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

Jun Nishio, Pamela A. Althof, Jacqueline M. Bailey, Ming Zhou, James R. Neff, Frederic G. Barr, David M. Parham, Lisa Teot, Stephen J. Qualman, Julia A. Bridge

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)547-556
Number of pages10
JournalLaboratory Investigation
Volume86
Issue number6
DOIs
StatePublished - Jun 1 2006

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Alveolar Rhabdomyosarcoma
Fluorescence In Situ Hybridization
Paraffin
Reverse Transcriptase Polymerase Chain Reaction
Karyotyping
Gene Fusion
P1 Bacteriophage Artificial Chromosomes
Yeast Artificial Chromosomes
Cosmids
Neoplasms
Clone Cells
RNA

Keywords

  • Alveolar rhabdomyosarcoma
  • Cytogenetics
  • FISH
  • RT-PCR

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

Cite this

Nishio, J., Althof, P. A., Bailey, J. M., Zhou, M., Neff, J. R., Barr, F. G., ... Bridge, J. A. (2006). Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma. Laboratory Investigation, 86(6), 547-556. https://doi.org/10.1038/labinvest.3700416

Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma. / Nishio, Jun; Althof, Pamela A.; Bailey, Jacqueline M.; Zhou, Ming; Neff, James R.; Barr, Frederic G.; Parham, David M.; Teot, Lisa; Qualman, Stephen J.; Bridge, Julia A.

In: Laboratory Investigation, Vol. 86, No. 6, 01.06.2006, p. 547-556.

Research output: Contribution to journalArticle

Nishio, J, Althof, PA, Bailey, JM, Zhou, M, Neff, JR, Barr, FG, Parham, DM, Teot, L, Qualman, SJ & Bridge, JA 2006, 'Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma', Laboratory Investigation, vol. 86, no. 6, pp. 547-556. https://doi.org/10.1038/labinvest.3700416
Nishio, Jun ; Althof, Pamela A. ; Bailey, Jacqueline M. ; Zhou, Ming ; Neff, James R. ; Barr, Frederic G. ; Parham, David M. ; Teot, Lisa ; Qualman, Stephen J. ; Bridge, Julia A. / Use of a novel FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of alveolar rhabdomyosarcoma. In: Laboratory Investigation. 2006 ; Vol. 86, No. 6. pp. 547-556.
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AU - Barr, Frederic G.

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N2 - 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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