of newer diagnostic immunohistochemical stains and their potential applications.

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MOLECULAR CLASSIFICATION OF MESENCHYMAL NEOPLASMS

Mesenchymal neoplasms are often classified according to the presence of distinct, recurrent genetic abnormalities as (1) chimeric fusion gene-associated; (2) oncogenic mutation-associated; or (3) complex karyotype.

Chimeric fusion gene-associated tumors account for approximately 20% of sarcomas,5,6 and to date include over 30 known subtypes of mesenchymal neoplasia (Table 1).7–15 Prototypical exemplars of this class of sarcoma include Ewing sarcoma/primitive neuroectodermal tumor, characterized by EWSR1-FLI1 translocation, and synovial sarcoma, characterized by SS18-SSX translocations. More recently, reciprocal translocations have been identified in both nodular fasciitis16 and primary aneurysmal bone cyst,17,18 thereby confirming these lesions as clonal neoplastic proliferations. Solitary fibrous tumor, long recognized to have a relatively simple genomic profile, has now been confirmed to have a 12q13 intrachromosomal inversion fusing NAB2 and STAT6.19,20 Newer entities have also been described, including primary pulmonary myxoid sarcoma, a sarcoma with histologic features similar to extraskeletal myxoid chondrosarcoma or myoepithelial tumor of soft tissue, but arising primarily within the lung and associated with EWSR1-CREB1 fusions.21 Malignant gastrointestinal neuroectodermal tumor, characterized by EWSR1-ATF1 or EWSR1-CREB1 translocations, was previously considered to be a variant of clear-cell sarcoma, but has now been proposed to represent a distinct entity with no evidence of melanocytic differentiation.22 Finally, novel recurrent translocations have been found in 2 separate subsets of EWSR1-rearrangment-negative Ewing sarcoma–like undifferentiated small cell sarcomas. CIC-DUX4 translocations have been reported in up to 68% of EWSR1-translocation-negative small round-cell sarcomas,23–26 whereas BCOR-CCNB3 rearrangements were found in 4% of primitive sarcomas negative for EWSR1, SYT, or FOXO1 rearrangements, including both round-cell and fusiform-to-spindle cell sarcomas.27

TABLE 1
TABLE 1
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For convenience, tumors with distinct chromosomal amplifications, for example, the atypical lipomatous tumor-dedifferentiated liposarcoma spectrum of tumors and low-grade osteosarcoma, both with 12q14∼15 amplification28–31 and postradiation angiosarcoma with MYC amplification32,33 may be classified together with translocation-associated sarcomas.

The archetypical oncogenic mutation-associated sarcoma is gastrointestinal stromal tumor (GIST), which is characterized by activating mutations in KIT or PDGFRA in about 85% of cases.34 Other tumors with distinctive recurrent mutations include the locally aggressive desmoid-type fibromatosis with mutations in CTNNB1, the gene locus for β-catenin protein,35,36 and rhabdoid tumors, with deletions of INI137 (Table 2). Similar to malignant rhabdoid tumor, epithelioid sarcoma demonstrates a loss of INI1 expression,38,39 although loss may be due to either deletion40 or epigenetic alterations.41 Activating mutations in codon 201 of GNAS1, the locus encoding a GSα g-protein subunit, are characteristic of intramuscular and cellular myxomas,42 a finding which may be diagnostically useful to distinguish these tumors from malignant mimics such as low-grade myxofibrosarcoma or low-grade fibromyxoid sarcoma (LGFMS). Identical GNAS1 mutations are present in fibrous dysplasia of bone,43,44 a feature which can be used to distinguish these tumors from low-grade osteosarcoma when radiographic or histologic features are insufficient, as may occur in small biopsy specimens.

TABLE 2
TABLE 2
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Complex karyotype sarcomas include many of the genomically unstable high-grade spindle cell and pleomorphic sarcomas, in particular leiomyosarcoma, myxofibrosarcoma, pleomorphic rhabdomyosarcoma, and undifferentiated pleomorphic sarcoma (UPS).45 Molecular testing is often less helpful diagnostically in these tumors, although mutational testing suggests possibilities for targeted therapy.46

The use of molecular testing in sarcoma diagnosis is a prevalent and well-accepted part of the tumor workup. However, choosing a test [fluorescence in situ hybridization (FISH) versus polymerase chain reaction (PCR)—either reverse-transcription (RT) or genomic] and correctly interpreting the result requires an understanding of the uses and limitations of each technique.

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FLUORESCENCE IN SITU HYBRIDIZATION

FISH is one of the most widespread molecular tests used for clinical diagnostic purposes, in large part because of its flexibility. FISH may be performed on formalin-fixed paraffin-embedded tissues, frozen tissue, or cytologic smears, using either disaggregated intact nuclei or thin tissue sections. Probes against specific chromosomal regions can be used to detect both rearrangements and amplifications. Detection of chromosomal rearrangements is usually performed using a breakapart probe corresponding to the most commonly rearranged partner. For instance, FISH confirmation of myxoid liposarcoma (characterized by FUS-DDIT3 or EWSR1-DDIT3 fusions) usually relies on detection of DDIT3 rearrangement as the more sensitive test, as use of probes for FUS rearrangement would fail to detect the approximately 10% of cases resulting from DDIT3-EWSR1 rearrangements.

Breakapart FISH probes consist of 2 adjacent probes flanking the region of interest. One probe is labeled with an orange/red chromophore, whereas the other is labeled with a green chromophore. When the locus is intact, the red and green signals are adjacent, and the overlapping signal thus produced is visualized as yellow fluorescence. When a rearrangement such as reciprocal translocation occurs, the locus is disrupted and the probes are seen as spatially distinct red and green signals. Locus-specific probes can be used to assess for genomic amplification, for example, 12q14∼15 in atypical lipomatous tumor. The addition of a centromeric probe for the chromosome in question allows for a more accurate distinction between aneuploidy, low-level copy gain, or high-level amplification.

Because FISH only requires knowledge of the most frequently rearranged translocation partner, it is a useful test for identification of sarcomas in which one of the fusion partners is present in most cases. Although FISH is generally reliable and sensitive, it is not ideal for tumors in which rare neoplastic cells may be admixed with large numbers of non-neoplastic cells, for example, inflammatory variant of well-differentiated liposarcoma. FISH may not be successful in some decalcified or poorly fixed tissues due to excessive DNA damage. Because of the large size of the probes utilized, FISH has a maximal resolution of 50 to 100 kb47 and may not be able to detect small, nonreciprocal chromosomal abnormalities, for example, insertion of a small amount of sequence to result in a fusion gene, or small intrachromosomal rearrangements such as was recently reported in solitary fibrous tumor.19 FISH is also difficult to interpret in advanced disease, as translocation-associated tumors may accumulate additional complex genomic rearrangements over time that further disrupt the regions of interest.

