Molecular Assays for Chromosomal Translocations in the Diagnosis of Pediatric Soft Tissue Sarcomas

Barr, Frederic G.; Chatten, Jane; D’Cruz, Celina M.; Wilson, Albert E.; Nauta, Lauren E.; Nycum, Lynn M.; Biegel, Jaclyn A.; Womer, Richard B.

doi:10.1001/jama.1995.03520310051029

Cited by 171 publications

(84 citation statements)

References 22 publications

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“…Similarly, several cases of ARMS have previously been reported that failed to demonstrate tumor-specific chromosomal translocations on cytogenetic analysis, but were shown to express PAX-FKHR fusion transcripts by RT-PCR and/or FISH. [24][25][26] Possible explanations for these findings are the difficulty of identifying the small derivative chromosome 13 or the presence of a cryptic translocation. FISH and RT-PCR analyses are valuable techniques that should be performed to detect the presence of tumor-specific chromosomal translocations or associated fusion gene transcripts even when cytogenetic analysis is successful.…”

Section: Discussionmentioning

confidence: 99%

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma

Nishio

Althof

Bailey

et al. 2006

Laboratory Investigation

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A valuable diagnostic adjunct and important prognostic parameter in alveolar rhabdomyosarcoma (ARMS) is the identification of translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), and the associated PAX3-FKHR and PAX7-FKHR fusion transcripts, respectively. Most RMS fusion gene type studies have been based on reverse transcriptase-polymerase chain reaction (RT-PCR) detection of the fusion transcript, a technique limited by RNA quality and failure of devised primer sets to detect unusual variants. As an alternative approach, we developed a fluorescence in situ hybridization (FISH) assay that can: (1) distinguish between the two most common ARMS-associated fusion genes; (2) identify potential unusual variant translocations; (3) assess histologic components in mixed alveolar/embryonal RMS; and (4) be performed on paraffinized tissue. FISH analyses of 75 specimens (40 ARMS, 16 ERMS, 8 mixed ARMS/ERMS, and 11 non-RMS tumors) using selected cosmid clone, bacterial, P1-derived, and yeast artificial chromosome probe sets were successful in all but two cases. Among specimens with informative results for both FISH and RT-PCR or standard karyotyping, PAX/ FKHR classification results were concordant in 94.6% (53/56). The three discordant cases included one exhibiting a t(2;13) by FISH that was subsequently confirmed by repeat RT-PCR, a second showing a rearrangement of the PAX3 locus only (consistent with the presence of a PAX3 variant translocation), and a third revealing a t(2;13) by FISH that lacked this translocation cytogenetically. Both alveolar and embryonal components of the mixed ARMS/ERMS subtype were negative for PAX3, PAX7, and FKHR rearrangements, a surprising finding confirmed by RT-PCR and/or conventional karyotyping. These data demonstrate that FISH with newly designed probe sets is a reliable and highly specific method of detecting t(1;13) and t(2;13) in routinely processed tissue and may be useful in differentiating ARMS from other small round cell tumors. The findings also suggest that FISH may be a more sensitive assay than RT-PCR in some settings, capable of revealing variant translocations.

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Section: Discussionmentioning

confidence: 99%

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma

Nishio

Althof

Bailey

et al. 2006

Laboratory Investigation

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“…EWS-WT1 fusion product was previously identified in all cases by RT-PCR using frozen tissue. 17 Tissues were fixed in 10% formalin for 12-24 h and embedded in paraffin for routine histologic evaluation. Of the 24 cases, 20 were large specimens of enbloc excision of the tumor in which a representative block containing large section of tumor tissue was available for immunohistochemical staining.…”

Section: Methodsmentioning

confidence: 99%

PDGF-A, PDGF-Rβ, TGFβ3 and bone morphogenic protein-4 in desmoplastic small round cell tumors with EWS-WT1 gene fusion product and their role in stromal desmoplasia: an immunohistochemical study

et al. 2005

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“…As the genetics and biology of the gene fusions in ARMS were elucidated, studies were initiated to explore clinical applications of these gene fusions (Arden et al, 1996;Barr et al, 1995;de Alava et al, 1995;Downing et al, 1995;Frascella et al, 1998;Reichmuth et al, 1996). Using RT ± PCR assays for the PAX3 ± FKHR and PAX7 ± FKHR transcripts, multiple tumors were assayed to determine the frequency of these fusions.…”

Section: Molecular Diagnostic and Therapeutic Studies Of Arms Gene Fumentioning

confidence: 99%

Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma

2001

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The chromosomal translocations t(2;13)(q35;q14) and t(1;13)(p36;q14) are characteristic of alveolar rhabdomyosarcoma, a pediatric soft tissue cancer related to the striated muscle lineage. These translocations rearrange PAX3 and PAX7, members of the paired box transcription factor family, and juxtapose these genes with FKHR, a member of the fork head transcription factor family. This juxtaposition generates PAX3 ± FKHR and PAX7 ± FKHR chimeric genes that are expressed as chimeric transcripts that encode chimeric proteins. The fusion proteins, which contain the PAX3/PAX7 DNA binding domain and the FKHR transcriptional activation domain, activate transcription from PAX-binding sites with higher potency than the corresponding wild-type PAX proteins. This increased function results from the insensitivity of the FKHR activation domain to inhibitory e ects of N-terminal PAX3/PAX7 domains. In addition to altered function, the fusion products are expressed in ARMS tumors at higher levels than the corresponding wild-type PAX products due to two distinct mechanisms. The PAX7 ± FKHR fusion is overexpressed as a result of in vivo ampli®cation while the PAX3 ± FKHR fusion is overexpressed due to a copy number-independent increase in transcriptional rate. Finally, though FKHR subcellular localization is regulated by an AKTdependent pathway, the fusion proteins are resistant to these signals and show exclusively nuclear localization. Therefore, these translocations alter biological activity at the levels of protein function, gene expression, and subcellular localization with the cumulative outcome postulated to be aberrant regulation of PAX3/PAX7 target genes. This aberrant gene expression program is then hypothesized to contribute to tumorigenic behavior by impacting on the control of growth, apoptosis, di erentiation and motility. Oncogene (2001) 20, 5736 ± 5746.

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Molecular Assays for Chromosomal Translocations in the Diagnosis of Pediatric Soft Tissue Sarcomas

Cited by 171 publications

References 22 publications

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma

PDGF-A, PDGF-Rβ, TGFβ3 and bone morphogenic protein-4 in desmoplastic small round cell tumors with EWS-WT1 gene fusion product and their role in stromal desmoplasia: an immunohistochemical study

Gene fusions involving PAX and FOX family members in alveolar rhabdomyosarcoma

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