Abstract
Purpose: To assess if molecular detection of minimal disseminated disease by real-time reverse transcription and polymerase chain reaction (RT-PCR) could contribute to a better treatment stratification in patients with rhabdomyosarcoma (RMS). Methods: Relative quantification of the tumor-mRNA present in serial samples of bone marrow (BM) and peripheral blood (PB) from 16 patients with RMS (7 alveolar and 9 embryonal) was performed by a real-time RT-PCR assay. Expression of MyoD1 and acetylcholine receptor (AChR) was analyzed in all samples, along with PAX3/7-FKHR in samples from alveolar tumors. Results: A good correlation was found between the expression of PAX3/7-FKHR and AChR, while MyoD1 was more sensitive but less specific. In this study, patients with positive PB at the end of treatment showed a poorer prognosis than patients with negative PB. Moreover, in this patient cohort, metastatic relapses were preceded by the detection of microcirculating disease in all cases. Conclusion: The detection of minimal circulating and micrometastatic disease by real-time RT-PCR, based on the expression of multiple genes, yields highly reproducible results. Patients with positive PB after treatment show poorer survival than patients without microcirculating disease.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Athale U, Shurtleff S, Jenkins J, Poquette C, Tan M, Downing J, Pappo A (2001) 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 23:99–104
Avigad S, Cohen IJ, Zilberstein J, Liberzon E, Goshen Y, Ash S, Meller I, Kollender Y, Issakov J, Zaizov R, Yaniv I (2004) The predictive potential of molecular detection in the nonmetastatic Ewing family of tumors. Cancer 100:1053–1058
Barr FG, Gallili N, Holich J, Biegel JA, Rovera G, Emanuel BS (1993) Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcomas. Nat Genet 3:113–117
Bridge JA, Liu J, Weibolt V, Suijkerbuijk R, Wenger G, Zhang J, Wan X, Baker KS, Sorensen P, Barr FG (2000) Novel genomic imbalances in embryonal rhabdomyosarcoma revealed by comparative genomic hybridization: an Intergroup Rhabdomyosarcoma Study. Genes Chromosomes Cancer 27:337–344
Burchill SA, Lewis IJ, Abrams KR, Riley R, Imeson J, Pearson ADJ, Pinkerton R, Selby P (2001) Circulating neuroblastoma cells detected by reverse transcriptase polymerase chain reaction for Tyrosine Hydroxylase mRNA are an independent poor prognostic indicator in stage 4 neuroblastoma in children over 1 year. J Clin Oncol 19:1795–1801
Davis RJ, D’Cruz CM, Lowell MA, Biegel JA, Barr FG (1994) Fusion of PAX7 to the FKHR by the variant t(1;13) (p36;q14) translocation in alveolar rhabdomyosarcomas. Cancer Res 54:2869–2872
Frascella E, Rosolen A (1998) Detection of the MyoD1 transcript in rhabdomyosarcoma cell lines and tumor samples by reverse transcription polymerase chain reaction. Am J Pathol 152:577–583
Gattenloehner S, Vincent A, Leuschner I, Tzartos S, Muller-Hermelink H, Kirchner T, Marx A (1998) The fetal form of acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors. Am J Pathol 152:437–444
Gattenloehner S, Dockhorn-Dworniczak, Leuschner I, Vincent A, Müller-Hermelink H, Marx A (1999) A comparison of MyoD1 and fetal acetylcholine receptor expression in childhood tumors and normal tissues: Implications for the molecular diagnosis of minimal disease in rhabdomyosarcomas. J Mol Diagn 1:23–31
Kelly KM, Womer RB, Barr FG (1996) Minimal disease detection in patients with alveolar rhabdomyosarcoma using a reverse transcriptase-polymerase chain reaction method. Cancer 78:1320–1327
Livak KJ, SchmittgenTD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25:402–408
Loh WE Jr, Scrable HJ, Livanos E, Arboleda MJ, Cavenee WK, Oshimura M, Weissman BE (1992) Human chromosome 11 contains two different growth suppressor genes for embryonal rhabdomyosarcoma. Proc Natl Acad Sci USA 89:1755–1759
Maurer HM, Beltangady M, Gehan EA, Crist W, Hammond D, Hays DM, Heyn R, Lawrence W, Newton W, Ortega J (1988) The Intergroup Rhabdomyosarcoma Study I. A final report. Cancer 61:209–220
Michelagnoli MP, Burchill SA, Cullinane C, Selby PJ, Lewis IJ (2003) Myogenin: a more specific target for RT-PCR detection of rhabdomyosarcoma than MyoD1. Med Pediatr Oncol 40:1–8
Mocellin S, Rossi CR, Pilati P, Nitti D, Marincola FM (2003) Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 9:189–195
Newton WA Jr, Gehan EA, Webber BL, Marsden HB, van Unnik AJ, Hamoudi AB, Tsokos MG, Shimada H, Harms D, Schmidt D (1995) Classification of rhabdomyosarcomas and related sarcomas. Pathologic aspects and proposal for a new classification—an Intergroup Rhabdomyosarcoma Study. Cancer 76:1073–1085
Tokunaga E, Maehara Y, Oki E, Koga T, Kakeji Y, Sugimachi K (2000) Application of quantitative RT-PCR using “TaqMan” technology to evaluate the expression of CK 18 mRNA in various cell lines. J Exp Clin Cancer Res 19:375–381
Thomson B, Hawkins D, Felgenhauer J, Radich (1999) RT-PCR evaluation of peripheral blood, bone marrow and peripheral blood stem cells in children and adolescents undergoing VACIME chemotherapy for Ewing’s sarcoma and alveolar rhabdomyosarcoma. Bone Marrow Transplant 24:527–533
Weigelt B, Bosma AJ, Hart AA, Rodenhuis S, van’t Veer LJ (2003) Marker genes for circulating tumour cells predict survival in metastasized breast cancer patients. Br J Cancer 88:1091–1094
Acknowledgements
The authors wish to thank Dr. Beverly Raney for his helpful critical review and Ms. Christine O’Hara for help with the English version of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
S. Gallego and A. Llort contributed equally to this work. This work was supported by a grant from the ISCIII (G089/03).
Rights and permissions
About this article
Cite this article
Gallego, S., Llort, A., Roma, J. et al. Detection of bone marrow micrometastasis and microcirculating disease in rhabdomyosarcoma by a real-time RT-PCR assay. J Cancer Res Clin Oncol 132, 356–362 (2006). https://doi.org/10.1007/s00432-006-0083-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00432-006-0083-y