—  SYMPOSIUM #21  —

The Role of Ancillary Techniques in the Assessment of Soft Tissue Tumors
Moderators: Dr. John R. Goldblum and Dr. Cyril Fisher

Section 2 - Molecular Diagnosis of Soft Tissue Tumors

Marc Ladanyi
Memorial Sloan-Kettering Cancer Center
New York , NY , USA


Overview
Certain sarcomas are characterized by specific recurrent chromosomal translocations, biologically similar to those seen in leukemias. These chromosomal translocations produce highly specific gene fusions, of which the specificity for and prevalence in selected sarcomas are such that they have come to define these entities [1, 2, 3, 4] . Two key concepts in translocation sarcomas are first, that these sarcomas contain their fusion gene from their earliest presentation and do not show benign or premalignant phase; and secondly, the fusion gene is present in all tumor cells and is expressed throughout the clinical course. The pathobiology of the oncogenic fusion proteins involves in almost all instances either transcriptional deregulation (most) or aberrant signaling (some). The fusion genes produced by these translocations most often encode chimeric transcription factors that cause transcriptional deregulation [2] . In broad terms, chimeric transcription factors are thought to deregulate the expression of specific repertoires of target genes, possibly providing multiple oncogenic hits analogous to the multistep process of epithelial carcinogenesis.

All of the major cytogenetically described translocations in sarcomas have by now been cloned [2] . A recent study that used network modeling to analyze the relationship of all known gene fusions in human cancer suggests that it is likely that all or most genes involved in multiple gene fusions (such as EWS, TFE3, MLL) have already been identified and that only rare or variant gene fusions remain to be identified [5] . Indeed, uncommon gene fusions, some of them variants of known fusions, continue to be described. For instance, rare "Ewing sarcoma-like tumors" have recently been reported with the following fusions: EWS-POU5F1 [6] , CIC-DUX4 [7] , and BRD4-NUT [8] . The latter fusion is not novel as it is already know to be associated with aggressive midline carcinomas [9] . In addition, we have recently identified a novel EWS-SP3 fusion in a " Ewing sarcoma-like tumor" (L. Wang, M. Ladanyi, et al., submitted). Other recently described variants of known sarcoma fusions include PAX-NCOA1 in alveolar rhabdomyosarcoma [10] and EWS-CREB1 in clear cell sarcoma [11] .

Regarding the perennial question of how and why translocations arise [12, 13] , an interesting recent bioinformatic analysis of the sequence and structure of all genes involved in translocations provides compelling new insights [14] . Comparing 268 genes involved in translocations to 9406 control genes, the authors found striking differences in overall gene size, average intron length, and length of the longest intron, all three of which were significantly greater in the former group. They did not find any differences in the presence of so-called recombinogenic sequence elements. These data support the concept that the intronic breaks that lead to specific recurrent chromosomal translocations in cancer are largely random events (risk of breaks is simply proportional to intron length) that become fixed through natural selection if they provide a growth advantage to the cell. Other factors that can be incorporated into this model include the increased "availability" of the genes for rearrangement that is created by the open chromatin conformation associated with gene transcription or replication [15] , and the unexpected proximity of some translocation partner genes due to the 3 dimensional arrangement of chromosomes in the nucleus. Evidence for the latter phenomenon has been presented in hematologic and thyroid cancers [16, 17] , but not yet in sarcomas.