Additional caveats about the use of FISH as a diagnostic test involve the promiscuity of particular chromosomal breakpoints in mesenchymal and epithelial neoplasia. The FET (EWSR1, FUS, TAFII68) and ETS (FLI1, ERG, ETV) gene families in particular, are involved in a wide variety of tumors (Fig. 1). EWSR1 rearrangements are present in an array of sarcomas of vastly different presentation and clinical behavior, as well as in epithelial tumors, specifically, hyalinizing clear-cell carcinoma of the salivary gland. The presence of an EWSR1 rearrangement in any tumor must therefore be interpreted only after taking into account clinical, histologic, and immunophenotypic features.

FIGURE 1
FIGURE 1
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An example of the dilemmas involved in selecting FISH probes for sarcoma diagnosis is extraskeletal myxoid chondrosarcoma, a rare tumor of uncertain lineage with a propensity to arise in the proximal limb girdles. This tumor may share similar morphologic and immunohistochemical features with both myoepithelial tumors of soft tissue and bone and primary pulmonary myxoid sarcoma. These tumors are all composed of deeply eosinophilic spindled to rounded tumor cells, may have a prominent myxoid stroma, and are characterized by EWSR1 rearrangements. The presence of an EWSR1 rearrangement in a morphologically compatible tumor supports this differential diagnosis but cannot distinguish between the 3 entities. FISH probes for the most common fusion partners, for example, NR4A3 in extraskeletal myxoid chondrosarcoma, POU5F1 in myoepithelial tumor, or CREB1 in pulmonary myxoid sarcoma are very specific, but not widely available outside of specialized referral centers, due to the impracticality and expense of maintaining a FISH probe library for such rare entities.

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REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION

RT-PCR offers an alternative to FISH for the identification of specific translocations, and may be more sensitive than FISH when only a few tumor cells are present. However, RT-PCR requires intact mRNA, which is less stable in tissues than the DNA required for FISH analysis. Although RT-PCR may be performed on formalin-fixed, paraffin-embedded tissues, RNA degradation limits potential amplicons to a maximal size of about 300 bp, with a limit of 150 bp being preferred. Rapidly fixed tissues such as small needle core biopsies may perform better than large sections from resected tumors. PCR may also be inhibited by the presence of hemosiderin in hemorrhagic or necrotic tumors, and care should be taken when selecting blocks from which to perform analysis.

In contrast to FISH, where only the most common fusion partner must be known, RT-PCR requires primers specific for both upstream (5′) and downstream (3′) fusion partners. Because breakpoints are usually intronic, RT-PCR is used to detect the fusion of specific exons from each translocation partner. The selection of primer pairs must take into account the heterogeneity of fusion products. Each partner may have multiple possible breakpoints involving different introns, thereby resulting in an array of potential chimeric genes involving different combinations of exons. Variant translocations which include an additional exon may be too large to amplify effectively, whereas alternative translocations that do not include the region the primer is designed for will not be detectable at all. Thus, as with FISH, a negative translocation by RT-PCR does not exclude a diagnosis.

PCR reactions can be multiplexed, with multiple primer sets present in 1 reaction, in order to detect the more common variant translocations or alternate fusion partners. Different fusion products are distinguished by the size of the PCR product, and sequenced for confirmation. Thus, a test to discriminate Ewing sarcoma from desmoplastic round-cell tumor might utilize a 5′ primer corresponding to EWSR1 and 3′ primers corresponding to FLI1, ERG, and WT1.

Although it has been postulated that variant translocations may predict prognosis in some sarcomas, including alveolar rhabdomyosarcoma,48,49 synovial sarcoma,50 and Ewing sarcoma,51 these findings remain somewhat controversial.52,53

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GENOMIC POLYMERASE CHAIN REACTION

PCR may be performed on genomic DNA in order to detect somatic mutations, such as KIT and PDGFRA mutations in GIST or CTNNB1 mutations in fibromatosis. As with RT-PCR, primers are designed to detect the most frequently affected sites. For GIST these are in KIT exon 11 (65%), exon 9 (10%), and PDGFRA exon 18 (10%)34,54 with an additional 5% of cases bearing mutations on other exons of these 2 genes. In fibromatosis, CTNNB1 mutations involve exons 41 and 45.36 However, wild-type sequences at these sites do not exclude the diagnosis, as mutations in other genes, such as adenomatosis polyposis coli, may result in the same phenotype. PCR can also be used to detect GNAS1 mutations in myxoma or fibrous dysplasia.42,44

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ROLE OF IMMUNOHISTOCHEMISTRY IN THE DIAGNOSIS OF MESENCHYMAL NEOPLASIA

Historically, the use of immunohistochemistry in mesenchymal tumors was, first and foremost, to exclude more common epithelial, melanocytic, or lymphoid malignancies (using stains for cytokeratin, epithelial membrane antigen, S100 protein, melanoma markers or CD45). A broad panel of mesenchymal antigens might then be required to establish a line of differentiation; for spindle cell tumors, such a panel often includes desmin, smooth muscle actin (SMA), and CD34; CD99 is added for round-cell tumors. Additional markers of differentiation might include the skeletal muscle antigens myogenin, myoglobin or myo-D1, or smooth muscle antigens h-caldesmon or smooth muscle myosin. Nonspecific markers of underlying cell-cycle abnormality, including Bcl-2 and p53, were added to diagnostic panels after they were found to be broadly associated with some subsets of sarcoma. However, these are of limited practical use.

Unfortunately, a panel of antibodies is required to discriminate between the differential diagnoses, and even then, a lack of specificity can be troublesome. Sarcomas may express keratin or epithelial membrane antigen, particularly epithelioid and pleomorphic variants of leiomyosarcoma and angiosarcoma, epithelioid sarcoma, or biphasic tumors such as synovial sarcoma and rare cases of malignant peripheral nerve sheath tumor (MPNST). S100, although strongly positive mainly in benign peripheral nerve sheath tumors, may also be seen in myoepithelial tumor, synovial sarcoma, or GIST, as well as in the micropthalmia-associated transcription factor (MiTF) family of tumors, among others. SMA and desmin are seen at least focally in nearly all tumors of fibroblastic or myofibroblastic phenotype as well as in GIST and leiomyosarcoma. Given this phenotypic overlap, it was clear that more specific markers of sarcoma subtype were needed in order to improve diagnostic accuracy.

