Relapsed disease remains a major obstacle following autologous haematopoietic SCT (HSCT) for non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). Studies regarding the importance of detectable tumour cells in PBSC collections have been inconclusive. Patients undergoing autologous HSCT for NHL and MM between 2001 and 2006 were enrolled (n=158). PBSC grafts were assessed for clonal IgH CDR3 gene rearrangements using qualitative semi-nested PCR. In comparison to patients with PCR-positive PBSC grafts, patients negative for detectable disease had no improvement in overall survival (OS) or PFS for MM (P=0.91 and 0.91) or NHL (P=0.82 and 0.85). Further, no significant difference in OS was observed between patients with PCR-positive compared with PCR-negative PBSC grafts with aggressive NHL histology (P=0.74) or indolent disease (P=0.29). Patients with contaminating tumour cells in autologous PBSCs do not have worsened OS or PFS in MM or NHL. Tumour cells detected by sensitive molecular methods in PBSC collections may be distinct from cells contaminating marrow and appear to have limited utility in identifying patients with MM and B-cell NHL who would benefit from purging strategies.
High-dose chemotherapy followed by autologous haematopoietic SCT (HSCT) remains a common approach in the treatment of multiple myeloma (MM)1, 2 and relapsed non-Hodgkin's lymphoma (NHL).3 The reinfusion of autologous BM or PBSCs after myeloablative conditioning allows for haematopoietic recovery and immune reconstitution. Relapsed disease, however, remains one of the most significant challenges in autologous HSCT, because of cells that are resistant to the high-dose therapy and possibly related to reinfusion of haematopoietic grafts that are contaminated with malignant cells.
The contribution of contaminating tumour cells in autologous grafts to relapse of disease following HSCT has been examined earlier for various haematological malignancies with mixed results. Most studies involving patients with NHL4, 5, 6, 7 and MM8, 9 have suggested that contaminating tumour cells in autologous grafts are correlated with decreased survival and worsened PFS. These studies have not examined this issue exclusively in PBSC grafts. Another study, however, found no difference in survival for NHL patients with autologous PBSC grafts with evidence of tumour contamination.10
In MM, the reinfusion of tumour-free autologous grafts following CD34+ cell selection did not result in improved overall survival (OS) or PFS as shown in several randomized trials.11, 12 The benefit of purging in follicular lymphoma continues to be debated. One study showed improved OS and PFS in patients with documented marrow involvement before transplantation who received purged autologous marrow grafts that were free of tumour contamination.13, 14 A randomized study involving unpurged and purged marrow grafting revealed no significant difference in OS or PFS in patients with follicular lymphoma regardless of marrow involvement at transplant.15 The importance of tumour cells detected in PBSCs and the function of purging PBSC collections remains under study.
With the recent development of more sensitive molecular testing methods, the impact of low levels of contaminating clonal B cells on clinical outcomes after HSCT remains unclear. Our study addresses the impact of detectable clonal B-cell populations using a sensitive molecular approach in PBSC grafts. The function of contaminating cells on OS and PFS is reported in patients with B-cell malignancies who underwent autologous HSCT using PBSC grafts.
Materials and methods
Patients with a diagnosis of MM or B-cell NHL who underwent autologous PBSC transplantation at The Ottawa Hospital between October 2002 and January 2006 were included. Patients provided informed consent for the use of medical information for research purposes and for the analysis of PBSCs for research, in accordance with a consent process approved by our institutional research ethics board. All patients with molecular analysis of IgH clonality in mononuclear cells from PBSCs before autologous transplantation were included. Patients who received BM grafts, and patients who had earlier undergone autologous HSCT were excluded from the analysis. IgH testing was performed systematically in a blinded manner in all patients undergoing transplantation at our centre. Personnel were not available to perform IgH testing for 8 patients with NHL and 10 patients with MM who underwent transplantation during the study period. These cases represent 8.2% and 12.6% of transplants performed for B-cell NHL and MM, respectively, during the study period. The data pertaining to these cases are not included in the analysis.
Molecular analysis for tumour contamination
Clonal B-cell populations were identified by molecular analysis of the third complementarity-determining region of the Ig heavy chain (IgH CDR3). Mononuclear cells from PBSCs underwent testing for rearrangements of IgH CDR3 using a semi-nested PCR as briefly described below. Genomic DNA from mononuclear blood cells (200 μl) was amplified initially with 0.4 μM of VHFR3 and 0.4 μM of LJH primers that were described earlier.16 The PCR products were amplified for 29 cycles using Taq DNA polymerase. A second PCR was performed using 1:40 volume of the first PCR reaction as template, as well as 0.4 μM of VHFR3 primer and 0.4 μM of VLJH primer. The second PCR was also completed for 29 cycles using Taq DNA polymerase. The semi-nested PCR products were resolved by gel electrophoresis in 8% polyacrylamide and visualized by ethidium bromide chelation and UV light detection. For patients undergoing PBSC collection on consecutive days and where multiple PCR analyses were performed, grafts were designated as positive if any of the collections were positive by PCR. PCR amplification that yielded a clonal band indicated the presence of a dominant clonal population of B-lymphocytes consistent with contaminating tumour cells or a polyclonal smear suggesting the absence of clonal tumour contamination (Figure 1).