A Broader Role for Gene Fusions in Human Cancers?
Specific intergenic rearrangements or gene fusions represent, in terms of numbers of different genes involved, the single most common type of somatic genetic alteration in human cancer. Over 75% of genes somatically altered in human tumors are translocated [18] . Biologically, these gene fusions operate either by promoter substitution or the formation of aberrant chimeric proteins. Although these types of translocations have been historically associated mainly with leukemias, lymphomas, and sarcomas, and have been less often detected in carcinomas, Mitelman and colleagues have provocatively argued that this skewed distribution may be at least partly artefactual [19] . Their argument is based on the observation that in every tumor type, the numbers of specific translocations described is proportional to the number of cases successfully karyotyped. Carcinomas, being far more difficult to karyotype than leukemias, lymphomas, and sarcomas, have so far yielded few specific translocations. Until recently, such translocations had only been described in select types of less common carcinomas, including thyroid cancers [20, 21] , certain types of renal adenocarcinomas [22, 23, 24, 25] , as well as a few other rare carcinomas [9, 26] . However, this field changed dramatically in late 2005 with the first report of recurrent fusions involving ERG and other ETS family genes in prostate cancer [27] . The most common fusion, TMPRSS2-ERG, is present in approximately 50% of prostate cancers, a striking proportion confirmed by others [28] . These findings have invigorated the notion proposed by Mitelman and colleagues that many more fusion genes remain to be found within the complex karyotypes of carcinomas [19] .

Molecular Diagnosis of Sarcoma Translocations – Practical Aspects
The necessity of molecular testing in modern sarcoma diagnosis has recently been the subject of instructive studies [29, 30]. Based on these studies, molecular confirmation of translocation status seems more often useful in diagnosing synovial sarcomas (useful in approx. 50%) than Ewing sarcomas (useful in approx. 10%). The two main diagnostic methods for sarcoma translocations are reverse-transcriptase PCR (RT-PCR) and fluorescent in situ hybridization (FISH) [1]. RT-PCR, as an RNA-based assay, is susceptible to failure due to poor RNA quality. As a PCR-based assay, it is also susceptible to false-positives due to PCR cross-contamination. It is critical to include two types of contamination controls: controls lacking only the template RNA (to detect contamination of the PCR reagents) and controls lacking only the reverse transcriptase (to detect contamination of the patient RNA sample). Although RT-PCR is more sensitive and provides more detailed fusion information than FISH, it is less adaptable to paraffin material and frozen tissue is definitely preferred. Another important consideration for RT-PCR assays is that they require extensive knowledge of the specific exons involved by the gene fusions and of the variability in exon composition of some fusions. Recently, real-time RT-PCR, which employs highly sensitive fluorescent detection of PCR products as they are generated ("in real-time"), has emerged as an improved strategy for RT-PCR detection of sarcoma fusion transcripts in archival pathology material [31], with the added benefit of avoiding the cross-contamination risks of other high sensitivity PCR approaches such as nested PCR.

FISH is a DNA-based assay. Given the relatively better preservation of DNA than RNA in paraffin material, it is generally more adaptable to paraffin material than RT-PCR, yet frozen tissue is also preferable for FISH. False-positives in FISH can arise from overinterpretation [32]. Split signal FISH assays can be overinterpreted because of occasional separation of the signals in some normal cells. Fused signal FISH assays are susceptible to false-positives due to occasional random juxtaposition of signals. In both types of FISH assays, conservative thresholds for positivity need to be carefully established [32]. Several sarcoma FISH probe pairs are commercially available, including EWS, FUS(TLS), CHOP, FKHR, and SYT (Abbott-Vysis , USA ). EWS and SYT probe pairs labeled for chromogenic in situ hybridization (CISH) are also on the market ( Invitrogen, USA). These probe pairs are all based on a split signal FISH assay design and thus only document gene rearrangement, not specific gene fusions.

RT-PCR and FISH are complementary methods for detecting sarcoma translocations. The use of one or the other as the first line approach often reflects differences in local expertise. Most studies comparing both techniques in the sarcoma setting suggest that optimal diagnostic accuracy can be achieved when both are available [32, 33, 34, 35]. The College of American Pathologists now offers regular proficiency testing for sarcoma translocation detection by FISH and RT-PCR.