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IMMUNOHISTOCHEMICAL MARKERS OF DIFFERENTIATION

Diagnostic immunohistochemical markers have long included antigens identified in mature tissues, for example, SMA, myoglobin, S100 protein, and CD31, as well as more recently identified markers such as FLI1 and ERG in vascular tumors.55,56 However, as with many lymphomas, most sarcomas demonstrate an immature or primitive tissue phenotype. Newer differentiation markers include factors expressed in earlier stages of differentiation, including transcription factors actively involved in promotion of lineage development, rather than antigens predominately seen in terminally differentiated mature tissues. Thus, myogenin and myo-D1, early transcriptional regulators of rhabdomyogenesis, have replaced myoglobin, expressed primarily in mature skeletal muscle, as more sensitive markers of rhabdomyosarcoma.57

Sox9 and Sox10 are transcriptional regulators of neural crest development. Sox9 was first identified as a marker of chondrogenesis and was purported to be useful for distinguishing mesenchymal chondrosarcoma from other small round blue cell tumors.58,59 Sox9 was later identified in other tumors, predominately those of neuroectodermal phenotype, including melanoma,60 neurofibroma, and MPNST, as well as synovial sarcoma and Ewing sarcoma.61–63 Rarely, Sox9 may be seen in extraskeletal myxoid chondrosarcoma,64 as well as in over 95% of chordoma,61 and other tumors with partial cartilaginous differentiation such as chondroblastic osteosarcoma.62 Given this expression pattern, Sox9 might not be ideal for identifying primitive cartilaginous tumors from mimics, but could potentially be used as part of a panel for the diagnosis of MPNST.

Sox10 is also frequently expressed in neuroectodermal tumors, including nearly a third of MPNST, and a majority of clear-cell sarcoma and desmoplastic melanoma, while being rare in non-neural crest-derived spindle cell tumors.65,66 Diagnostically, Sox10 could be used as a secondary marker in S100-positive tumors; Sox10 positivity would exclude such tumors as synovial sarcoma, or Ewing sarcoma, and could be used to support a diagnosis of MPNST or melanoma.

Brachyury, a transcriptional regulator of notochordogenesis, was first identified in 2006 as a novel, sensitive marker for axial chordoma.67 It is also expressed in hemangioblastoma,68 and peripheral and soft-tissue chordoma,69 but not in mimics such as chondrosarcoma,70 myoepithelial tumors,71 or metastatic carcinomas or germ cell tumors.69,72 Brachyury is therefore considered to be very specific for chordoma and hemangioblastoma.

Although not a specific marker of lipogenesis, adipophilin is a sensitive marker for intracellular lipid vacuoles73 which is induced early in adipocytic differentiation. Currently, the diagnostic role of adipophilin is limited, although it has been suggested as a marker for sebaceous carcinoma.74 Potential uses for adipophilin in sarcoma include distinguishing small round-cell tumors on needle core biopsy. While such tumors as Ewing sarcoma would be negative, primitive round-cell liposarcoma contains minute adipophilin-positive vacuoles of fat.75,76 Alternatively, adipophilin could be used to distinguish between genuine lipoblasts and the pseudolipoblasts seen in myxofibrosarcoma or myxoma. Care must be taken with the interpretation of adipophilin. Positive stain takes the form of a “champagne bubble” pattern because adipophilin stains proteins in the membrane of intracytoplasmic lipid vacuoles; a granular cytoplasmic stain may be seen in histiocytes, and should not be considered to be a positive result.74

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IMMUNOHISTOCHEMICAL MARKERS DIRECTLY DERIVED FROM GENOMIC/GENETIC ALTERATIONS

Some immunohistochemical markers are derived directly from known underlying genetic abnormalities.

Immunohistochemical stains for MDM2 and CDK4 in liposarcoma77,78 and well-differentiated osteosarcoma79 or c-myc in postradiation angiosarcoma80,81 reflect amplification of these gene loci. Occasionally, MDM2 expression is immunohistochemically detected in the absence of gene locus amplification. This may be particularly troublesome in the diagnosis of retroperitoneal spindle cell sarcomas. Leiomyosarcoma and UPS have been shown to express MDM2 in a small percentage of cases,82–85 whereas dedifferentiated liposarcomas may focally express myogenic markers.86–88 In such cases, where definitive diagnosis cannot be supported by morphology and immunohistochemistry alone, a positive MDM2 immunohistochemical study may prompt reflex molecular FISH testing to verify locus amplification.

ALK1 immunohistochemistry is a useful adjunct study in inflammatory myofibroblastic tumors (IMT). Positive ALK1 stain in IMT is associated with rearrangements of ALK gene,89 and may be seen in up to 60% of cases.90 Therefore, in morphologically suspected IMT, immunohistochemistry represents a cost effective screen for tumors likely to harbor ALK rearrangements. ALK immunohistochemistry is less useful as a standalone diagnostic marker because of low sensitivity and specificity. While many mimics of IMT are negative for ALK, including nodular fasciitis, GIST, and fibromatosis,90 ALK expression can be seen in other malignant spindle cell tumors including MPNST, leiomyosarcoma, and UPS91,92 and small cell tumors such as Ewing sarcoma and alveolar rhabdomyosarcoma.91,93,94 These cases reflect dysregulation of ALK expression or increased gene copy number, rather than translocation.91–93

Loss of expression of INI1 in rhabdoid tumors and epithelioid sarcomas also functions as an immunohistochemically detectable diagnostic marker.39,95,96 Care must be taken when interpreting INI1 stain to evaluate internal positive control cells, including stromal fibroblasts, endothelial cells, or lymphocytes, to verify true loss of expression in tumor cells rather than technical failure of the stain. INI1 loss has also been reported in a subset of epithelioid MPNST, extraskeletal myxoid chondrosarcoma, synovial sarcoma, myoepithelial tumor, and chordoma.95–97 Of note, weak positive stain (as compared to intratumoral endothelial cells) has been reported as a potentially diagnostic feature in synovial sarcoma,98 although subtle staining variations due to technical issues can make interpretation of “reduced expression” difficult.

Translocations involving TFE3 are shared by alveolar soft-part sarcoma and some translocation-associated renal cell carcinomas. Fusion of ASPCR1-TFE3 results in dysregulation and aberrant nuclear expression of TFE3 in both of these neoplasms.99 TFE3 is a transcription factor related to MiTF, and functions in a similar manner. MiTF has been called the “master regulator” of melanocytic differentiation and is expressed in perivascular epithelioid cell tumors (PEComas), angiomyolipomas, and a subset of clear-cell sarcoma of soft tissue, as well as in true melanocytic neoplasms. Because TFE3 and MiTF are related, some investigators have suggested that alveolar soft-part sarcoma, clear-cell sarcoma, and PEComa belong to the same family of tumors. Support for this argument is buttressed by the finding that PEComa family tumors and clear-cell sarcoma express TFE3,100 although usually at lower levels than seen in alveolar soft-part sarcoma. Unrelated sarcoma subtypes are largely negative for this marker.99 Moreover, PEComa with strong TFE3 expression have been found to have rearrangements in TFE3 in place of the more common TSC2 inactivating mutations,101–103 further bolstering a histogenic link between these entities.

