A Rational Approach to Genetic Testing for Sarcoma : Diagnostic Molecular Pathology

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00019606-200903000-00001ReviewDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2009 Lippincott Williams & Wilkins, Inc.18March 2009 p 1-10A Rational Approach to Genetic Testing for SarcomaReview ArticleGulley, Margaret L. MD; Kaiser-Rogers, Kathleen A. PhDDepartment of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NCReprints: Margaret L. Gulley, MD, Department of Pathology and Laboratory Medicine, 913 Brinkhous-Bullitt Building, University of North Carolina, Chapel Hill, NC 27599-7525 (e-mail: [email protected]).AbstractDiagnosis of sarcoma increasingly relies on identifying genetic defects using modern molecular technologies. Each analytic method has unique advantages and specimen requirements that should be considered when allocating tissue for downstream testing. Karyotype on fresh tissue represents a genome-wide screen of gross chromosomal alterations, whereas fluorescence in situ hybridization and polymerase chain reaction detect specific defects that are characteristic of a given tumor type such as t(11;22) EWSR1-FLI1 in Ewing family tumors, t(X;18) SS18-SSX1 in synovial sarcoma, t(2;13) PAX3-FOXO1A in alveolar rhabdomyosarcoma, and MYCN gene amplification in neuroblastoma. Identifying a clonal genetic defect also provides a tumor marker that could help stage the extent of spread of the neoplasm or monitor the efficacy of therapy. In research laboratories, array-based methods identify genes and biochemical pathways contributing to tumor growth and maintenance, opening avenues for pharmacogenetic tests that predict which therapy is likely to overcome the biochemical defects with minimal toxicity. Array-based discoveries are also spurring validation of smaller test panels that rely on conventional technologies such as immunohistochemistry and reverse transcription polymerase chain reaction. The pathologist's expertize is critical in: (1) consulting with clinicians about specimen collection and handling; (2) preserving tissue for immediate testing and for any downstream testing that is indicated once morphology and immunophenotype are known; (3) performing tests that maximize outcome on the basis of the strengths and limitations of each assay in each available specimen type; and (4) conveying results to the rest of the healthcare team using proper gene nomenclature and interpreting the findings in a way that facilitates optimal clinical management.Many subtypes of sarcoma harbor translocations or other characteristic genetic defects. Identification of these alterations assists in diagnosis of each tumor type and, in some cases, imparts prognostic or predictive information influencing clinical management. The most informative genetic test methods are karyotype, fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (rtPCR), and direct DNA sequencing. Results are increasingly being incorporated into modern classification schemes for sarcomas, especially as targeted therapies are developed that thwart the underlying biochemical defect responsible for tumor growth.1Tumor-specific translocations are helpful in supporting a diagnosis of cancer. Laboratory tests that detect the translocation potentially provide a convenient biomarker for staging the extent of spread of disease and for monitoring success of therapy. Specific gene defects impart prognostic information for certain tumors including Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma, and neuroblastoma.2,3 Gene expression profiles show promise for facilitating classification of tumors of uncertain histogenesis and as prognosticators or predictors of outcome for pediatric small round cell tumors.4–9 The relative merits of karyotype, FISH, rtPCR, and other molecular tests relate to the type of genetic alteration that one seeks to identify and the specimen types available for laboratory analysis.KARYOTYPEKaryotyping is the most comprehensive laboratory method for spotting the various translocations and other numeric or structural defects that characterize sarcomas. Fresh tumor tissue is required so that cultured cells can be arrested in metaphase to allow visualization of G-bands in each chromosome. Both numerical and structural abnormalities are then documented for each chromosome in at least 20 cells. Results should be interpreted in the context of what is known about the clinical setting and the lesion's histopathology and immunophenotype, and in the context of the vast literature on chromosomal defects associated with particular subtypes of cancer. A helpful website that summarizes the literature and displays images of cytogenetic findings is the Atlas of Genetics and Cytogenetics in Oncology and Haematology, http://atlasgeneticsoncology.org. Karyotype results may be falsely negative for a variety of reasons, such as preferential growth of surrounding stromal cells, absent tumor growth secondary to necrosis or a poor mitotic index, or presence of an occult rearrangement that is visible only at the molecular level. Additionally, in the setting of a very complex karyotype, a clinically relevant chromosome rearrangement may be masked by concurrent rearrangements of the involved chromosomes.It should be mentioned that every human cancer is thought to contain genetic alterations that are responsible for proliferation, resistance to apoptosis, and/or immune evasion. Although gross genetic defects are easily seen in metaphase spreads of malignant cells dividing in vitro, we are only just beginning to identify the point mutations, microdeletions, and other subtle defects that characterize cancer at the molecular level.FISHFISH is perhaps the most helpful genetic technology for identifying a specific gene rearrangement because FISH can be used to assay either interphase (nondividing) or metaphase (dividing) cells. A break-apart probe strategy permits detection of any rearrangement involving a particular gene regardless of which translocation partner might be involved. From a technical perspective, FISH is most informative when it is applied to intact cells obtained from cultured fresh tissue, touch preparations, or smears. Touch preparations or smears may be air dried and saved unstained at 4°C for days to weeks before FISH testing, thus allowing for completion of the morphologic and immunophenotypic workup before deciding which FISH tests to order, if any. Paraffin