Translating Molecular Diagnostic Markers into Ihc Markers
Many translocations can be converted into IHC assays based on the phenomenon of discordance in the expression levels of the amino- and carboxy-terminal ends of the product of gene B in tumors with A-B gene fusions, or the markedly aberrant expression of the carboxy-terminal encoded by gene B in the context of these fusion proteins, or their expression in aberrant cell types or aberrant cellular compartments. This has been confirmed by westerns and has been used as a basis for the IHC detection of oncogenic fusion proteins [36]. For instance, we have established the latter approach for the detection of the EWS-WT1 protein [37] and fusions involving TFE3 and TFEB [25, 38]. Inflammatory myofibroblastic tumors harbor a variety of ALK gene fusions in many or most cases, and IHC for the ALK carboxy-terminal end has become a widely used adjunct in diagnosis [39, 40, 41]. Another approach for converting molecular translocation detection into an IHC assay is provided by certain gene fusions where a 5' exon of gene B that is not normally translated becomes translated in the context of the fusion protein and therefore represents a novel peptide sequence for antibody generation. This approach has recently been demonstrated for TLS-CHOP and EWS-CHOP detection in myxoid liposarcomas [42].

Other Types of Molecular Diagnostic Markers in Sarcomas
Although recurrent translocations are by far the most widely used markers, certain other genetic alterations are so closely associated with specific sarcomas that they can also form the basis for confirmatory assays. This is the case with KIT (and PDGFRA) mutations in GISTs [43]. However, KIT immunoreactivity in GIST is related to tumor cell lineage more than to the underlying mutation. Co-amplification of MDM2 and CDK4 in well-differentiated and dedifferentiated liposarcomas due to 12q amplification has also emerged as a potentially useful marker in certain settings [44, 45]. Recently, this has been translated into a robust IHC assay [46, 47]. Finally, mutations involving the WNT pathway (APC or beta-catenin) are characteristic of (but not entirely specific for) aggressive desmoid-type fibromatosis. These result in nuclear beta-catenin accumulation readily detected by IHC [48, 49].

References
  1. Ladanyi M, Bridge JA. Contribution of molecular genetic data to the classification of sarcomas. Hum Pathol 2000;31:532-8.

  2. Xia SJ, Barr FG. Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors. Eur J Cancer 2005;41:2513-27.

  3. Antonescu CR. The role of genetic testing in soft tissue sarcoma. Histopathology 2006;48:13-21.

  4. Bennicelli JL, Barr FG. Chromosomal translocations and sarcomas. Curr Opin Oncol 2002;14:412-9.

  5. Hoglund M, Frigyesi A, Mitelman F. A gene fusion network in human neoplasia. Oncogene 2006;25:2674-8.

  6. Yamaguchi S, Yamazaki Y, Ishikawa Y, Kawaguchi N, Mukai H, Nakamura T. EWSR1 is fused to POU5F1 in a bone tumor with translocation t(6;22)(p21;q12). Genes Chromosomes Cancer 2005;43:217-22.

  7. Kawamura-Saito M, Yamazaki Y, Kaneko K, Kawaguchi N, Kanda H, Mukai H, Gotoh T, Motoi T, Fukayama M, Aburatani H, Takizawa T, Nakamura T. 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 (in press)

  8. Mertens F, Wiebe T, Adlercreutz C, Mandahl N, French CA. Successful treatment of a child with t(15;19)-positive tumor. Pediatr Blood Cancer 2006;.

  9. French CA, Miyoshi I, Kubonishi I, Grier HE, Perez-Atayde AR, Fletcher JA. BRD4-NUT fusion oncogene: a novel mechanism in aggressive carcinoma. Cancer Res 2003;63:304-7.

  10. Wachtel M, Dettling M, Koscielniak E, Stegmaier S, Treuner J, Simon-Klingenstein K, Buhlmann P, Niggli FK, Schafer BW. Gene expression signatures identify rhabdomyosarcoma subtypes and detect a novel t(2;2)(q35;p23) translocation fusing PAX3 to NCOA1. Cancer Res 2004;64:5539-45.

  11. Antonescu CR, Nafa K, Segal NH, Dal Cin P, Ladanyi M. EWS-CREB1: A recurrent variant fusion in clear cell sarcoma. Association with gastrointestinal location and absence of melanocytic differentiation. Clin Cancer Res 2006 (in press).

  12. Zucman-Rossi J, Legoix P, Victor JM, Lopez B, Thomas G. Chromosome translocation based on illegitimate recombination in human tumors. Proc Natl Acad Sci U S A 1998;95:11786-91.

