Tumour cell contamination in PBSC products that are reinfused after potentially curative HDCT has become an important issue in a number of haematological malignancies and solid tumours. In this study, we have analysed tumour cell contamination of PBSC collections in a large series of patients with ET and correlated these findings with clinical outcome.
Our data show that the incidence of tumour cell contamination of PBSC collections as determined by RT–PCR is very low (seven out of the 88 patients included in this study; 8%) in this group of patients with a high-risk ET. Taking into account all interpretable results of PBSC analysis by RT–PCR, the incidence was even lower (seven out of 171 cases; 4.1%), This finding is in contrast to the frequency of PBSC contamination observed in some previously published studies. In a recently published series all 11 patients had contaminating tumour cells in their PBSC harvests (Yaniv et al, 2004
). 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.
First, it is of concern that tumour cell detection by RT–PCR may yield false-positive and/or false-negative results. In our study, to avoid false-positive results generated by cross-contamination linked to the use of PCR amplification techniques, several strict precautions were taken. Samples were processed at different sites for RNA isolation, PCR amplification, and electrophoresis of PCR products and the two rounds of nested PCR amplification were processed without opening the tube (Peter et al, 1995
). The observation that the type of fusion transcript was consistent between primary tumour and PBSC collection provided a good control for the specificity of the technique. Following the introduction of real-time PCR in 1999, the risk of carry-over contamination was even more reduced as no PCR products are manipulated with this method (Peter et al, 2001
). Moreover since that time the GeneAmp RNA PCR kit has been used which contains an enzyme (Uracil N-glycosylase) in order to degrade specifically PCR products from previous PCR amplifications (Longo et al, 1990
). To avoid false-negative results owing to an error during the procedure resulting in degraded RNAs, an internal control was established. The quality of RNA was controlled by a test amplification of the ubiquitously expressed EWS
gene with identical PCR conditions. In the current study, a total of 34 patients were not included as no interpretable results of PBSC RT–PCR analysis could be obtained owing to poor RNA quality.
Second, the time of harvesting might influence the incidence of tumour cell contamination of PBSC collections. In a previous study, one patient had a positive harvest after two and three cycles of induction chemotherapy, whereas his harvest was negative after four cycles (Toretsky et al, 1995
). More recently, another study reported that in two patients a first PBSC collection had had tumour contamination detected by RT–PCR but subsequently cleared after two additional cycles of chemotherapy (Thomson et al, 1999
). In our study, all PBSC collections were performed after a median of five cycles of induction chemotherapy (a median of four cycles in the group of patients with and five cycles in the group of patients without tumour cell contamination of PBSC collection), most likely contributing to the observed low incidence of tumour cell contamination of PBSC collection. No positive samples were observed in the group of patients treated according to the EuroEwing 99 protocol (nonrandomised arms), which consists of a more intensive induction chemotherapy (six cycles of Vincristine, Ifosphamide, Doxorubicin, and Etoposide) in comparison to the previous EW 88 and EW 93–97 protocols. This could suggest that more intensive induction chemotherapy might eliminate circulating cells. However, owing to the small number of positive samples and because of the inclusion of only nonrandomised patients of the EuroEwing 99 protocol, the relevance of these findings with regard to current clinical practice needs to be interpreted with caution.
Third, the status of disease at the time of PBSC collection might also influence the incidence of tumour cell contamination. In our series, of the seven patients with positive harvest, only one patient (patient 7) had BM disease at the time of harvesting and all the patients with a negative harvest were free of disease in BM as detected histologically at that time. Unfortunately for none of the seven patients with tumour cell contamination of PBSC collection in this study, samples of BM for the detection of micrometastases at the time of PBSC collection could be studied. In a recently published study, six out of the six PBSC-positive patients tested had BM micrometastases at the time of harvesting (Yaniv et al, 2004
). In two other studies tumour cells in BM detected by RT–PCR were observed in two out of the three PBSC-positive patients tested and in three out of the three PBSC-positive patients tested but also in the only PBSC-negative patient, respectively (Toretsky et al, 1995
; Leung et al, 1998
). Interestingly, in the two series with a low incidence of tumour cell contamination of PBSC collection, no cases of BM micometastases at the time of PBSC collection were observed in the first and only one case in the second study (Fischmeister et al, 1999
; Thomson et al, 1999
). This observation might suggest that micrometastatic disease in BM should be sought for before collecting PBSC.
Tumour cells present in autografts have been shown to be associated with poor outcome in patients with haematological malignancies or solid tumours. In a recently published study, the presence of occult tumour cells in aphaeresis products was correlated with worse EFS and OS in patients with high-risk primary breast cancer and with worse EFS in patients with metastatic breast cancer (Nieto et al, 2004
). Another study concluded that breast cancer patients with more than three contaminating cells in their aphaeresis products represent a poor prognosis group (Syme et al, 2003
). In malignant germ-cell tumours, the presence of contaminating tumour cells in PBSC harvests seems to predict a poor EFS and OS in patients undergoing HDCT and autologous PBSC reinfusion (Hildebrandt et al, 2000
). As demonstrated in a recently published trial, myeloma patients with graft contamination of more than 4.5 × 105
plasma cells kg−1
have a high risk of early disease progression after HDCT (Vogel et al, 2005
To date, the prognostic significance of tumour cell contamination of PBSC collection from patients with ET has only been analysed in small series of patients. In a series of 11 patients, seven out of the 11 patients with contaminated PBSC harvest relapsed after graft and a possible correlation between the number of contaminating cells in the harvest and relapse after transplantation was suggested (Yaniv et al, 2004
). In our study, no statistically significant difference in EFS or OS after graft could be found between patients with and without tumour cell contamination of PBSC collection. Owing to the low incidence of tumour cell contamination of PBSC collections, these results should be interpreted with caution. Furthermore, the prognostic significance of the contamination of PBSC collection might not be the same in patients with different clinical indications for HDCT. However, when the subgroup of patients with a primary indication of HDCT was analysed separately, no significant correlation could be found between tumour cell contamination of the PBSC collection and poorer EFS or OS.
The role of reinfused contaminating tumour cells for disease progression remains unclear. In our study, of the four patients with a positive harvest who relapsed, two patients relapsed in prior metastatic sites, indicating that remaining systemic disease might have been the primary cause of relapse rather than a direct effect of reinfusion of tumour cells. This suggests an insufficient cytoreductive capacity of HDCT rather than a direct effect of tumour cells contaminating the graft. Indeed, the benefit of HDCT has been questioned especially for patients with BM metastases (Oberlin et al, 2006
). It might be possible that PBSC contamination reflects only a greater overall tumour burden, with reinfused cancer cells having no major role in disease relapse. Prior analyses in advanced breast cancer have suggested that contaminating tumour cells in PBSC collections may be a marker of widespread micrometastatic disease rather than a direct cause of recurrence after transplantation (Nieto et al, 2004
). Furthermore, the biological significance of contaminating tumour cells in PBSC collection remains to be established. Indeed, two patients in our study did not relapse after graft and are still in CR, which might suggest that the autograft did not contain tumorigenic tumour cells with a metastatic potential. Several investigators have performed qualitative tests on micrometastatic tumour cells, including the determination of the viability and growth potential of these cells and the assessment of the invasive capacity in various solid tumours (Ross et al, 1993
; Moss et al, 1994
; Sharp et al, 1996
). In ET, the oncogenic potential of tumour cells detected by RT–PCR in PBSC collection, although likely, remains to be proven.
In conclusion, our study shows that the incidence of tumour cell contamination of PBSC collection from patients with high-risk ET is low and that it does not seem to have an additional negative impact on outcome in this population already at risk.