The sensitivity of the semi-nested PCR assay was evaluated using DNA from a clonal B-cell line obtained from InviviScribe Technologies (San Diego, CA, USA). One microgram of monoclonal DNA was diluted using a 10-fold dilution series into mononuclear cell DNA obtained from healthy donors.
Analysis of survival
Survival was determined from day 0 of the first transplant in all patients. Data were obtained from the Blood and Marrow Transplant Program Database at The Ottawa Hospital. The survival status of all patients was confirmed and further updated by reviewing all medical records in February 2007 before analysis. OS was determined for each patient. Further, non-relapse mortality by day 100 was recorded. Survival estimates were determined according to the method of Kaplan and Meier.17
Analysis of PFS
The effect of tumour contamination in PBSC grafts on PFS was examined for patients with MM and NHL. Only patients with MM associated with a serum monoclonal protein were included in the analysis of disease progression as defined using criteria developed by Durie et al.18 Patients with NHL were defined as having progression of disease based on clinical and radiological information extracted from medical records. Patients with NHL underwent regular CT scanning 1–4 times annually and were followed 2–4 times annually in the ambulatory clinics of the transplant programme. Patients with MM were reviewed every 1–2 months and had blood work performed at each visit, including serum protein electrophoresis.
A total of 158 patients with B-cell malignancies had molecular analysis for IgH gene rearrangements performed on PBSC mononuclear cells. Of these patients, 89 had a diagnosis of NHL and 69 had MM. Patient characteristics are summarized in Table 1 for those with PCR-positive compared with PCR-negative PBSC grafts.
Among the 89 patients with NHL, 32 (36%) had evidence of tumour contamination detected in the PBSC grafts, whereas the remaining 57 (64%) were negative by PCR. All patients with NHL were further analysed according to the histological subtype (see Table 1). The proportion of patients with graft contamination was not statistically different between aggressive and indolent NHL (P=0.20).
Among the 69 patients with MM, 17 (25%) had evidence of tumour contamination versus 52 (75%) without evidence of clonal IgH rearrangements (see Table 2). There were no significant differences in the proportion of patients with graft contamination between subtypes of MM.
Analysis of OS
Considering all patients included in the study (n=158), 55 (35%) had evidence of tumour contamination in PBSC grafts and 111 (65%) were PCR-negative for clonal IgH rearrangements. OS was not different between patients with or without tumour contamination. A log-rank test comparing the two survival curves revealed no significant difference (P=0.58).
Survival among patients with NHL (n=89) who had no detectable tumour contamination in PBSC grafts before autologous HSCT was not improved compared to those with positive PCR results (P=0.82). Moreover, there was no survival difference when aggressive and indolent histology NHL were analysed separately (P=0.74 and 0.29).
Overall survival among patients who had MM (n=69) was also evaluated. Patients without detectable tumour contamination in PBSC grafts did not have improved survival compared with those with tumour contamination (P=0.91).
With regard to transplant-related deaths occurring within 100 days of the transplant, non-relapse mortality was higher (5.6%, five deaths) for NHL compared with MM (0%, P=0.045). Non-relapse mortality for patients with NHL and tumour contamination was 6.3% (two patients) compared with 5.3% (three patients) for NHL patients without graft tumour contamination (P=0.85).
Analysis of PFS
The effect of tumour-free PBSC grafts on PFS was addressed. Eighty-three patients with NHL had sufficient data available for analysis of disease progression (data missing in six patients followed outside of our centre). PFS of patients with NHL and tumour-free PBSC grafts was not different when compared with patients with graft contamination (P=0.85, see Figure 2). Separate analysis of patients with aggressive or indolent histology NHL revealed no difference in PFS between those with tumour-free compared with contaminated PBSC grafts (P=NS). PFS was also evaluated among patients with follicular lymphoma (grades I–III and including clinical or histological transformation). No improvement in PFS was associated with tumour-free PBSC grafts compared with grafts with evidence of tumour contamination (P=0.89).
Analysis of PFS in MM was performed in 53 patients who had detectable serum monoclonal protein. Sixteen additional patients with MM were not included in this analysis because of a diagnosis of light-chain disease or non-secretory myeloma, and time to progression could not be determined reliably. No difference in PFS was observed when comparing patients with tumour contamination and those without detectable IgH rearrangements (P=0.77, Figure 3).