  13. Aplan PD. Causes of oncogenic chromosomal translocation. Trends Genet 2006;22:46-55.

  14. Novo FJ, Vizmanos JL. Chromosome translocations in cancer: computational evidence for the random generation of double-strand breaks. Trends Genet 2006;22:193-6.

  15. Chuang CH, Belmont AS. Close encounters between active genes in the nucleus. Genome Biol 2005;6:237.

  16. Roix JJ, McQueen PG, Munson PJ, Parada LA, Misteli T. Spatial proximity of translocation-prone gene loci in human lymphomas. Nat Genet 2003;34:287-91.

  17. Nikiforova MN, Stringer JR, Blough R, Medvedovic M, Fagin JA, Nikiforov YE. Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science 2000;290:138-41.

  18. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, Rahman N, Stratton MR. A census of human cancer genes. Nat Rev Cancer 2004;4:177-83.

  19. Mitelman F, Johansson B, Mertens F. Fusion genes and rearranged genes as a linear function of chromosome aberrations in cancer. Nat Genet 2004;36:331-4.

  20. Tallini G, Asa SL. RET oncogene activation in papillary thyroid carcinoma. Adv Anat Pathol 2001;8:345-54.

  21. Kroll TG, Sarraf P, Pecciarini L, Chen CJ, Mueller E, Spiegelman BM, Fletcher JA. PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma. Science 2000;289:1357-60.

  22. Argani P, Antonescu CR, Illei PB, Lui MY, Timmons CF, Newbury R, Reuter VE, Garvin AJ, Perez-Atayde AR, Fletcher JA, Beckwith JB, Bridge JA, Ladanyi M. Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumor entity previously included among renal cell carcinomas of children and adolescents. Am J Pathol 2001;159:179-92.

  23. Argani P, Antonescu CR, Couturier J, Fournet JC, Sciot R, biec-Rychter M, Hutchinson B, Reuter VE, Boccon-Gibod L, Timmons CF, Hafez N, Ladanyi M. PRCC-TFE3 renal tumors: morphologic, immunohistochemical, ultrastructural and molecular analysis of an entity associated with the t(X;1)(p11.2;q21). Am J Surg Pathol 2002;26:1553-66.

  24. Argani P, Lui MY, Couturier J, Fournet JC, Fournet JC, Ladanyi M. A novel CLTC-TFE3 gene fusion in pediatric renal adenocarcinoma with t(X;17)(p11.2;q23). Oncogene 2003;22:5374-8.

  25. Argani P, Lae M, Hutchinson B, Reuter VE, Collins MH, Perentesis J, Tomaszewski JE, Brooks JS, Acs G, Bridge JA, Vargas SO, Davis IJ, Fisher DE, Ladanyi M. Renal Carcinomas With the t(6;11)(p21;q12): Clinicopathologic Features and Demonstration of the Specific Alpha-TFEB Gene Fusion by Immunohistochemistry, RT-PCR, and DNA PCR. Am J Surg Pathol 2005;29:230-40.

  26. Tonon G, Modi S, Wu L, Kubo A, Coxon AB, Komiya T, O'Neil K, Stover K, El Naggar A, Griffin JD, Kirsch IR, Kaye FJ. t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nat Genet 2003;33:208-13.

  27. Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, Varambally S, Cao X, Tchinda J, Kuefer R, Lee C, Montie JE, Shah RB, Pienta KJ, Rubin MA, Chinnaiyan AM. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005;310:644-8.

  28. Soller MJ, Isaksson M, Elfving P, Soller W, Lundgren R, Panagopoulos I. Confirmation of the high frequency of the TMPRSS2/ERG fusion gene in prostate cancer. Genes Chromos Cancer 2006;.

  29. Coindre JM, Pelmus M, Hostein I, Lussan C, Bui BN, Guillou L. Should molecular testing be required for diagnosing synovial sarcoma? A prospective study of 204 cases. Cancer 2003;98:2700-7.

  30. Folpe AL, Goldblum JR, Rubin BP, Shehata BM, Liu W, Dei Tos AP, Weiss SW. Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases. Am J Surg Pathol 2005;29:1025-33.