In Ewing sarcoma, EWSR1 translocations usually lead to dysregulation and overexpression of chimeric fusion genes with domains from either FLI1 or ERG. Immunohistochemical staining for FLI1 or ERG can therefore be used to detect Ewing sarcoma with rearrangement of these loci.104,105 Antibodies against FLI1 (particularly against the Ets domain) may also cross-react to detect both ERG and FLI1-rearranged tumors.105 Neither FLI1 nor ERG expression is confined to Ewing sarcoma. ERG rearrangement is detectable by immunohistochemistry in prostatic carcinoma,55 and both FLI1 and ERG are normally expressed in vascular endothelium,56 with frequent retention in vascular neoplasia.55,56 Of note, FLI1 expression is also seen in lymphoblastic lymphoma, a CD99-positive morphologic mimic of Ewing sarcoma.104 Although FLI1 or ERG expression can be used to support a diagnosis of Ewing sarcoma, after exclusion of mimics such as lymphoma, in actual practice, FLI1 is more frequently used as a diagnostic marker in poorly differentiated or epithelioid angiosarcoma, whereas ERG is used in prostatic carcinoma.

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IMMUNOHISTOCHEMICAL MARKERS IDENTIFIED BY GENE EXPRESSION PROFILING

Gene expression profiling can be used to identify differentially regulated biomarkers in morphologically similar, but biologically distinct tumor types. Although validation is ongoing for many markers thus identified, several have joined the ranks of clinically accepted diagnostic immunohistochemical studies (Table 3). Given the relative cost efficiency and technical ease of immunohistochemical studies compared to molecular analyses, it is hoped that in future such studies will continue to identify additional markers for a greater variety of sarcoma types.

TABLE 3
TABLE 3
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Comparison of gene expression profiles of synovial sarcomas and morphologically similar MPNST identified TLE1, a transcriptional regulator of neuronal and epithelial differentiation, as a specific marker of synovial sarcoma.106 Strong, diffuse, nuclear staining has since been reported in 80% to 100% of synovial sarcoma.107,108 TLE1 is not entirely specific, and weak to moderate nuclear staining may occasionally be seen in mimics of synovial sarcoma such as solitary fibrous tumor, mesothelioma, and MPNST among others, with a reported specificity of 75%.106,108 Studies on TLE1 have been predominately confined to spindle cell mimics of synovial sarcoma. TLE1 has not been extensively validated in round-cell malignancies, and should therefore not be used to distinguish poorly differentiated, round-cell variants of synovial sarcoma from other poorly differentiated small cell malignancies. Indeed, myxoid/round-cell liposarcoma may express TLE1,108 emphasizing the need to interpret immunophenotype in conjunction with clinicopathologic data.

LGFMS is a bland spindle cell sarcoma characterized by FUS-CREB translocations. On histology alone, particularly on small biopsies, LGFMS may be easily mistaken for benign or low-grade mesenchymal tumors, such as cellular myxoma, perineurioma, or low-grade myxofibrosarcoma. Gene expression profiling identified Muc4 as a potentially specific marker for LGFMS. Immunohistochemical analysis of Muc4 in LGFMS and a variety of histologic mimics, including nerve sheath tumors (neurofibroma, schwannoma, perineurioma), myxofibrosarcoma, and dermatofibrosarcoma protuberans, among others, confirmed that Muc4 was very specific for LGFMS, with 100% of cases demonstrating strong, diffuse cytoplasmic staining, while mimics were negative.109

Sclerosing epithelioid fibrosarcoma (SEF) may share overlapping features with LGFMS, including the presence of FUS-CREB3L2 fusion, as well as Muc4 positivity in up to 78% of cases,110 both in tumors with an LGFMS-like component and in pure SEF. Notably, Muc4-negative SEF were also negative for FUS rearrangement, suggesting that Muc4 expression might be specifically regulated by FUS-CREB3L2 fusion product.110 Muc4 expression is seen in a variety of carcinomas, and was reported in synovial sarcoma; 30% of monophasic tumors showed some degree of positivity and 90% of biphasic synovial sarcomas, with staining predominately confined to the epithelial component. Focal reactivity was also reported in some ossifying fibromyxoid tumors, epithelioid GIST, and myoepithelial tumors.110 These findings suggest that Muc4 may be most helpful as a diagnostic marker in predominately spindle cell tumors, but slightly less useful in tumors with an epithelioid component.

Although the majority of GISTs express strong, diffuse c-kit, about 10% of cases—those with PDGFRA mutation or wild-type KIT/PDGFRA—may be negative. Gene expression profiling studies identified the ion channel anoctamin-1 (DOG1) as a specific marker for GIST.111 Subsequent validation studies confirmed DOG1 positivity in 80% to 95% of cases, depending on the antibody used, and that DOG1 expression was present in both kit-positive and kit-negative tumors.112,113 DOG1 has since been reported to have a higher sensitivity for GIST than c-kit, particularly in unusual subtypes such as pediatric GIST.114 Among mesenchymal tumors, DOG1 is highly specific for GIST, with only rare reports of DOG1 expression in leiomyosarcoma, synovial sarcoma, and melanoma.112,113,115 DOG1 is also expressed in some carcinomas and some normal epithelial cell types. The presence of DOG1 expression in tumors classified as uterine leiomyosarcoma or peritoneal leiomatosis112,113,116 does raise the possibility that these tumors may represent extragastrointestinal GIST with extensive myoid differentiation. Notably, while DOG1 is a very sensitive and specific diagnostic marker, it does not convey therapeutic and prognostic information that may be gained from KIT and PDGFRA mutational analysis.

Gene expression profiling has also been used to elucidate specific prognostic subtypes within established tumor types. In leiomyosarcoma, gene expression signatures identified 5 markers of smooth muscle differentiation with prognostic importance.117 These 5 markers included smooth muscle gamma actin, calsequestrin 2, human muscle cofilin 2, myosin light chain kinase, and sarcolemmal membrane associated protein; coexpression of any 3 or more markers was considered to represent a “muscle-enriched” subtype associated with improved prognosis.117 Moreover, a small subset of UPS (2/29, 7%) was later found to meet criteria for inclusion in this class,118 and likely represented the pleomorphic variant of leiomyosarcoma. Although these 5 markers have not yet been validated or introduced into routine clinical practice, their potential utility as both markers of myogenic phenotype and outcome should not be overlooked.

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SECOND-GENERATION SEQUENCING

Second-generation sequencing technologies are increasing being used to investigate the pathogenesis of sarcoma. Second-generation techniques, including whole-exome and transcriptomic sequencing, were used to discover both the NAB2-STAT6 fusion in solitary fibrous tumor,19,20 as well as the BCOR-CCNB3 translocation of primitive small round blue cell tumors.27 Second-generation sequencing has the advantage of being able to identify chromosomal rearrangements too small to be resolved by karyotypic or FISH, and also detects sequence mutations at the same time. No a priori knowledge or suspicion of involved loci is required for mutation discovery. Second-generation sequencing can be applied to whole genomic DNA to evaluate alterations in noncoding sites, such as promoters or other regulatory elements, or can evaluate exomic alterations. When applied to mRNA, second-generation techniques may be used to establish gene expression levels as well as structural alterations in the coding sequence. Combinatorial use of these technologies in conjunction with other techniques such as chromatin immunoprecipitation have been used to map out promoter sequences that bind specific fusion proteins, correlate these findings with expression levels of the targeted gene to determine which factors are aberrantly upregulated or downregulated, and thereby identify potential therapeutic targets for sarcomas such as alveolar rhabdomyosarcoma and alveolar soft-part sarcoma.119 Thus, although such studies are still in their infancy and require extensive validation, it is hoped that the advent of these powerful technologies will result in the identification of both improved diagnostic and therapeutic markers for sarcoma.