sections are somewhat less informative as only part of each nucleus is represented in thin histologic sections. To overcome this disadvantage, some laboratories have validated FISH assays on whole nuclei disaggregated from thick (50 um) paraffin sections. FISH probes are readily available for a variety of relevant gene targets including EWSR1, ALK, SS18, FOXO1A, DDIT3, and FUS.The sensitivity of a FISH assay to low level disease depends on the quality of the specimen, the number of intact cells that are scored, and the probe design, as all of these factors influence the cutoff value for reliable assignment of an abnormal result. A typical interphase FISH assay scores 200 cells and can reliably detect tumor involving as few as 5% of cells in the specimen. In general, sensitivity of FISH is comparable with that of a 20-cell karyotype. Karyotype, however, is dependent on the growth rate of tumor cells compared with stromal cells in the specimen, and sarcoma cells are notorious for their finicky proliferation in culture media. When dividing tumor cells are available, metaphase FISH may be applied, typically involving analysis of 20 cells. Such analysis of metaphase chromosomes is quite helpful in resolving complex karyotypes, identifying partner chromosomes, and finding marker chromosomes that may harbor cryptic translocations.A variant FISH method often referred to as spectral karyotyping (SKY FISH) or 24-color multiplex FISH (M-FISH) employs whole-chromosome paint procedures to apply uniquely colored sets of probes targeting nearly the full length of each of the 24 chromosomes. Accurate interpretation of these multicolored karyotypes requires parallel analysis of G-banded metaphase cells. This strategy can be useful for identifying translocations hidden in complex karyotypes or involving marker chromosomes.POLYMERASE CHAIN REACTIONThe most sensitive analytic test is polymerase chain reaction (PCR) because it is capable of copying DNA or cDNA a billion-fold to the point where it can be readily detected or further analyzed for tumor-related defects. Sensitivity levels of 1 in a 100,000 cells are typically achieved, potentially allowing for submicroscopic detection of metastasis or for identification of minimal residual disease after therapy. The method is amenable to fresh, frozen, or paraffin-embedded tissue and body fluids.Several groups have developed primers to amplify DNA across the translocation breakpoints in Ewing family sarcomas, synovial sarcoma, alveolar rhabodmyosarcoma, and desmoplastic round cell tumor.10–12 Although rtPCR can be carried out on paraffin-embedded tissue, it is much more reliable in fresh or frozen tissue. It is recommended that touch preparations (for interphase FISH) and frozen tissue (for rtPCR or expression profiles) be saved on any biopsy that is likely to require downstream genetic testing.13Examples of the relative merits of each genetic technology in various specimen types and clinical settings are described below. Table 1 displays a list of genetic defects that are associated with histopathologic subtypes of sarcoma. Note that correct cytogenetic and gene nomenclature is used to depict each characteristic genetic defect.14 Current gene names and symbols may be searched for in the database of the Human Gene Organization Gene Nomenclature Committee, www.genenames.org..JOURNAL/dimp/04.03/00019606-200903000-00001/table1-1/v/2021-02-17T200000Z/r/image-tiff Common Genetic Abnormalities in Sarcomas of Bone and Soft TissueEWING FAMILY OF SARCOMASEwing sarcoma is a high grade, small blue cell tumor that primarily affects long bones or the vertebral area in young adults and children. Histologically, the proliferating cells may form Homer-Wright rosettes and similar rosette structures. Prominent rosetting and immunohistochemical evidence of neuroendocrine differentiation are critical histopathologic criteria for subclassifying a tumor as a primitive neuroectodermal tumor (PNET).15 However, the distinction between Ewing sarcoma and PNET is no longer considered to be critical for clinical management as prognosis and therapy are similar.Ewing sarcomas and PNETs comprise the Ewing family tumors that characteristically harbor a recurring t(11;22)(q24;q12) that juxtaposes the FLI1 and EWSR1 genes to encode a chimeric RNA and protein. About 10% of Ewing family tumors have an alternate translocation involving the EWSR1 gene, implying that disruption of EWSR1 is the critical molecular event underlying tumorigenesis.Most breaks within the EWSR1 gene occur in the intron 7 to 10 region, resulting in juxtaposition of exons 1 through 7 with a segment of the partner gene (Fig. 1). This causes replacement of a putative RNA polymerase II binding region of EWSR1 with the DNA binding domain of the partner gene, thus encoding a fusion protein whose expression is controlled by the ubiquitously active EWSR1 promoter. Each of the partner genes encodes an ETS-family transcription factor that is important in embryologic development. The resulting aberrant transcription factor is responsible, at least in part, for sarcoma pathogenesis.16 Deletion of CDKN2A or mutation of TP53 is a secondary event imparting a poor prognosis.17JOURNAL/dimp/04.03/00019606-200903000-00001/figure1-1/v/2021-02-17T200000Z/r/image-jpeg Molecular anatomy of the EWSR1-FLI1 translocation. Among cases of Ewing sarcoma, breakpoints (shown as blue arrows) within EWSR1 are clustered in introns 7 to 10, whereas the FLI1 breakpoints are variable but commonly involve introns 4 or 5. At the DNA level, PCR amplification across the translocated breakpoint would require many primer sets to detect all possible translocation variants. However, after transcription and processing to splice out the introns and form mature mRNA, there are relatively limited breakpoint variants that can be tested for by rtPCR. The most common fusion transcripts are detected by extracting RNA from the patient specimen, reverse transcribing the RNA to cDNA, and then applying rtPCR using a forward primer targeting EWSR1 exon 7 and a reverse primer targeting FLI1 exon 6. PCR indicates polymerase chain reaction; rtPCR, reverse transcription polymerase chain reaction.Karyotype is an excellent analytic method for the initial workup of a suspected Ewing family tumor because the characteristic t(11;22) is evident in approximately 90% of these tumors, and less common alternative translocations are also evident. Furthermore, if the tumor is not a