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

Abstract

Summary:Peripheral blood stem cell support allows dose intensification of multiple cycle chemotherapy for metastatic tumors, including pediatric sarcomas. The VACIME protocol (vincristine, adriamycin, cyclophosphamide, ifosfamide, mesna and etoposide) utilizes peripheral blood stem cells (PBSC) collected following the treatment cycle as support for subsequent dose-and timeintensive chemotherapy. A critical assumption is that PBSC collected in this manner will be purged of residual tumor cells in vivo. We tested this assumption using sensitive reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of the characteristic translocations of the Ewing's sarcoma family of tumors (ESFT) and alveolar rhabdomyosarcoma (ARMS), t(11;22), and t(2;13), respectively. We used RT-PCR to evaluate 122 samples of peripheral blood (PB), bone marrow (BM) and PBSC collected from 12 pediatric patients with metastatic ESFT and ARMS. The samples included pre-therapy BM and PB, as well as BM, PB, and PBSC collections at various times in the VACIME treatment course. Molecular evidence of tumor contamination was detected in 1/40 PBSC collections from 12 patients. In all patients, we documented clearance of disease by RT-PCR in peripheral blood and bone marrow by week 9 of the VACIME protocol. In vivo purging in combination with the intensive VAC-IME regime appears to be effective in removing tumor cells from PBSC, bone marrow, and peripheral blood as detected by RT-PCR.

Incidence and prognostic value of tumour cells detected by RT–PCR in peripheral blood stem cell collections from patients with Ewing tumour

“…Two other studies have also reported a high incidence of contamination by tumour cells, occurring in either marrow or PBSC grafts of all five patients in both studies (Toretsky et al, 1995;Leung et al, 1998). The incidence of tumour cell contamination of PBSC products in the present study (8%) is more consistent with the findings of three other studies which reported a proportion of patients with tumour cell contamination of PBSC samples of one out of 15 (6.7%), one out of nine (11.1%), and three out of 15 (20%), respectively (Fischmeister et al, 1999;Montanaro et al, 1999;Thomson et al, 1999). This important variability in incidence of tumour cell contamination in PBSC collections in previous reports might be due to different sensitivity and specificity of the techniques of detection, to different times of harvesting and to different disease status at the time of PBSC collection.…”

“…Previous studies on small series of patients with ET have shown that tumour cells might contaminate peripheral sites, including autologous stem cell products from the patient (Toretsky et al, 1995;Leung et al, 1998;Fischmeister et al, 1999;Thomson et al, 1999;Montanaro et al, 1999;Yaniv et al, 2004). Data on the incidence and prognostic value of tumour cell contamination of peripheral blood stem cell (PBSC) collections have been controversial.…”

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force

“…These studies have resulted in the introduction of new interventions to target this disease with the expectation of improved patient outcome (Faderl et al, 1999;Foroni et al, 1999;Hochhaus et al, 2000;Roman et al, 2000;Campana et al, 2001). Amplification of known tumour-specific gene rearrangements has also provided a powerful tool to detect potential significant MD in solid cancers including those of the Ewing's sarcoma family of tumours (de Alava et al, 1998;Zoubek et al, 1998;Schleiermacher et al, 2003;Avigad et al, 2004), alveolar rhabdomyosarcoma (Thomson et al, 1999;Athale et al, 2001;Gallego et al, 2006) and desmoplastic small round cell tumours (Athale et al, 2001).…”

Ewing Sarcoma tumor cells express CD34: implications for autologous stem cell transplantation

“…19 We and others have previously studied tumor contamination in PBSC harvests from EFT patients and revealed a high incidence of contamination. [15][16][17][18][19] Toretsky et al 15 identified the chimeric transcript in the blood progenitor cell collections of all five of their EFT patients studied. Leung et al 16 studied five high-risk EFT patients and all were reverse transcription (RT)-PCR positive, either in marrow or blood grafts.…”

“…At least one positive collection was identified in one out of nine and 10 out of 15 high-risk EFT patients. 17,18 In our previous study, tumor-contaminated harvests were detected by RT-PCR in all 11 patients in at least one of the collections. 19 In this study, we investigated tumor cell contamination in harvests from EFT patients prior and after CD34 þ selection by RT-PCR and flow cytometry (FC) analyses and studied primary tumor cells for mRNA expression of CD34 by RT-PCR.…”