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MOLECULAR ALTERNATIVES TO HISTOPATHOLOGIC DIAGNOSIS

The pathologic identification of distinct histologic subtypes is based on the premise that outcomes and therapeutic responses can be predicted from such classifications. Many of the specific molecular abnormalities and diagnostic immunohistochemical studies described above are useful mainly for their incremental contribution to accurate sarcoma classification. However, an ever-growing body of literature posits that it is not the histologic phenotype that matters as much as the underlying genomic, genetic, and transcriptomic alterations that are present. Newer classification studies rely heavily on techniques such as gene expression profiling, comparative genomic hybrizidization, and second-generation deep sequencing to generate dense data profile that can bypass traditional histologic classification and grading in outcome prediction. Although currently not feasible for routine clinical application, it is important to be aware of such tools and their implications for the field of pathology.

The power of expression array technology when creatively applied to sarcomas is exemplified by the development of the complexity index in sarcoma (CINSARC) score. Complex karyotype sarcomas were initially classified by genomic profile into 3 categories: coamplifications consistent with dedifferentiated liposarcoma, simple alterations involving chromosome or chromosomal arm loss, or highly complex profiles corresponding primarily to leiomyosarcoma and UPS.120 Although the genomic alterations did not predict outcome, they were correlated with tumor grade, implying a link between chromosomal instability and aggressive disease. A gene expression signature (CINSARC score) was then developed, based on factors thought to contribute to genomic instability. The CINSARC score outperformed the Fédération Nationale des Centres de Lutte Contre le Cancer sarcoma grading score as a prognostic measure of metastasis-free survival, regardless of histologic phenotype.120 Furthermore, CINSARC score was prognostic in tumors as varied as GIST, breast carcinoma, and diffuse large B-cell lymphoma, raising the possibility that someday histologic classification and grading might be rendered obsolete in favor of purely molecular classification schemes.

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CONCLUSIONS

The diagnosis of mesenchymal neoplasia continues to be a rapidly evolving field. In an age of tumor-specific targeted therapies, it is essential to correctly identify malignant sarcoma and exclude mimics from the diagnosis. Both immunohistochemical and molecular techniques can help with this process. However, in order to apply these techniques to daily practice in a practical and robust manner, one must be aware of the limitations and pitfalls associated with new markers and selected technologies. No single study has yet shown to be 100% specific or sensitive in clinical practice. Nevertheless, by careful selection and correlation of all aspects of a case—clinical, histologic, immunophenotypic, and molecular—an accurate diagnosis may still be achieved.

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REFERENCES

1. Demetri GD, Antonia S, Benjamin RS, et al .Soft tissue sarcoma.J Natl Compr Canc Netw. 2010; 8:630–674.

2. Jemal A, Siegel R, Xu J, et al .Cancer statistics, 2010.CA Cancer J Clin. 2010; 60:277–300.

3. Fletcher CDM, Rydholm A, Singer S, et al .Fletcher CDM, Unni KK, Mertens F .Soft tissue tumours: epidemiology, clinical features, histopathological typing and grading.World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone. 2002; Lyon:IARC Press; 9–18.

4. Thway K, Fisher C .Histopathological diagnostic discrepancies in soft tissue tumours referred to a specialist centre.Sarcoma. 2009; 2009:

Article ID: 741975


5. Ducimetiere F, Lurkin A, Ranchere-Vince D, et al .Incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing.PLoS One. 2011; 6:e20294

6. Mitelman F .Recurrent chromosome aberrations in cancer.Mutat Res. 2000; 462:247–253.

7. Dal Cin P, Sciot R, De Smet L, et al .Translocation 2;11 in a fibroma of tendon sheath.Histopathology. 1998; 32:433–435.

8. Demicco EG, Lazar AJ .Clinicopathologic considerations: how can we fine tune our approach to sarcoma.Semin Oncol. 2011; 38:S3–S18.

9. Endo M, Hasegawa T, Tashiro T, et al .Bizarre parosteal osteochondromatous proliferation with a t(1;17) translocation.Virchows Arch. 2005; 447:99–102.

10. Nilsson M, Domanski HA, Mertens F, et al .Molecular cytogenetic characterization of recurrent translocation breakpoints in bizarre parosteal osteochondromatous proliferation (Nora’s lesion).Hum Pathol. 2004; 35:1063–1069.

11. Thway K, Flora RS, Fisher C .Chondroid lipoma: an update and review.Ann Diagn Pathol. 2012; 16:230–234.

12. Mertens F, Moller E, Mandahl N, et al .The t(X;6) in subungual exostosis results in transcriptional deregulation of the gene for insulin receptor substrate 4.Int J Cancer. 2011; 128:487–491.

13. Storlazzi CT, Wozniak A, Panagopoulos I, et al .Rearrangement of the COL12A1 and COL4A5 genes in subungual exostosis: molecular cytogenetic delineation of the tumor-specific translocation t(X;6)(q13-14;q22).Int J Cancer. 2006; 118:1972–1976.

14. Zambrano E, Nose V, Perez-Atayde AR, et al .Distinct chromosomal rearrangements in subungual (Dupuytren) exostosis and bizarre parosteal osteochondromatous proliferation (Nora lesion).Am J Surg Pathol. 2004; 28:1033–1039.

15. Isphording A, Ali RH, Irving J, et al .YWHAE-FAM22 endometrial stromal sarcoma: diagnosis by reverse transcription-polymerase chain reaction in formalin-fixed, paraffin embedded tumor.Hum Pathol. 2013; 44:837–843.

16. Erickson-Johnson MR, Chou MM, Evers BR, et al .Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion.Lab Invest. 2011; 91:1427–1433.

17. Oliveira AM, Hsi BL, Weremowicz S, et al .USP6 (Tre2) fusion oncogenes in aneurysmal bone cyst.Cancer Res. 2004; 64:1920–1923.

18. Oliveira AM, Perez-Atayde AR, Inwards CY, et al .USP6 and CDH11 oncogenes identify the neoplastic cell in primary aneurysmal bone cysts and are absent in so-called secondary aneurysmal bone cysts.Am J Pathol. 2004; 165:1773–1780.

19. Robinson DR, Wu YM, Kalyana-Sundaram S, et al .Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing.Nat Genet. 2013; 45:180–185.

20. Chmielecki J, Crago AM, Rosenberg M, et al .Whole-exome sequencing identifies a recurrent NAB2-STAT6 fusion in solitary fibrous tumors.Nat Genet. 2013; 45:131–132.

21. Thway K, Nicholson AG, Lawson K, et al .Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity.Am J Surg Pathol. 2011; 35:1722–1732.