Ewing family tumor, karyotyping might reveal a different rearrangement that helps categorize the lesion. Given a strong suspicion of Ewing sarcoma/PNET and a normal karyotype, FISH should be considered as an additional test for the genetic defects that characterize Ewing family tumors. FISH, using a break-apart probe targeting the EWSR1 gene, is an excellent method to identify or exclude an EWSR1 gene rearrangement. FISH may be carried out on metaphase cells (requiring fresh tissue) or on interphase cells (feasible on a wide variety of sample types including fine needle aspirates, touch preparations, smears, or paraffin-embedded tissue) (Fig. 2).18JOURNAL/dimp/04.03/00019606-200903000-00001/figure2-1/v/2021-02-17T200000Z/r/image-jpeg FISH detects rearrangement of the EWSR1 gene at 22q12. The diagram on the left depicts the EWSR1 gene and a break-apart probe strategy whereby the 2 probes flanking this gene are labeled with red and green fluorochromes, respectively. In a normal cell, these 2 probes colocalize such that the red and green signals merge and appear yellow at each of the 2 normal EWSR1 alleles. In the presence of EWSR1 gene rearrangement, however, these probes separate to form independent red and green fluorescent signals that represent the derivative chromosome 22 and the derivative partner chromosome, respectively. Not shown is metaphase FISH analysis in which the reciprocal translocation partner (such as 11q24 where the FLI1 gene resides) is pinpointed by the green probe in G-banded chromosomes. In yet another type of analysis not shown (called the “single fusion” probe strategy), a red EWSR1 probe similar to that depicted above and a green probe targeting the FLI1 locus are cohybridized such that, in the presence of the 11;22 translocation, 1 red signal and 1 green signal join to produce a yellow fusion signal on the abnormal (derivative) chromosome 22. Further description of “single fusion” or “dual fusion” FISH probe strategies is not relevant to the current discussion as these analyses are generally only available in research laboratories. FISH indicates fluorescence in situ hybridization.It is important to recognize that translocations of 22q12 are not entirely sensitive nor specific for Ewing family tumors.8,19–22 As shown in Table 1, 5 other subtypes of sarcoma commonly harbor an EWSR1 translocation, emphasizing a commonality in biochemistry despite differences in histogenesis, anatomic site, and other clinicopathologic features that distinguish the various bone and soft tissue sarcomas. It is not clear if a different partner gene or a different cell of origin is most critical in driving the clinical phenotype. In any case, it is important to correlate genetic test results with morphology, immunophenotype, and clinical features to optimally categorize each tumor. Multiple complementary genetic tests may be required to characterize a given tumor.The various translocation partners for EWSR1 may be identified using karyotype, rtPCR, or FISH.10,23 An advantage unique to rtPCR is defining which of 2 prognostically relevant EWSR1-FLI1 transcript structures is present: type 1 fusion transcripts are not transcribed as actively and carry a good prognosis compared with the alternative fusions.24 Although prognostic information is helpful, patient management decisions do not tend to rely on this information at this time. Another advantage of rtPCR is its exquisite sensitivity to low level transcripts, which may be helpful in staging the degree of spread to sites that are only minimally involved by tumor.25 Minimal residual disease after therapy can be measured using quantitative rtPCR,26 but the implications of detecting low level transcripts remain to be defined in clinical trials.A downside of rtPCR is that separate hybridizations must be carried out for each of the alternate EWSR1 rearrangements, thus requiring significant investment in assay validation and ongoing proficiency testing. Variability in breakpoint sites and technical problems in obtaining high quality RNA (especially from paraffin-embedded tissue) and avoiding amplicon contamination all confound rtPCR and contribute to the overall utility of FISH strategies compared with rtPCR methods.27Meticulous care is required to validate, perform, and interpret any of the various laboratory assays that are used to detect tumor-specific genetic alterations.28 As none of these analytic tests are foolproof, it is useful to employ more than 1 complementary method, and to correlate genetic test results with other clinicopathologic findings. Algorithms have been proposed to guide the use of immunostains and genetic tests in evaluating soft tissue tumors.1,15,29–31 Immunohistochemical analysis of CD99 and FLI1 reveal overexpression in most Ewing/PNET tumors harboring a t(11;22).32 Likewise, a positive WT1 immunostain marks desmoplastic round cell tumor,33 and a positive KIT immunostain distinguishes gastrointestinal stromal tumor (GIST) from similar appearing spindle cell neoplasms.SYNOVIAL SARCOMADespite its name, synovial sarcoma does not arise from synovium. It does, however, often arise deep in the soft tissue near a joint in the extremity of a young adult patient. Identification of a t(X;18)(p11.2;q11.2) is quite sensitive and specific for synovial sarcoma, and, therefore, is among the most helpful genetic tests for classifying sarcomas. There are 2 common histologic and genetic variants of synovial sarcoma: a monophasic variant comprised of vimentin-expressing spindle cells carrying SS18-SSX2 translocation and a biphasic variant comprised of a mixture of vimentin-expressing spindle cells and keratin-expressing glandular epithelial cells harboring the SS18-SSX1 translocation. The biphasic variant may resemble adenocarcinoma or more typically carcinosarcoma and carries a worse prognosis, at least in early stage patients. The 2 translocations are indistinguishable using traditional cytogenetics, but at the molecular level, the alternate gene partners on the X chromosome are evident.Karyotype is helpful when it is positive, but negative results could reflect failure of the tumor cells to divide sufficiently in culture, or other mechanisms of false negative results as alluded to above. FISH using an SS18 break-apart probe is quite helpful for demonstrating t(X;18), but it cannot distinguish which partner gene is involved for prognostic purposes. RNA from either frozen or paraffin-embedded tissue is suitable for