  31. Hostein I, Menard A, Bui BN, Lussan C, Wafflart J, Delattre O, Peter M, Benhattar J, Guillou L, Coindre JM. Molecular detection of the synovial sarcoma translocation t(X;18) by real-time polymerase chain reaction in paraffin-embedded material. Diagn Mol Pathol 2002;11:16-21.

  32. Friedrichs N, Kriegl L, Poremba C, Schaefer KL, Gabbert HE, Shimomura A, Paggen E, Merkelbach-Bruse S, Buettner R. Pitfalls in the Detection of t(11;22) Translocation by fluores-cence in situ hybridization and RT-PCR. A single-binded study. Diag Mol Pathol 2006;15:83-9.

  33. Qian X, Jin L, Shearer BM, Ketterling RP, Jalal SM, Lloyd RV. 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;14:23-8.

  34. Bridge RS, Rajaram V, Dehner LP, Pfeifer JD, Perry A. 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.

  35. Nishio J, Althof PA, Bailey JM, Zhou M, Neff JR, Barr FG, Parham DM, Teot L, Qualman SJ, Bridge JA. Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma. Lab Invest 2006;86:547-56.

  36. Falini B, Mason DY. Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry. Blood 2002;99:409-26.

  37. Gerald WL, Ladanyi M, de Alava E, Cuatrecasas M, Kushner BH, La Quaglia MP, Rosai J. Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round-cell tumor and its variants. J Clin Oncol 1998;16:3028-36.

  38. Argani P, Lal P, Hutchinson B, Lui MY, Reuter VE, Ladanyi M. Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions. A sensitive and specific immunohistochemical assay. Am J Surg Pathol 2003;23:750-61.

  39. Coffin CM, Patel A, Perkins S, Elenitoba-Johnson KS, Perlman E, Griffin CA. ALK1 and p80 expression and chromosomal rearrangements involving 2p23 in inflammatory myofibroblastic tumor. Mod Pathol 2001;14:569-76.

  40. Cook JR, Dehner LP, Collins MH, Ma Z, Morris SW, Coffin CM, Hill DA. Anaplastic lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: a comparative immunohistochemical study. Am J Surg Pathol 2001;25:1364-71.

  41. Li XQ, Hisaoka M, Shi DR, Zhu XZ, Hashimoto H. Expression of anaplastic lymphoma kinase in soft tissue tumors: an immunohistochemical and molecular study of 249 cases. Hum Pathol 2004;35:711-21.

  42. Oikawa K, Ishida T, Imamura T, Yoshida K, Takanashi M, Hattori H, Ishikawa A, Fujita K, Yamamoto K, Matsubayashi J, Kuroda M, Mukai K. Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas. Am J Surg Pathol 2006;30:351-6.

  43. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol 2004;22:3813-25.

  44. Dei Tos AP, Doglioni C, Piccinin S, Sciot R, Furlanetto A, Boiocchi M, Dal CP, Maestro R, Fletcher CD, Tallini G. Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours. J Pathol 2000;190:531-6.

  45. Hostein I, Pelmus M, Aurias A, Pedeutour F, Mathoulin-Pelissier S, Coindre JM. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas. J Pathol 2004;202:95-102.

  46. Binh MB, Sastre-Garau X, Guillou L, de PG, Terrier P, Lagace R, Aurias A, Hostein I, Coindre JM. 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;29:1340-7.

  47. Binh MB, Garau XS, Guillou L, Aurias A, Coindre JM. Reproducibility of MDM2 and CDK4 staining in soft tissue tumors. Am J Clin Pathol 2006;125:693-7.

  48. Ng TL, Gown AM, Barry TS, Cheang MC, Chan AK, Turbin DA, Hsu FD, West RB, Nielsen TO. Nuclear beta-catenin in mesenchymal tumors. Mod Pathol 2005;18:68-74.

  49. Bhattacharya B, Dilworth HP, Iacobuzio-Donahue C, Ricci F, Weber K, Furlong MA, Fisher C, Montgomery E. Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions. Am J Surg Pathol 2005;29:653-9.