Residual neoplastic lymphocytes in hematopoietic progenitor cells reinfused in B-NHL patients during autologous transplantation may be an important cause of relapse. It has already been demonstrated by others that tumor cells contaminating BM harvest used in autologous transplantation can contribute to disease recurrence [4
], and some recent reports indicate that this may be true also when using PBPCs [19
Although BM and PB analysis at the end of the treatment has a well defined role in predicting lymphoma relapse [6
], it is still a matter of debate whether the same tissues are adequate for predicting MRD in PBPCs. Some recent papers have already given interesting insights about this isssue, although for different types of NHLs [20
] or after different therapeutic strategies [21
]. Thus, additional indications about this issue may be helpful to prospectively guide PBPC harvesting procedures, in particular when exvivo purging strategies have to be considered. In the present study, we tried to qualitatively evaluate if data obtained from BM and PB are predictive for the molecular status of PBPCs. To this aim, in a series of NHL patients who underwent high-dose chemotherapy, we analyzed PB and BM samples obtained at the time of PBPC collection: among patients who had a PCR-negative BM, 29% still had positive PBPCs. Discordance was also evident comparing data obtained from PB and PBPCs: PCR-positive PBPCs were harvested in 30% of PCR-negative PB patients. In addition, the comparison of the results obtained by immunophenotyping and by PCR, showed that 2 out of 7 patients (28%) who scored negative at FACS analysis were found to have positive PB and PBPCs when analyzed by PCR. These findings question the value of PB immunophenotyping as a predictive assay for the evaluation of PBPCs.
PCR amplification is widely being used to detect lymphoma cells in BM and PB. In this respect, our results confirm that at diagnosis, when tumor burden is relevant, there is a concordance between BM and PB histological and molecular data, as reported by other groups [22
]. However, a note of caution has to be made about the interpretation of these data when chemotherapy is administered: in this case, there is a decrease in tumor burden, blood-marrow barrier is altered [23
] and cytokines probably modify the regulation of cell adhesion molecules [24
]. In this situation PCR sensitivity becomes critical: in fact, depending on the marker used, the percentage of positive BM samples collected after chemotherapy, at the time of PBPC harvest, could range from 66% (bcl-2/IgH or bcl-1/IgH) to 16% (patient specific oligonucleotide). These data are in accordance with those obtained by other groups [26
In the case of a PCR-negative BM, harvesting PCR-positive PBPCs may have different explanations such as i)an enrichment in neoplastic lymphocytes during PBPC harvest and ii)a negativity of BM samples for failure to aspirate the diagnostic cells due to residual reticulin fibrosis often associated with a lymphomatous infiltrate. However, it cannot be completely excluded that the detection of MRD in PBPCs may be due to the expansion and release in the bloodstream of the lymphoma cell population as a result of hematopoietic growth factor stimulation and their action on the growth rate and adhesion receptors of lymphoma cells as already suggested by other authors [24
An explanation for a PCR-negative PB patient whose harvest scores PCR-positive may be the administration of rituximab, sufficient to clear the former but not the latter from neoplastic cells. In this respect, it is thus not surprising that nearly 50% of these cases have a positive BM, since the clearance of B-cells might be more rapid in PB than in BM.
Finally, an interesting issue that remains to be solved is about the cases with PCR-positive PBPCs who score negative both at BM and PB level (cases 8, 15, 17, 22): this could be due to recruitment of neoplastic cells in PBPCs from other sites (i.e. spleen, liver) by unknown mechanism, in a way similar to intra-apheresis CD34+ cell recruitment described by some authors [27
Taken together our data suggest that, in patients with indolent and mantle cell NHL, bcl-2/IgH and bcl-1/IgH PCR is superior to PCR based on CDIII sequences in terms of sensitivity, and that this technique has to be applied to PBPCs, and not to PB or BM, to assess neoplastic contamination of the harvests. As the number of patients is small, final conclusions about the clinical importance of our findings awaits further study. In addition, in the near future, quantitative rather than qualitative assays for the evaluation of MRD in PBPCs are desirable to evaluate and optimize chemo-immunotherapy protocols and PBPC mobilization regimens.