rtPCR to detect and to distinguish the 2 common translocation variants.34 From a mechanistic standpoint, the translocation creates a chimeric gene encoding a fusion protein that redirects the transcription factor function of SS18. Relevant downstream targets include CCND1 (cyclin D1) that enhances cell cycle progression.RHABDOMYOSARCOMARhabdomyosarcoma is the most common soft tissue sarcoma in children and young adults, often presenting as a muscle mass in the extremities, paranasal sinus, or retroperitoneum. In alveolar rhabdomyosarcoma, expression profiling has identified 3 further subsets of patients having good, intermediate, and poor outcomes.4 Histologically, there is striated muscle differentiation with concomitant expression of vimentin, muscle-specific actin, desmin, myogenin, and MyoD1. PAX3 and PAX7 are transcription factors that initiate myogenesis in muscle stem cells, and the aberrant fusion of their DNA binding domain with the transactivation domain of FOXO1A creates a potent transcription factor that stimulates myogenesis and resists apoptosis. Prognosis is poor compared with embryonal rhabdomyosarcoma, justifying the effort to distinguish these 2 tumor types.In alveolar rhabdomyosarcomas, FISH using a break-apart probe identifies rearrangement of the FOXO1A gene.35,36 Karyotype typically reveals either t(2;13)(q35;q14) PAX3-FOXO1A or t(1;13)(p36;q14) PAX7-FOXO1A, with the latter being less common but imparting a better prognosis. It should be noted that alveolar rhabdomyosarcomas tend to have a poor prognosis overall, especially when presenting with disseminated disease, so genetic testing may be moot in stage IV patients. Testing is most useful when the histologic features are not classic (eg, mixed alveolar and embryonal patterns).DESMOPLASTIC ROUND CELL TUMORDesmoplastic round cell tumors usually harbor a recurring t(11;22) involving the EWSR1 gene on chromosome 22 and the WT1 (Wilms tumor 1) gene on the short arm of chromosome 11 (11p13). In contrast, the t(11;22) described above in association with Ewing sarcoma involves the FLI1 gene in the long arm of chromosome 11 (11q24). The appearance of the derivative chromosome 11, as analyzed by karyotype, can therefore distinguish these alterative gene partners. Another method to distinguish the 2 alternate gene partners for EWSR1 is cDNA amplification across the breakpoint of each fusion transcript. The fused EWSR1-WT1 chimeric protein is apparently expressed under control of the 5′ portion of the EWSR1 gene, whereas the 3′ end of WT1 functions as a transcription factor up-regulating oncogenic factors such as platelet-derived growth factor (PDGF).Clinically, desmoplastic round cell tumor tends to occur in the abdomen of adolescents and young adults where it behaves very aggressively despite an initial good response to chemotherapy. The fibrosis (ie, desmoplasia) that is typical of this subtype of sarcoma is probably a consequence of PDGF-related recruitment of fibroblasts, and PDGF inhibitor therapy is being explored as a novel treatment strategy. The immunophenotype can be confusing: multilineage differentiation can result in positivity for multiple immunohistochemical markers including WT1. When desmin is expressed, it can be especially difficult to distinguish this tumor from rhabdomyosarcoma unless genetic testing reveals the underlying gene defect.NEUROBLASTOMANeuroblastoma commonly presents as an adrenal or paraspinal mass. Prognosis relates to MYCN gene amplification status, which, in turn, influences the amount of MYCN protein produced by tumor cells. Patients with MYCN gene amplification (over 10 extra copies/cell; can be as high as 1000 copies/cell) are generally placed in a high-risk prognostic group, although other factors, such as age, stage, histology, and DNA ploidy, also impact prognosis. MYCN amplification can be detected by karyotype (double minutes or a homogeneously staining region), FISH, or quantitative PCR. Polysomy for chromosome 2 should be taken into account when determining if 10 extra copies of the MYCN gene are present in tumor cells. Concurrent or alternate genetic defects are common in neuroblastomas: deletion of 1p36 is associated with a poor prognosis, whereas aneuploidy confers a good prognosis.VIRUS-RELATED SARCOMASSome sarcomas harbor viral infection and are potentially treatable using antiviral strategies. For example, Kaposi sarcoma cells contain the human herpesvirus 8 (HHV8) genome, also known as Kaposi sarcoma-associated herpesvirus. Epstein-Barr virus (EBV) is found within the neoplastic cells of some cases of follicular dendritic cell sarcoma (particularly those arising in liver, spleen, or lymph node) and also in its benign counterpart, inflammatory myofibroblastic tumor. Expression of CD21, which functions as the surface receptor for EBV, by follicular dendritic cells suggests a route by which the virus enters the cell. Another subset of sarcomas strongly associated with EBV is leiomyosarcoma arising in immunocompromised individuals.37,38 Monoclonality of the EBV genomic structure in sarcoma tissue implies that viral infection occurred before malignant transformation, implying a role for EBV in sarcomagenesis.39,40In each of these sarcomas, localization of the virus to the malignant cells is confirmed by histochemical assays, such as HHV8 latency-associated nuclear antigen (LANA) immunohistochemistry or EBV EBER in situ hybridization.38,41–43 Infusion of virus-specific cytotoxic T cells that target infected cells for immune destruction is one potential therapeutic strategy, whereas drugs like ganciclovir or sirolimus (rapamycin) seem to operate by capitalizing on virus-induced biochemical effects.44–46 Quantitative PCR to measure viral load in blood samples is being explored as an indicator of tumor burden that facilitates early diagnosis, monitoring efficacy of therapy, and predicting relapse.47,48THERAPEUTIC IMPLICATIONS AND INNOVATIONSNew treatment approaches are being explored that take advantage of the unique fusion protein produced by various subtypes of sarcoma. In some studies, fusion protein-specific cytotoxic T cells were shown to lyse sarcoma cells, whereas interfering RNA has been used to inhibit transcription of the fusion gene.49,50 Other approaches are based on overcoming downstream biochemical pathways. For example, an activating mutation of the KIT gene (encoding CD117), or less commonly the PDGFRA gene in GIST, results in overactive tyrosine