Bone Marrow Transplantation, (1999) 24 , 405–409  1999 Stockton Press All rights reserved 0268–3369/99 $15.00 http:/ / www.stockton-press.co.uk/ bmt Low incidence of molecular evidence for tumour in PBPC harvests from patients with high risk Ewing tumours G Fischmeister, 1 A Zoubek, 1 D Jugovic, 1 V Witt, 1 R Ladenstein 1 , G Fritsch 1 , P Ho ¨cker 2 , H Gadner 1 and H Kovar 1 1 St Anna Children’s Hospital and Children’s Cancer Research Institute, Vienna; and 2 Department of Transfusion Medicine, University of Vienna, Austria Summary: Reverse transcriptase polymerase chain reaction (RT- PCR) was applied to evaluate the frequency of tumour cells in PBPC products from 15 high risk Ewing tumour (ET) patients who were treated according to EICESS 92 with high-dose chemotherapy (HDC) and stem cell rescue. Initial tumour cell contamination of the bone marrow (BM) detected by light microscopy was found in five and by RT-PCR in eight cases. RT-PCR was per- formed on each PBPC sample repeatedly at a sensitivity comparable to 20–100 highly EWS-Fli1 expressing tumour cells per 10 ml of fresh blood. Irrespective of the extent of BM involvement at diagnosis, all BM samples obtained before harvest were RT-PCR negative. Among 12 of 35 analysed apheresis products with single positive RT-PCR results only one sample tested reproducibly positive for tumour cell contamination in independent determinations. These preliminary data suggest that tumour cell contamination of PBPC is rarely found in patients with ET. Keywords: Ewing tumour; minimal residual disease; apheresis products: RT-PCR; EWS-FLI1 Current multimodal therapy may be curative for approxi- mately 70% of patients with localised Ewing tumours (ET). However, for patients with metastatic disease at diagnosis and for about one-third of patients with localised disease, prognosis remains poor, due to relapse after first-line treat- ment. 1–4 Thus, minimal numbers of potentially metastatic tumour cells are postulated to be present in the circulation of almost every patient, even in the absence of clinically overt disseminated disease at diagnosis. For solid tumours, viable tumour cells in the peripheral blood have been demonstrated in patients with neuroblas- toma 5 and breast cancer, 6 and transiently in melanoma patients. 7 Since these circulating tumour cells retained their clonogenic potential they may contribute to relapse after autologous reconstitution. Gene marking studies have revealed reinfused tumour cells in the metastases of neuro- blastoma and breast cancer patients. 8–10 Correspondence: Dr H Kovar, Children’s Cancer Research Institute, St Anna Children’s Hospital, Kinderspitalgasse 6, A-1090 Vienna, Austria Received 18 January 1999; accepted 4 April 1999 High-dose chemotherapy (HDC) followed by autologous peripheral blood progenitor cell (PBPC) transplantation is effective for the treatment of a number of malignant dis- orders including metastatic or relapsed ET. 11 Preliminary data from non-paediatric malignancies suggest that patients receiving tumour cell contaminated autografts do worse than patients transplanted with autografts free of detectable minimal residual disease (MRD). 12 Previously, it has been demonstrated for patients with neuroblastoma, that PBPC harvests are less contaminated by tumour cells than bone marrow 13 and different purging methods have been estab- lished to reduce the tumour load in stem cell prep- arations. 8,14 Little is known about the mobilisation of tumour cells by growth factors. Data from multiple mye- loma patients suggest a concomitant mobilisation of mye- loma cells with chemotherapy and G-CSF. 15 This obser- vation was not confirmed in patients with breast cancer. 16 Overall, methods for sensitive tumour cell monitoring are warranted to guarantee autograft quality. Based on the pres- ence of unique chimeric transcripts in ET cells the feasi- bility of reverse transcriptase polymerase chain reaction methodology (RT-PCR) for follow-up of MRD in ET patients has been demonstrated recently by several groups. 17–19 In addition, we have previously reported on transient mobilisation of RT-PCR detectable numbers of ET cells into the peripheral blood during tumour biopsies and extensive surgical procedures. 20 The targets for RT- PCR result from alternative gene fusions between the EWS gene and the closely related ETS transcription factor genes FLI1, ERG or FEV, in approximately 87%, 10% and less than 1% of tumours, or ETV1 or E1AF in rare cases, respectively. 21 Thus, EWS gene rearrangements can be detected in at least 98% of ET patients. In the present study we report on the incidence of RT- PCR detectable tumour cell contamination in PBPC samples from patients with localised and metastatic ET. Materials and methods Patients The study cohort comprised 15 patients from a single insti- tution (10 female, five male) between 8 months and 25 years of age (median age 16 years) (Table 1). They were classified as high risk ET because of metastatic disease or large pelvic tumour mass and were scheduled for stem cell