Materials and methods

Patients

Clinical and laboratory data were retrospectively acquired for 23 allogeneic stem cell transplants (19 T-cell-depleted (TCD) related match, one T-cell-replete (TCR) unrelated (UR) partial match, one TCD-UR match, one TCR-twin, one TCR-UR match) at the Medical College of Wisconsin from 1989 to 2002. The TCR grafts were used for patients who received transplantation before 1990. After 1990, the TCD grafts have become the standard procedure for transplantation in our institution. All transplants were performed using bone marrow as the stem cell source. Post transplant immune suppression was with cyclosporin A. Clinical information was obtained for all of the transplants and included transplantation type, time to engraftment, graft-versus-host disease (GVHD), relapse of FL, and survival. Delayed engraftment was defined as absolute neutrophil count (ANC) <500 10⁶/l beyond day 25 and platelet count <20 10⁹/l beyond day 40.

RQ-PCR

RQ-PCR was performed retrospectively on mononuclear cells prepared from cryopreserved peripheral blood (PB) and/or bone marrow (BM) samples from all 23 donors and 20 recipients pre-transplant, and 14 recipients at various time intervals post transplant (Tables 1 and 2). All the samples were collected as part of routine clinical care and thus the intervals varied among patients. The bone marrow samples from donors were collected at the same time as grafts but did not go through T-cell-depletion process. This RQ-PCR assay was developed to detect and quantitate cells carrying t(14;18) (BCL-2/IGH fusion) at the major breakpoint region (MBR) and reported in detail previously.¹⁶ The RQ-PCR assay showed the sensitivity of detecting a single cell carrying t(14;18) in the background of 40 000 normal cells (ie a tumor load of 0.0025%). The linear measuring range of the standard curve of quantitation of the assay was between tumor loads of 0.01 to 10%. Detectable t(14;18) below 0.01% was expressed as <0.01%. An interassay coefficient of variation (CV) of 10.6% and an intra-assay CV of 6.9% were achieved for reproducibility. Our results indicate that the sensitivity, reproducibility, and linearity of this assay are the same, if not better, than other recently reported RQ-PCR assays for detecting t(14;18).^{18, 19, 20, 21} Patients' samples were tested in duplicate initially. If both runs were negative, an additional two runs were performed. The tumor load was calculated as the average of all runs performed on a given sample. For each patient sample, the -globin gene was amplified separately using another RQ-PCR assay reported previously.¹⁶ This was to ensure that all samples contained similar amounts of DNA and were free of PCR inhibitors. Use of these samples was approved by the Institutional Review Board of Medical College of Wisconsin.

Table 1 - Recipient information for t(14;18)-positive donors.

Full table

Table 2 - Recipient information for t(14;18)-negative donors.

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Statistical analysis

Patient characteristics were compared between recipients receiving grafts from donors positive for t(14:18) and those receiving grafts from donors negative for t(14:18) using the Student t test for continuous variables and Fisher's Exact test for categorical outcomes. Probabilities of survival and disease-free survival (DFS) were estimated using the Kaplan–Meier method, while rates of relapse were estimated using cumulative incidence. Survival, DFS, and rates of relapse were compared between the two groups using the log-rank test. Proportions of patients experiencing delayed engraftment, acute GVHD, or chronic GVHD were compared between the two groups using Fisher's Exact test.

Results

Of the 23 donors, 11 had a PB and/or BM sample positive for t(14;18) (BCL2/IGH fusion) at low levels (<one t(14;18) cell in 10K total cells). Four of the 11 donors had only PB samples available. Six (86%) of seven donors with both PB and BM samples available for testing had t(14;18) cells in both PB and BM samples and just one donor had t(14;18) cells only in PB sample. As the BM samples of donors were collected as part of the grafts prior to T-cell-depletion process, the above findings suggested that the majority of grafts from these 11 donors likely contained t(14;18) cells. T-cell depletion using antibody and complement at our institution²² resulted in only a 0.4 log loss of B cells from the graft (unpublished data, Carolyn A Keever-Taylor). For the 75 kg patients, they would have received 5.76 10⁶ B cells/kg without TCD and 3.1 10⁶ B cells/kg after TCD. Data regarding detection and quantitation of t(14;18) by RQ-PCR in the recipients, pre- and post transplant, are summarized in Tables 1 and 2.