kinase signaling that can be overcome by treating with Gleevec, the same drug that is effective in chronic myelogenous leukemia.50,51 Translocations involving PDGFRA or PDGFRB can likewise result in Gleevec responsiveness in chronic myelomonocytic leukemia or chronic eosinophilic leukemia. These data illustrate how tumors of diverse histogenesis share common biochemical mechanisms and targeted treatments. Dermatofibrosarcoma protuberans is also Gleevec-responsive as a consequence of the COL1A1-PDGFB translocation that is visible on karyotype as either a t(17;22) or a supernumerary ring chromosome that harbors the translocation as verified by rtPCR or FISH.52Another example of the same biochemical pathway being altered in tumors of diverse histogenesis is the FUS-ERG translocation seen in rare Ewing sarcomas and in some myeloid leukemias. Interestingly, both the FUS and EWSR1 genes belong to the TET gene family, whereas their translocation partner genes, ERG and FLI1, encode ETS-like transcription factors. These parallels suggest that substitution of similar-functioning motifs in chimeric proteins can result in similar downstream effects. As the field of pharmacogenetics advances, it is likely that our concept of tumor classification will evolve as shared biochemical pathways and drug sensitivities are found among tumors of diverse morphology and anatomic site.53 In this regard, the pathologist may continue to rightly claim a critical role on the healthcare team by evaluating each lesion in a way that defines the particular therapeutic options that are most likely to benefit each patient.Analysis of tumor RNA using arrays that simultaneously test for expression of thousands of genes have helped define the biochemical pathways that are altered in various clinicopathologic subtypes of sarcoma. Such studies have identified novel tumor markers that assist in diagnosis when more practically analyzed by conventional methods such as immunohistochemistry or rtPCR.29,30,54 Furthermore, the biochemical pathways that are abnormal in a given tumor specimen might suggest an effective therapy. For example, ERBB2 (HER2) is expressed in a significant proportion of synovial sarcomas, so the effect of trastuzumab (Herceptin) anti-ERBB2 antibody therapy is worth exploring. Downstream activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway in MYCN-amplified tumors, including some neuroblastomas and alveolar rhabdomyosarcomas, may be overcome by mTOR inhibitors such as rapamycin.55 Likewise, Kaposi sarcoma is thought to respond to rapamycin because of HHV8-induced activation of the same PI3K/AKT/mTOR pathway.56,57 The convergence of multiple etiologies for cancer into a limited number of downstream pathways that can be targeted with existing drugs will accelerate the clinical trials process that is required to test the efficacy of selected drugs in patients whose laboratory tests suggest they are likely to respond.Although the genes located at translocation breakpoints are generally easy to find and characterize, much remains to be learned about the contribution of gene dosage to disease pathogenesis. Indeed, frequent detection of aneuploidy in sarcomas suggests that numeric gains or losses of certain genes contribute to disease pathogenesis. In fact, it is likely that multiple genetic defects accumulate during sarcomagenesis, and only the gross changes have been identified using standard laboratory procedures. Technologic advances in comparative genomic hybridization technology will allow for comprehensive array-based detection of not only the gross copy number changes, but also the subtle gene copy number variants that previously eluded us. These arrays, in combination with methylation arrays designed to detect epigenetic silencing of genes, will pinpoint the location of additional candidate genes contributing to tumor development or maintenance. This research will undoubtedly lead to improved diagnosis, prognosis, and therapy of affected patients.TISSUE ALLOCATION FOR MORPHOLOGY, IMMUNOPHENOTYPE, AND GENETIC TESTSHistologic examination is the mainstay of diagnostic pathology, and it is important that adequate tissue be submitted for light microscopy before allocating tissue for ancillary tests. When sarcoma is in the differential diagnosis, fresh tissue should be submitted for karyotype, if possible, as karyotype not only identifies clonal changes implying that neoplasia is present, but also provides a genome-wide screen of gross chromosomal alternations that may elucidate the pathologic subtype. During gross examination, tissue sterility should be maintained as much as possible. Tissue submitted for karyotype should be kept moist and unfixed until it can be submerged in an antibiotic-containing transport medium. It should then be maintained at room temperature until delivery to the cytogenetics laboratory, ideally within 24 hours of collection.Any residual cells remaining after karyotype have been fixed in Carnoy solution (methanol and acetic acid) and can be refrigerated so that further testing is possible once karyotype results are known. These residual alcohol-fixed cells are amenable to FISH or rtPCR days to years later. It is, therefore, worth finding out if such residual cells are available for subsequent molecular analysis.Touch preparations should be made, especially when sample size is limited, so that interphase FISH can be applied once it is clear which FISH assay(s) are most relevant. It is important that touch preparations be made carefully to ensure that single cell layers are present. Interphase FISH is easier to interpret on touch preparations because whole nuclei are at hand, as compared with paraffin sections in which parts of nuclei are trimmed away in the process of histologic sectioning. Caution is advised, however, when assaying touch preparations made from a tissue containing a small focal area of suspicious cells. In this instance, it may be more informative to assay a paraffin section in which the area(s) of interest have been identified by marking an adjacent hematoxylin and eosin-stained slide.Modern digital scanning methods have made it feasible to interpret immunohistochemistry and FISH assays performed sequentially on a single slide or smear, thus allowing direct comparison of protein expression and probe hybridization results in scanned images.58 Sequential staining is useful when limited slides are available for analysis or when the target cells