22. Stockman DL, Miettinen M, Suster S, et al .Malignant gastrointestinal neuroectodermal tumor: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of 16 cases with a reappraisal of clear cell sarcoma-like tumors of the gastrointestinal tract.Am J Surg Pathol. 2012; 36:857–868.

23. Italiano A, Sung YS, Zhang L, et al .High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas.Genes Chromosomes Cancer. 2012; 51:207–208.

24. Graham C, Chilton-MacNeill S, Zielenska M, et al .The CIC-DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas.Hum Pathol. 2012; 43:180–189.

25. Yoshimoto M, Graham C, Chilton-MacNeill S, et al .Detailed cytogenetic and array analysis of pediatric primitive sarcomas reveals a recurrent CIC-DUX4 fusion gene event.Cancer Genet Cytogenet. 2009; 195:1–11.

26. Kawamura-Saito M, Yamazaki Y, Kaneko K, et al .Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13) translocation.Hum Mol Genet. 2006; 15:2125–2137.

27. Pierron G, Tirode F, Lucchesi C, et al .A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.Nat Genet. 2012; 44:461–466.

28. Dal Cin P, Kools P, Sciot R, et al .Cytogenetic and fluorescence in situ hybridization investigation of ring chromosomes characterizing a specific pathologic subgroup of adipose tissue tumors.Cancer Genet Cytogenet. 1993; 68:85–90.

29. Pedeutour F, Suijkerbuijk RF, Van Gaal J, et al .Chromosome 12 origin in rings and giant markers in well-differentiated liposarcoma.Cancer Genet Cytogenet. 1993; 66:133–134.

30. Mejia-Guerrero S, Quejada M, Gokgoz N, et al .Characterization of the 12q15 MDM2 and 12q13-14 CDK4 amplicons and clinical correlations in osteosarcoma.Genes Chromosomes Cancer. 2010; 49:518–525.

31. Wunder JS, Eppert K, Burrow SR, et al .Co-amplification and overexpression of CDK4, SAS and MDM2 occurs frequently in human parosteal osteosarcomas.Oncogene. 1999; 18:783–788.

32. Guo T, Zhang L, Chang NE, et al .Consistent MYC and FLT4 gene amplification in radiation-induced angiosarcoma but not in other radiation-associated atypical vascular lesions.Genes Chromosomes Cancer. 2011; 50:25–33.

33. Manner J, Radlwimmer B, Hohenberger P, et al .MYC high level gene amplification is a distinctive feature of angiosarcomas after irradiation or chronic lymphedema.Am J Pathol. 2010; 176:34–39.

34. Liegl-Atzwanger B, Fletcher JA, Fletcher CD .Gastrointestinal stromal tumors.Virchows Arch. 2010; 456:111–127.

35. Lazar AJ, Tuvin D, Hajibashi S, et al .Specific mutations in the beta-catenin gene (CTNNB1) correlate with local recurrence in sporadic desmoid tumors.Am J Pathol. 2008; 173:1518–1527.

36. Amary MF, Pauwels P, Meulemans E, et al .Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.Am J Surg Pathol. 2007; 31:1299–1309.

37. Biegel JA, Tan L, Zhang F, et al .Alterations of the hSNF5/INI1 gene in central nervous system atypical teratoid/rhabdoid tumors and renal and extrarenal rhabdoid tumors.Clin Cancer Res. 2002; 8:3461–3467.

38. Sakharpe A, Lahat G, Gulamhusein T, et al .Epithelioid sarcoma and unclassified sarcoma with epithelioid features: clinicopathological variables, molecular markers, and a new experimental model.Oncologist. 2011; 16:512–522.

39. Hornick JL, Dal Cin P, Fletcher CD .Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma.Am J Surg Pathol. 2009; 33:542–550.

40. Sullivan LM, Folpe AL, Pawel BR, et al .Epithelioid sarcoma is associated with a high percentage of SMARCB1 deletions.Mod Pathol. 2013; 26:385–392.

41. Kohashi K, Izumi T, Oda Y, et al .Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor.Hum Pathol. 2009; 40:349–355.

42. Delaney D, Diss TC, Presneau N, et al .GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma.Mod Pathol. 2009; 22:718–724.

43. de Sanctis L, Delmastro L, Russo MC, et al .Genetics of McCune-Albright syndrome.J Pediatr Endocrinol Metab. 2006; 19:suppl 2 577–582.

44. Lietman SA, Ding C, Levine MA .A highly sensitive polymerase chain reaction method detects activating mutations of the GNAS gene in peripheral blood cells in McCune-Albright syndrome or isolated fibrous dysplasia.J Bone Joint Surg Am. 2005; 87:2489–2494.

45. Guillou L, Aurias A .Soft tissue sarcomas with complex genomic profiles.Virchows Arch. 2010; 456:201–217.

46. Demicco EG, Maki RG, Lev DC, et al .New therapeutic targets in soft tissue sarcoma.Adv Anat Pathol. 2012; 19:170–180.

47. Trask BJ, Allen S, Massa H, et al .Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization.Cold Spring Harb Symp Quant Biol. 1993; 58:767–775.

48. Kazanowska B, Reich A, Stegmaier S, et al .Pax3-fkhr and pax7-fkhr fusion genes impact outcome of alveolar rhabdomyosarcoma in children.Fetal Pediatr Pathol. 2007; 26:17–31.

49. Sorensen PH, Lynch JC, Qualman SJ, et al .PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children’s oncology group.J Clin Oncol. 2002; 20:2672–2679.

50. Ladanyi M, Antonescu CR, Leung DH, et al .Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma: a multi-institutional retrospective study of 243 patients.Cancer Res. 2002; 62:135–140.

51. de Alava E, Kawai A, Healey JH, et al .EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing’s sarcoma.J Clin Oncol. 1998; 16:1248–1255.

52. Stegmaier S, Poremba C, Schaefer KL, et al .Prognostic value of PAX-FKHR fusion status in alveolar rhabdomyosarcoma: a report from the cooperative soft tissue sarcoma study group (CWS).Pediatr Blood Cancer. 2011; 57:406–414.

53. van Doorninck JA, Ji L, Schaub B, et al .Current treatment protocols have eliminated the prognostic advantage of type 1 fusions in Ewing sarcoma: a report from the Children’s Oncology Group.J Clin Oncol. 2010; 28:1989–1994.

54. Steigen SE, Eide TJ, Wasag B, et al .Mutations in gastrointestinal stromal tumors—a population-based study from Northern Norway.APMIS. 2007; 115:289–298.

55. Miettinen M, Wang ZF, Paetau A, et al .ERG transcription factor as an immunohistochemical marker for vascular endothelial tumors and prostatic carcinoma.Am J Surg Pathol. 2011; 35:432–441.

56. Folpe AL, Chand EM, Goldblum JR, et al .Expression of Fli-1, a nuclear transcription factor, distinguishes vascular neoplasms from potential mimics.Am J Surg Pathol. 2001; 25:1061–1066.