The results of our study suggest that tumour cells in autologous PBSCs detected by PCR amplification of the IgH CDR3 region do not influence the risk of relapse or OS for patients with B-cell NHL or myeloma. The reinfusion of PBSCs with detectable clonal tumour cells does not appear to contribute significantly to relapse of disease. Dominant factors associated with an increased risk of relapse most likely remain chemosensitivity of the underlying malignancy and burden of disease at the time of transplant.19, 20
Our results examining the function of PBSC tumour contamination in B-cell NHL showed no effect on survival or disease relapse even when aggressive and indolent histologies were considered separately. In addition, detecting clonal B-cells in autologous PBSC grafts in patients with follicular lymphoma did not influence the rate of relapsed disease. Although earlier studies have reported adverse outcomes in patients with diffuse large B-cell lymphoma or follicular lymphoma with detectable tumour cells in autologous marrow harvests,4, 5 the mobilization of tumour cells collected in PBSCs may be less relevant. It may be important to distinguish the presence of tumour cells in marrow harvests from PBSC collections. For example, in a separate study, sensitive molecular methods used to detect follicular lymphoma cells in autologous PBSCs revealed that some of these patients became free of any molecular evidence of disease after transplantation.21 It is possible that cytokine stimulation or repeated apheresis procedures induces migration of a sub-population of tumour cells into the PBSC collection without reflecting any meaningful difference in disease burden.22 The proliferative capacity and tumorigenicity of malignant lymphoma cells collected in PBSC may be reduced when compared to cells contaminating the marrow compartment, although experimental evidence for this is lacking.
The high-dose treatment regimen used in the treatment of lymphoma may be insufficient for some patients, with the role of tumour cells in the graft becoming less important. The effectiveness of transplant regimens is an ongoing area of active research. In particular, the high-dose treatment regimen used for the treatment of follicular lymphoma and other indolent NHL subtypes at our centre includes TBI and has been associated with favourable long-term results.23 A TBI-containing regimen may have been sufficient for overcoming possible differences in disease burden in our patients with follicular lymphoma who were positive by PCR. Aggressive histology lymphomas such as diffuse large B-cell lymphoma and Burkitt-like lymphoma may remain sufficiently sensitive to the conditioning regimen to achieve long-term disease control, independent of potentially sensitive markers of disease burden such as graft populations of clonal B cells.
An additional possibility is that malignant cells, although collected from peripheral blood during mobilization, are not capable of homing to favourable sites for disease propagation following reinfusion. The presence of cells in PBSC should be distinguished from earlier studies that addressed clonal tumour cells in peripheral blood or BM. It is possible that the mobilization regimen with chemotherapy and G-CSF released clonal tumour cells detectable only in PBSCs. It is also possible that the clonal B cells detected in our study may represent pre-malignant cells that are clonally related to the underlying lymphoma but are insufficient to cause relapse of the disease.
The molecular technique used in this study was moderately sensitive and able to detect 1:100 000 cells. Binding of CDR3 PCR primers may be imperfect in some cases and we may have detected only 70–80% of true cases where a clonal B-cell population was present.24, 25 Perhaps some of the grafts characterized as negative for tumour contamination in our study have undetectable levels of tumour that are still relevant. It is unclear whether a more quantitative assessment of tumour contamination would be informative, although one study involving 43 patients using quantitative PCR failed to identify a relationship with OS or PFS.26
Earlier literature has suggested a benefit with autologous graft purging in patients with follicular lymphoma who have detectable bcl2 in peripheral blood or BM.7, 13, 14 The benefits of graft purging in patients with follicular lymphoma who lack evidence of detectable molecular disease are less clear,15 and the function of both ex vivo and in vivo purging strategies in patients with aggressive histology lymphoma undergoing PBSC transplantation is under active investigation. Our results suggest that detecting tumour cells in PBSC grafts may not be the ideal method of selecting patients who would benefit most from a purging strategy. Patients may need to undergo tests using peripheral blood or BM before PBSC collection to identify appropriate candidates for purging protocols.
With regard to the function of contaminating myeloma cells in PBSC grafts, several studies have reported decreased survival and worsened PFS in patients with molecular evidence of tumour contamination in stem cell grafts.8, 9 Gene-marking studies using autologous marrow harvests in small numbers of patients have suggested that reinfused tumour cells may contribute to subsequent disease relapse.27, 28 One study involving gene-marking of CD34-selected PBSCs reports that contaminating cells do not contribute to relapses.29 Given the continuous risk of relapse following autologous transplantation observed in several large randomized studies,1, 2 it appears likely that high-dose melphalan is not able to eliminate residual disease in most patients. This observation most likely dominates the future risk of relapse and survival in these patients. In addition, purging studies in MM have failed to show improved OS or PFS when patients were reinfused with tumour-free PBSC grafts.11, 12
In summary, our study provides new insight suggesting the limited relevance of contaminating tumour cells detected by sensitive molecular methods in PBSC grafts in patients undergoing autologous transplantation for B-cell NHL and MM. A key issue in our study may be the exclusive analysis of PBSC grafts as compared to previous research addressing tumour contamination in BM harvests. Given some of the limitations discussed, we suggest that additional prospective studies would be appropriate to confirm our observations and to better define patients who would benefit from purging strategies.
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We acknowledge the support of Canadian Blood Services (CBS). DSA is an Adjunct Scientist at CBS. The database of the Blood and Marrow Transplant Program at The Ottawa Hospital received support from The Ottawa Hospital Foundation, BMT Research and Education Fund.
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Ho, J., Yang, L., Banihashemi, B. et al. Contaminating tumour cells in autologous PBSC grafts do not influence survival or relapse following transplant for multiple myeloma or B-cell non-Hodgkin's lymphoma. Bone Marrow Transplant 43, 223–228 (2009). https://doi.org/10.1038/bmt.2008.318
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