are difficult to discern by fluorescence microscopy, but are readily identified by bright field microscopy using traditional histochemical methods.58 Dual immunofluorescent stains for both probe and antibody targets are also feasible.59Bright field (chromogenic) in situ hybridization methods are gaining popularity as a replacement for FISH, especially as the technology improves to permit multiplexing of 2 or more probes each of which has its own color signal and enhancements to reliably detect single-copy gene targets. The primary strength of this approach is the ability to simultaneously examine both genetic and cytopathologic or histopathologic findings. Bright field in situ hybridization may be less costly to perform as a standard microscope is used rather than a fluorescence microscope equipped with multiple filters. Although fluorescent signals fade, the chromogenic stain is permanent.An advantage of rtPCR is the exquisite sensitivity to low level disease, which could be helpful in staging the extent to which a tumor has spread or in monitoring minimal residual disease after therapy (Fig. 3). Fresh, frozen, and paraffin-embedded tissue and body fluids and fine needle aspirates are all amenable to rtPCR analysis to detect particular fusion transcripts. The same specimen types are amenable to DNA sequencing to confirm the specificity of an amplicon or to detect, for example, point mutations of KIT or PDGFRA in GIST. For any PCR or rtPCR test, fresh or frozen tissue is preferable to fixed tissue as formalin fixation crosslinks DNA and RNA, thus making it less available for hybridization.60JOURNAL/dimp/04.03/00019606-200903000-00001/figure3-1/v/2021-02-17T200000Z/r/image-jpeg Primitive neuroectodermal tumor characteristically harbors t(11;22) as shown in this case arising in the rib of a 17-year-old boy. A, The touch preparation of the rib mass stained with Diff-Quick reveals undifferentiated tumor cells. B, Pleural fluid contains cells suspicious for but not diagnostic of tumor involvement. C, Karyotype of the rib mass reveals t(11;22) and additional defects, for example, +8 and +12, that are presumably secondary events. D, FISH using an EWSR1 break-apart probe strategy reveals that the EWSR1 gene is rearranged in some pleural fluid cells (A, 400×; B, 100×; and D, 100×). FISH indicates fluorescence in situ hybridization.REPORTING GENETIC TEST RESULTSIt is important that ancillary test results (eg, karyotype, molecular, immunophenotype, and electron microscopy) be correlated with morphologic and clinical information to resolve discrepancies and to maximize the value of the consulting pathologist's findings.14 This synthesis is generally carried out by the ordering physician who is usually the surgical pathologist to whom the tissue specimen was initially referred by the clinician. A molecular genetic pathologist is also well suited to synthesize the diverse clinicopathologic findings. In reporting the findings, it is important to describe the clinical significance of any genetic test result in terms of the impact on diagnosis, prognosis, or prediction of outcome in response to therapy. Reports should list the genetic technologies that were applied and use correct nomenclature for describing the gene targets and results.14 Because modern nomenclature may be difficult for a clinician to decipher, a written explanation of the findings and their significance is essential. When pertinent, comment on limitations of the assays and on recommendations for resolving important gaps. The rapid progress that is being made in applying and interpreting genetic tests highlights the role of pathologists in managing sarcoma patients.REFERENCES1. Chang CC, Shidham VB. Molecular genetics of pediatric soft tissue tumors: clinical application. J Mol Diagn. 2003;5:143–154.[Context Link][CrossRef][Medline Link]2. Ladanyi M. Translocation-based molecular diagnosis of sarcomas. Am J Surg Pathol. 2003;27:414–415; author reply 415–416.[Context Link]3. 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.[Context Link][Medline Link]4. Davicioni E, Finckenstein FG, Shahbazian V, et al. Identification of a PAX-FKHR gene expression signature that defines molecular classes and determines the prognosis of alveolar rhabdomyosarcomas. Cancer Res. 2006;66:6936–6946.[Context Link][CrossRef][Medline Link]5. Schofield D, Triche TJ. cDNA microarray analysis of global gene expression in sarcomas. Curr Opin Oncol. 2002;14:406–411.[Context Link][Full Text][CrossRef][Medline Link]6. Hancock JD, Lessnick SL. A transcriptional profiling meta-analysis reveals a core EWS-FLI gene expression signature. Cell Cycle. 2008;7:250–256.[Context Link][CrossRef][Medline Link]7. Dumur CI, Lyons-Weiler M, Sciulli C, et al. Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers. J Mol Diagn. 2008;10:67–77.[Context Link][CrossRef][Medline Link]8. Singer S, Socci ND, Ambrosini G, et al. Gene expression profiling of liposarcoma identifies distinct biological types/subtypes and potential therapeutic targets in well-differentiated and dedifferentiated liposarcoma. Cancer Res. 2007;67:6626–6636.[Context Link][CrossRef][Medline Link]9. Tschoep K, Kohlmann A, Schlemmer M, et al. Gene expression profiling in sarcomas. Crit Rev Oncol Hematol. 2007;63:111–124.[Context Link][CrossRef][Medline Link]10. Jin L, Majerus J, Oliveira A, et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and rt-PCR. Diagn Mol Pathol. 2003;12:224–230.[Context Link][Full Text][CrossRef][Medline Link]11. Lewis TB, Coffin CM, Bernard PS. Differentiating Ewing's sarcoma from other round blue cell tumors using a RT-PCR translocation panel on formalin-fixed paraffin-embedded tissues. Mod Pathol. 2007;20:397–404.[Context Link][CrossRef][Medline Link]12. Athale UH, Shurtleff SA, Jenkins JJ, et al. Use of reverse transcriptase polymerase chain reaction for diagnosis and staging of alveolar rhabdomyosarcoma, Ewing sarcoma family of tumors, and desmoplastic small round cell tumor. J Pediatr Hematol Oncol. 2001;23:99–104.[Context Link][Full Text][Medline Link]13. Carpentieri DF, Qualman SJ, Bowen J, et al. Protocol for the examination of specimens from pediatric and adult patients with osseous and extraosseous Ewing sarcoma family of tumors, including peripheral primitive neuroectodermal tumor and Ewing sarcoma. Arch Pathol Lab Med. 2005;129:866–873.[Context Link][CrossRef][Medline Link]14. Gulley ML, Braziel RM, Halling KC, et al. Clinical laboratory reports in molecular pathology. Arch Pathol Lab Med. 2007;131:852–863.