57. Fisher C .Immunohistochemistry in diagnosis of soft tissue tumours.Histopathology. 2011; 58:1001–1012.

58. Wehrli BM, Huang W, De Crombrugghe B, et al .Sox9, a master regulator of chondrogenesis, distinguishes mesenchymal chondrosarcoma from other small blue round cell tumors.Hum Pathol. 2003; 34:263–269.

59. Fanburg-Smith JC, Auerbach A, Marwaha JS, et al .Reappraisal of mesenchymal chondrosarcoma: novel morphologic observations of the hyaline cartilage and endochondral ossification and beta-catenin, Sox9, and osteocalcin immunostaining of 22 cases.Hum Pathol. 2010; 41:653–662.

60. McKay KM, Deavers MT, Prieto VG .Chondroid melanoma metastatic to lung and skin showing SOX-9 expression: a potential diagnostic pitfall.Int J Surg Pathol. 2012; 20:169–172.

61. Oakley GJ, Fuhrer K, Brachyury Seethala RR .SOX-9, and podoplanin, new markers in the skull base chordoma vs chondrosarcoma differential: a tissue microarray-based comparative analysis.Mod Pathol. 2008; 21:1461–1469.

62. Cajaiba MM, Jianhua L, Goodman MA, et al .Sox9 expression is not limited to chondroid neoplasms: variable occurrence in other soft tissue and bone tumors with frequent expression by synovial sarcomas.Int J Surg Pathol. 2010; 18:319–323.

63. Carbonnelle-Puscian A, Vidal V, Laurendeau I, et al .SOX9 expression increases with malignant potential in tumors from patients with neurofibromatosis 1 and is not correlated to desert hedgehog.Hum Pathol. 2011; 42:434–443.

64. Demicco EG, Wang WL, Madewell JE, et al .Osseous myxochondroid sarcoma: a detailed study of 5 cases of extraskeletal myxoid chondrosarcoma of the bone.Am J Surg Pathol. 2013; 37:752–762.

65. Gershon TR, Oppenheimer O, Chin SS, et al .Temporally regulated neural crest transcription factors distinguish neuroectodermal tumors of varying malignancy and differentiation.Neoplasia. 2005; 7:575–584.

66. Karamchandani JR, Nielsen TO, van de Rijn M, et al .Sox10 and S100 in the diagnosis of soft-tissue neoplasms.Appl Immunohistochem Mol Morphol. 2012; 20:445–450.

67. Vujovic S, Henderson S, Presneau N, et al .Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas.J Pathol. 2006; 209:157–165.

68. Barresi V, Vitarelli E, Branca G, et al .Expression of brachyury in hemangioblastoma: potential use in differential diagnosis.Am J Surg Pathol. 2012; 36:1052–1057.

69. Tirabosco R, Mangham DC, Rosenberg AE, et al .Brachyury expression in extra-axial skeletal and soft tissue chordomas: a marker that distinguishes chordoma from mixed tumor/myoepithelioma/parachordoma in soft tissue.Am J Surg Pathol. 2008; 32:572–580.

70. Jambhekar NA, Rekhi B, Thorat K, et al .Revisiting chordoma with brachyury, a “new age” marker: analysis of a validation study on 51 cases.Arch Pathol Lab Med. 2010; 134:1181–1187.

71. Rekhi B, Sable M, Jambhekar NA .Histopathological, immunohistochemical and molecular spectrum of myoepithelial tumours of soft tissues.Virchows Arch. 2012; 461:687–697.

72. Sangoi AR, Karamchandani J, Lane B, et al .Specificity of brachyury in the distinction of chordoma from clear cell renal cell carcinoma and germ cell tumors: a study of 305 cases.Mod Pathol. 2011; 24:425–429.

73. Heid HW, Moll R, Schwetlick I, et al .Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.Cell Tissue Res. 1998; 294:309–321.

74. Ostler DA, Prieto VG, Reed JA, et al .Adipophilin expression in sebaceous tumors and other cutaneous lesions with clear cell histology: an immunohistochemical study of 117 cases.Mod Pathol. 2010; 23:567–573.

75. Straub BK, Herpel E, Singer S, et al .Lipid droplet-associated PAT-proteins show frequent and differential expression in neoplastic steatogenesis.Mod Pathol. 2010; 23:480–492.

76. Hoffman A, Ghadimi MP, Demicco EG, et al .Localized and metastatic myxoid/round cell liposarcoma: clinical and molecular observations.Cancer. 2013; 119:1868–1877.

77. Binh MB, Garau XS, Guillou L, et al .Reproducibility of MDM2 and CDK4 staining in soft tissue tumors.Am J Clin Pathol. 2006; 125:693–697.

78. Binh MB, Sastre-Garau X, Guillou L, et al .MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data.Am J Surg Pathol. 2005; 29:1340–1347.

79. Dujardin F, Binh MB, Bouvier C, et al .MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary fibro-osseous lesions of the bone.Mod Pathol. 2011; 24:624–637.

80. Fernandez AP, Sun Y, Tubbs RR, et al .FISH for MYC amplification and anti-MYC immunohistochemistry: useful diagnostic tools in the assessment of secondary angiosarcoma and atypical vascular proliferations.J Cutan Pathol. 2012; 39:234–242.

81. Mentzel T, Schildhaus HU, Palmedo G, et al .Postradiation cutaneous angiosarcoma after treatment of breast carcinoma is characterized by MYC amplification in contrast to atypical vascular lesions after radiotherapy and control cases: clinicopathological, immunohistochemical and molecular analysis of 66 cases.Mod Pathol. 2012; 25:75–85.

82. Ragazzini P, Gamberi G, Pazzaglia L, et al .Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma.Histol Histopathol. 2004; 19:401–411.

83. Patterson H, Gill S, Fisher C, et al .Abnormalities of the p53 MDM2 and DCC genes in human leiomyosarcomas.Br J Cancer. 1994; 69:1052–1058.

84. Rao UN, Finkelstein SD, Jones MW .Comparative immunohistochemical and molecular analysis of uterine and extrauterine leiomyosarcomas.Mod Pathol. 1999; 12:1001–1009.

85. Hall KL, Teneriello MG, Taylor RR, et al .Analysis of Ki-ras, p53, and MDM2 genes in uterine leiomyomas and leiomyosarcomas.Gynecol Oncol. 1997; 65:330–335.

86. Henricks WH, Chu YC, Goldblum JR, et al .Dedifferentiated liposarcoma: a clinicopathological analysis of 155 cases with a proposal for an expanded definition of dedifferentiation.Am J Surg Pathol. 1997; 21:271–281.

87. Russell MJ, Flynt FL, Harroff AL, et al .Dedifferentiated liposarcoma of the retroperitoneum with extensive leiomyosarcomatous differentiation and beta-human chorionic gonadotropin production.Sarcoma. 2008; 2008:

Article ID: 658090


88. Evans HL, Khurana KK, Kemp BL, et al .Heterologous elements in the dedifferentiated component of dedifferentiated liposarcoma.Am J Surg Pathol. 1994; 18:1150–1157.