[Context Link][Medline Link]15. Olsen SH, Thomas DG, Lucas DR. Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma. Mod Pathol. 2006;19:659–668.[Context Link][CrossRef][Medline Link]16. Xia SJ, Barr FG. Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors. Eur J Cancer. 2005;41:2513–2527.[Context Link][CrossRef][Medline Link]17. Huang HY, Illei PB, Zhao Z, et al. Ewing sarcomas with p53 mutation or p16/p14ARF homozygous deletion: a highly lethal subset associated with poor chemoresponse. J Clin Oncol. 2005;23:548–558.[Context Link][Full Text][CrossRef][Medline Link]18. Kilpatrick SE, Bergman S, Pettenati MJ, et al. The usefulness of cytogenetic analysis in fine needle aspirates for the histologic subtyping of sarcomas. Mod Pathol. 2006;19:815–819.[Context Link][CrossRef][Medline Link]19. Patel RM, Downs-Kelly E, Weiss SW, et al. Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma. Mod Pathol. 2005;18:1585–1590.[Context Link][CrossRef][Medline Link]20. Wang L, Bhargava R, Zheng T, et al. Undifferentiated small round cell sarcomas with rare EWS gene fusions: identification of a novel EWS-SP3 fusion and of additional cases with the EWS-ETV1 and EWS-FEV fusions. J Mol Diagn. 2007;9:498–509.[Context Link][CrossRef][Medline Link]21. Bode-Lesniewska B, Frigerio S, Exner U, et al. Relevance of translocation type in myxoid liposarcoma and identification of a novel EWSR1-DDIT3 fusion. Genes Chromosomes Cancer. 2007;46:961–971.[Context Link][CrossRef][Medline Link]22. Barr FG, Womer RB. Molecular diagnosis of ewing family tumors: too many fusions? J Mol Diagn. 2007;9:437–440.[Context Link][CrossRef][Medline Link]23. Qian X, Jin L, Shearer BM, et al. Molecular diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization. Diagn Mol Pathol. 2005;14:23–28.[Context Link][Full Text][CrossRef][Medline Link]24. Lin PP, Brody RI, Hamelin AC, et al. Differential transactivation by alternative EWS-FLI1 fusion proteins correlates with clinical heterogeneity in Ewing's sarcoma. Cancer Res. 1999;59:1428–1432.[Context Link][Medline Link]25. Schleiermacher G, Peter M, Oberlin O, et al. Increased risk of systemic relapses associated with bone marrow micrometastasis and circulating tumor cells in localized Ewing tumor. J Clin Oncol. 2003;21:85–91.[Context Link][Full Text][CrossRef][Medline Link]26. Vermeulen J, Ballet S, Oberlin O, et al. Incidence and prognostic value of tumour cells detected by RT-PCR in peripheral blood stem cell collections from patients with Ewing tumour. Br J Cancer. 2006;95:1326–1333.[Context Link][CrossRef][Medline Link]27. Bridge RS, Rajaram V, Dehner LP, et al. Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors. Mod Pathol. 2006;19:1–8.[Context Link][CrossRef][Medline Link]28. Friedrichs N, Kriegl L, Poremba C, et al. Pitfalls in the detection of t(11;22) translocation by fluorescence in situ hybridization and RT-PCR: a single-blinded study. Diagn Mol Pathol. 2006;15:83–89.[Context Link][Full Text][CrossRef][Medline Link]29. Bahrami A, Truong LD, Ro JY. Undifferentiated tumor: true identity by immunohistochemistry. Arch Pathol Lab Med. 2008;132:326–348.[Context Link][CrossRef][Medline Link]30. Heim-Hall J, Yohe SL. Application of immunohistochemistry to soft tissue neoplasms. Arch Pathol Lab Med. 2008;132:476–489.[Context Link][CrossRef][Medline Link]31. Downs-Kelly E, Goldblum JR, Patel RM, et al. The utility of fluorescence in situ hybridization (FISH) in the diagnosis of myxoid soft tissue neoplasms. Am J Surg Pathol. 2008;32:8–13.[Context Link][Full Text][Medline Link]32. Hameed M. Small round cell tumors of bone. Arch Pathol Lab Med. 2007;131:192–204.[Context Link][Medline Link]33. Wang LL, Perlman EJ, Vujanic GM, et al. Desmoplastic small round cell tumor of the kidney in childhood. Am J Surg Pathol. 2007;31:576–584.[Context Link][Full Text][CrossRef][Medline Link]34. Hill DA, Riedley SE, Patel AR, et al. Real-time polymerase chain reaction as an aid for the detection of SYT-SSX1 and SYT-SSX2 transcripts in fresh and archival pediatric synovial sarcoma specimens: report of 25 cases from St. Jude Children's Research Hospital. Pediatr Dev Pathol. 2003;6:24–34.[Context Link][Full Text][CrossRef][Medline Link]35. Mehra S, de la Roza G, Tull J, et al. Detection of FOXO1 (FKHR) gene break-apart by fluorescence in situ hybridization in formalin-fixed, paraffin-embedded alveolar rhabdomyosarcomas and its clinicopathologic correlation. Diagn Mol Pathol. 2008;17:14–20.[Context Link][Full Text][CrossRef][Medline Link]36. Matsumura T, Yamaguchi T, Seki K, et al. Advantage of FISH analysis using FKHR probes for an adjunct to diagnosis of rhabdomyosarcomas. Virchows Arch. 2008;452:251–258.[Context Link][CrossRef][Medline Link]37. Yamamoto H, Kohashi K, Oda Y, et al. Absence of human herpesvirus-8 and Epstein-Barr virus in inflammatory myofibroblastic tumor with anaplastic large cell lymphoma kinase fusion gene. Pathol Int. 2006;56:584–590.[Context Link][Full Text][CrossRef][Medline Link]38. Gulley ML. Molecular diagnosis of Epstein-Barr virus-related diseases. J Mol Diagn. 2001;3:1–10.[Context Link][CrossRef][Medline Link]39. Shek TW, Ho FC, Ng IO, et al. Follicular dendritic cell tumor of the liver. Evidence for an Epstein-Barr virus-related clonal proliferation of follicular dendritic cells. Am J Surg Pathol. 1996;20:313–324.[Context Link][Full Text][CrossRef][Medline Link]40. Jenson HB, Leach CT, McClain KL, et al. Benign and malignant smooth muscle tumors containing Epstein-Barr virus in children with AIDS. Leuk Lymphoma. 1997;27:303–314.[Context Link][CrossRef][Medline Link]41. Ryan JL, Fan H, Glaser SL, et al. Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments: a novel approach to screen for the virus in paraffin-embedded tissue and plasma. J Mol Diagn. 2004;6:378–385.[Context Link][CrossRef][Medline Link]42. Mergan F, Jaubert F, Sauvat F, et al. Inflammatory myofibroblastic tumor in children: clinical review with anaplastic lymphoma kinase, Epstein-Barr virus, and human herpesvirus 8 detection analysis. J Pediatr Surg. 2005;40:1581–1586.[Context Link][CrossRef][Medline Link]43. Cesarman E, Mesri EA. Kaposi sarcoma-associated herpesvirus and other viruses in human lymphomagenesis. Curr Top Microbiol Immunol. 2007;312:263–287.[Context Link][Medline Link]44. Bonatti H, Hoefer D, Rogatsch H, et al. Successful management of recurrent Epstein-Barr virus-associated multilocular leiomyosarcoma after cardiac transplantation. Transplant Proc. 2005;37:1839–1844.