89. Coffin CM, Patel A, Perkins S, et al .ALK1 and p80 expression and chromosomal rearrangements involving 2p23 in inflammatory myofibroblastic tumor.Mod Pathol. 2001; 14:569–576.

90. Cook JR, Dehner LP, Collins MH, et al .Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: a comparative immunohistochemical study.Am J Surg Pathol. 2001; 25:1364–1371.

91. Li XQ, Hisaoka M, Shi DR, et al .Expression of anaplastic lymphoma kinase in soft tissue tumors: an immunohistochemical and molecular study of 249 cases.Hum Pathol. 2004; 35:711–721.

92. Cessna MH, Zhou H, Sanger WG, et al .Expression of ALK1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal mimics: a study of 135 cases.Mod Pathol. 2002; 15:931–938.

93. Yoshida A, Shibata T, Wakai S, et al .Anaplastic Lymphoma Kinase status in rhabdomyosarcomas.Mod Pathol. 2013;

[Epub ahead of print]


94. van Gaal JC, Flucke UE, Roeffen MH, et al .Anaplastic lymphoma kinase aberrations in rhabdomyosarcoma: clinical and prognostic implications.J Clin Oncol. 2012; 30:308–315.

95. Mularz K, Harazin-Lechowska A, Ambicka A, et al .Specificity and sensitivity of INI-1 labeling in epithelioid sarcoma. Loss of INI1 expression as a frequent immunohistochemical event in synovial sarcoma. Loss of INI1 expression as a frequent immunohistochemical event in synovial sarcoma.Pol J Pathol. 2012; 63:179–183.

96. Hollmann TJ, Hornick JL .INI1-deficient tumors: diagnostic features and molecular genetics.Am J Surg Pathol. 2011; 35:e47–e63.

97. Mobley BC, McKenney JK, Bangs CD, et al .Loss of SMARCB1/INI1 expression in poorly differentiated chordomas.Acta Neuropathol. 2010; 120:745–753.

98. Arnold MA, Arnold CA, Li G, et al .A unique pattern of INI1 immunohistochemistry distinguishes synovial sarcoma from its histologic mimics.Hum Pathol. 2013; 44:881–887.

99. Argani P, Lal P, Hutchinson B, et al .Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay.Am J Surg Pathol. 2003; 27:750–761.

100. Dickson BC, Brooks JS, Pasha TL, et al .TFE3 expression in tumors of the microphthalmia-associated transcription factor (MiTF) family.Int J Surg Pathol. 2011; 19:26–30.

101. Malinowska I, Kwiatkowski DJ, Weiss S, et al .Perivascular epithelioid cell tumors (PEComas) harboring TFE3 gene rearrangements lack the TSC2 alterations characteristic of conventional PEComas: further evidence for a biological distinction.Am J Surg Pathol. 2012; 36:783–784.

102. Ohe C, Kuroda N, Hes O, et al .A renal epithelioid angiomyolipoma/perivascular epithelioid cell tumor with TFE3 gene break visualized by FISH.Med Mol Morphol. 2012; 45:234–237.

103. Argani P, Aulmann S, Illei PB, et al .A distinctive subset of PEComas harbors TFE3 gene fusions.Am J Surg Pathol. 2010; 34:1395–1406.

104. Folpe AL, Hill CE, Parham DM, et al .Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing’s sarcoma/primitive neuroectodermal tumor.Am J Surg Pathol. 2000; 24:1657–1662.

105. Wang WL, Patel NR, Caragea M, et al .Expression of ERG, an Ets family transcription factor, identifies ERG-rearranged Ewing sarcoma.Mod Pathol. 2012; 25:1378–1383.

106. Terry J, Saito T, Subramanian S, et al .TLE1 as a diagnostic immunohistochemical marker for synovial sarcoma emerging from gene expression profiling studies.Am J Surg Pathol. 2007; 31:240–246.

107. Rekhi B, Basak R, Desai SB, et al .Immunohistochemical validation of TLE1, a novel marker, for synovial sarcomas.Indian J Med Res. 2012; 136:766–775.

108. Kosemehmetoglu K, Vrana JA, Folpe AL .TLE1 expression is not specific for synovial sarcoma: a whole section study of 163 soft tissue and bone neoplasms.Mod Pathol. 2009; 22:872–878.

109. Doyle LA, Moller E, Dal Cin P, et al .MUC4 is a highly sensitive and specific marker for low-grade fibromyxoid sarcoma.Am J Surg Pathol. 2011; 35:733–741.

110. Doyle LA, Wang WL, Dal Cin P, et al .MUC4 is a sensitive and extremely useful marker for sclerosing epithelioid fibrosarcoma: association with FUS gene rearrangement.Am J Surg Pathol. 2012; 36:1444–1451.

111. West RB, Corless CL, Chen X, et al .The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status.Am J Pathol. 2004; 165:107–113.

112. Espinosa I, Lee CH, Kim MK, et al .A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors.Am J Surg Pathol. 2008; 32:210–218.

113. Miettinen M, Wang ZF, Lasota J .DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study of 1840 cases.Am J Surg Pathol. 2009; 33:1401–1408.

114. Liegl B, Hornick JL, Corless CL, et al .Monoclonal antibody DOG1.1 shows higher sensitivity than KIT in the diagnosis of gastrointestinal stromal tumors, including unusual subtypes.Am J Surg Pathol. 2009; 33:437–446.

115. Hemminger J, Iwenofu OH .Discovered on gastrointestinal stromal tumours 1 (DOG1) expression in non-gastrointestinal stromal tumour (GIST) neoplasms.Histopathology. 2012; 61:170–177.

116. Sah SP, McCluggage WG .DOG1 immunoreactivity in uterine leiomyosarcomas.J Clin Pathol. 2013; 66:40–43.

117. Beck AH, Lee CH, Witten DM, et al .Discovery of molecular subtypes in leiomyosarcoma through integrative molecular profiling.Oncogene. 2010; 29:845–854.

118. Mills AM, Beck AH, Montgomery KD, et al .Expression of subtype-specific group 1 leiomyosarcoma markers in a wide variety of sarcomas by gene expression analysis and immunohistochemistry.Am J Surg Pathol. 2011; 35:583–589.

119. Taylor BS, Barretina J, Maki RG, et al .Advances in sarcoma genomics and new therapeutic targets.Nat Rev Cancer. 2011; 11:541–557.

120. Chibon F, Lagarde P, Salas S, et al .Validated prediction of clinical outcome in sarcomas and multiple types of cancer on the basis of a gene expression signature related to genome complexity.Nat Med. 2010; 16:781–787.

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International Journal of Surgical Pathology
The Utility of Immunohistochemistry for Providing Genetic Information on Tumors
Chan, JKC; Ip, YT; Cheuk, W
International Journal of Surgical Pathology, 21(5): 455-475.
10.1177/1066896913502529
CrossRef
Keywords:

sarcoma; diagnosis; molecular classification; immunohistochemistry; fluorescence in situ hybridization

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