[Context Link][CrossRef][Medline Link]45. Stallone G, Schena A, Infante B, et al. Sirolimus for Kaposi's sarcoma in renal-transplant recipients. N Engl J Med. 2005;352:1317–1323.[Context Link][Full Text][CrossRef][Medline Link]46. Israel BF, Kenney SC. Virally targeted therapies for EBV-associated malignancies. Oncogene. 2003;22:5122–5130.[Context Link][Full Text][CrossRef][Medline Link]47. Pan L, Milligan L, Michaeli J, et al. Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma. J Mol Diagn. 2001;3:32–38.[Context Link][CrossRef][Medline Link]48. Fan H, Gulley ML. Epstein-Barr viral load measurement as a marker of EBV-related disease. Mol Diagn. 2001;6:279–289.[Context Link][Full Text][CrossRef][Medline Link]49. Hu-Lieskovan S, Heidel JD, Bartlett DW, et al. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res. 2005;65:8984–8992.[Context Link][CrossRef][Medline Link]50. Borden EC, Baker LH, Bell RS, et al. Soft tissue sarcomas of adults: state of the translational science. Clin Cancer Res. 2003;9:1941–1956.[Context Link][Medline Link]51. Uren A, Toretsky JA. Pediatric malignancies provide unique cancer therapy targets. Curr Opin Pediatr. 2005;17:14–19.[Context Link][Full Text][CrossRef][Medline Link]52. Patel KU, Szabo SS, Hernandez VS, et al. Dermatofibrosarcoma protuberans COL1A1-PDGFB fusion is identified in virtually all dermatofibrosarcoma protuberans cases when investigated by newly developed multiplex reverse transcription polymerase chain reaction and fluorescence in situ hybridization assays. Hum Pathol. 2008;39:184–193.[Context Link][CrossRef][Medline Link]53. Barr FG, Zhang PJ. The impact of genetics on sarcoma diagnosis: an evolving science. Clin Cancer Res. 2006;12:5256–5257.[Context Link][Medline Link]54. Chen QR, Vansant G, Oades K, et al. Diagnosis of the small round blue cell tumors using multiplex polymerase chain reaction. J Mol Diagn. 2007;9:80–88.[Context Link][CrossRef][Medline Link]55. Johnsen JI, Segerstrom L, Orrego A, et al. Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo. Oncogene. 2008;27:2910–2922.[Context Link][Full Text][CrossRef][Medline Link]56. Montaner S. Akt/TSC/mTOR activation by the KSHV G protein-coupled receptor: emerging insights into the molecular oncogenesis and treatment of Kaposi's sarcoma. Cell Cycle. 2007;6:438–443.[Context Link][CrossRef][Medline Link]57. Dittmer DP, Krown SE. Targeted therapy for Kaposi's sarcoma and Kaposi's sarcoma-associated herpesvirus. Curr Opin Oncol. 2007;19:452–457.[Context Link][Full Text][CrossRef][Medline Link]58. Bedell V, Forman SJ, Gaal K, et al. Successful application of a direct detection slide-based sequential phenotype/genotype assay using archived bone marrow smears and paraffin embedded tissue sections. J Mol Diagn. 2007;9:589–597.[Context Link][CrossRef][Medline Link]59. Bzorek M Sr, Petersen BL, Hansen L. Simultaneous phenotyping and genotyping (FICTION-methodology) on paraffin sections and cytologic specimens: a comparison of 2 different protocols. Appl Immunohistochem Mol Morphol. 2008;16:279–286.[Context Link][Full Text][CrossRef][Medline Link]60. Hill DA, O'Sullivan MJ, Zhu X, et al. Practical application of molecular genetic testing as an aid to the surgical pathologic diagnosis of sarcomas: a prospective study. 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Among cases of Ewing sarcoma, breakpoints (shown as blue arrows) within EWSR1 are clustered in introns 7 to 10, whereas the FLI1 breakpoints are variable but commonly involve introns 4 or 5. At the DNA level, PCR amplification across the translocated breakpoint would require many primer sets to detect all possible translocation variants. However, after transcription and processing to splice out the introns and form mature mRNA, there are relatively limited breakpoint variants that can be tested for by rtPCR. The most common fusion transcripts are detected by extracting RNA from the patient specimen, reverse transcribing the RNA to cDNA, and then applying rtPCR using a forward primer targeting EWSR1 exon 7 and a reverse primer targeting FLI1 exon 6. PCR indicates polymerase chain reaction; rtPCR, reverse transcription polymerase chain reaction. FISH detects rearrangement of the EWSR1 gene at 22q12. The diagram on the left depicts the EWSR1 gene and a break-apart probe strategy whereby the 2 probes flanking this gene are labeled with red and green fluorochromes, respectively. In a normal cell, these 2 probes colocalize such that the red and green signals merge and appear yellow at each of the 2 normal EWSR1 alleles. In the presence of EWSR1 gene rearrangement, however, these probes separate to form independent red and green fluorescent signals that represent the derivative chromosome 22 and the derivative partner chromosome, respectively. Not shown is metaphase FISH analysis in which the reciprocal translocation partner (such as 11q24 where the FLI1 gene resides) is pinpointed by the green probe in G-banded chromosomes. In yet another type of analysis not shown (called the “single fusion” probe strategy), a red EWSR1 probe similar to that depicted above and a green probe targeting the FLI1 locus are cohybridized such that, in the presence of the 11;22 translocation, 1 red signal and 1 green signal join to produce a yellow fusion signal on the abnormal (derivative) chromosome 22. Further description of “single fusion” or “dual fusion” FISH probe strategies is not relevant to the current discussion as these analyses are generally only available in research laboratories. FISH indicates fluorescence in situ hybridization. Primitive neuroectodermal tumor characteristically harbors t(11;22) as shown in this case arising in the rib of a 17-year-old boy. A, The touch preparation of the rib mass stained with Diff-Quick reveals undifferentiated tumor cells. B, Pleural fluid contains cells suspicious for but not diagnostic of tumor involvement. C, Karyotype of the rib mass reveals t(11;22) and additional defects, for example, +8 and +12, that are presumably secondary events. D, FISH using an EWSR1 break-apart probe strategy reveals that the EWSR1 gene is rearranged in some pleural fluid cells (A, 400×; B, 100×; and D, 100×). FISH indicates fluorescence in situ hybridization.A Rational Approach to Genetic Testing for SarcomaGulley Margaret L. MD; Kaiser-Rogers, Kathleen A. PhDReview ArticleReview Article118p 1-10

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