There were no significant differences in pre-transplant clinical parameters (age at diagnosis or at transplantation, status of disease at transplant) between recipients receiving t(14;18)-positive and t(14;18)-negative donor stem cells (Table 3). We did not observe any significant differences in time to engraftment or development of acute and chronic GVHD between recipients receiving grafts from donors positive for t(14:18) and those receiving grafts from donors negative for t(14:18) (Table 3). There were no differences in rates of relapse (Figure 1a), (DFS Figure 1b), or overall survival (OS, Figure 1c) between recipients receiving donor t(14;18)-positive stem cells and those receiving t(14;18)-negative stem cells (P=0.551, 0.980, and 0.767, respectively, log-rank test).

Figure 1.

There are no differences in rates of relapse (a), DFS (b), and OS (c) between recipients (shown as black line) receiving grafts from donors with t(14;18) cells and those (shown as gray line) receiving grafts from donors without t(14;18) cells (P=0.551, 0.980 and 0.767, respectively, log-rank test).

Full figure and legend (49K)

Table 3 - Clinical parameters of recipients studied.

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Seven (four without detectable t(14;18) pre-transplant, three with detectable t(14;18) pre-transplant) of the 11 recipients receiving stem cells from t(14;18)-positive donors had post-transplant samples available for RQ-PCR study allowing for study of the effect on transplant outcomes of the donor t(14;18) cells in the recipients (Table 1). Two (case# 923 and 345, Table 1) of the four recipients who did not have detectable t(14;18) pre-transplant had detectable t(14;18) (quantitative levels <0.01%) at 2 and 11 years post transplant (Table 4), after receiving donor stem cells positive for t(14;18). Neither of these two recipients developed FL post transplant at 3.5 and 13 years post transplant, 1.5 and 2 years after the emergence of the t(14;18) cells that were possibly of donor origin. The remaining two recipients continued to have no detectable t(14;18) cells post transplant and remained free of disease.

Table 4 - Percent of t(14;18)-positive cells in recipients of donor positive for t(14;18).

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Three recipients (Case# 762, 342, and 573) who had detectable t(14;18) before transplantation received grafts from t(14;18)-positive donors, thus creating a scenario in which the donor's t(14;18) clone could confound post transplant MRD determination. One (case# 762) of these three cases had relapsed disease of the same clone of recipient origin as indicated by the same band size of the IgH/Bcl-2 PCR products before and after transplant (data not shown). The second case (case#342) had detectable t(14;18) at the level of <0.01%, possibly of donor origin, after transplant. This clone was detected as early as 6 months after transplant (Table 4). The recipient remains in complete remission at 145 months post transplant (Table 4). The third case (case#573), reported previously,¹⁷ had detectable t(14;18) of donor origin with eight serial determinations over a 5-year period (Table 4). This patient never developed FL with follow-up of 109 months.

Discussion

We found that a significant proportion (48%) of donors used to transplant FL patients carried t(14;18) cells. The percentage of donors carrying t(14;18) in this study is comparable to that found in the literature. The reported mean prevalence of t(14;18) in peripheral blood of healthy individuals is 36%, ranging from 8 to 100%, depending on the sensitivity of the PCR assays used to detect t(14;18).^{4, 5, 6, 7, 8, 9} In our study, quantitative levels of t(14;18) in healthy donors were low (<0.01%). This finding also correlates well with other studies that have shown a low quantity of cells carrying t(14;18) in individuals without FL.^{4, 5, 6, 7, 8, 9}

Our results suggest that the presence of t(14;18) in the donors may not significantly alter the overall clinical outcome of recipients, although the sample size is relatively small. Differences in engraftment, incidence or severity of GVHD, rates of relapse, overall or DFS were not identified based on donor's t(14:18) status. In our previous case report, we suggested that detectable t(14;18) cells in donors should not result in donor exclusion for alloSCT, even for recipients with FLs. The lack of an adverse effect of infused donor-derived t(14;18) cells in this cohort of patients further supports this suggestion. This is of clinical importance given that t(14;18) is commonly detected in healthy individuals. In addition, donor clones carrying t(14;18) that may be given to the recipient did not result in the development of FL even after a prolonged period of observation. This supports the concept as previously expressed in a nontransplant setting that genetic abnormalities in addition to t(14;18) are required for lymphomagenesis.²³ Our findings further support that the need for multiple genetic abnormalities in the lymphoma clone remains valid in the recipients who by virtue of their diseases may have a genetic susceptibility to develop FL and who are immunosuppressed secondary to the transplantation regimen. The need for multiple genetic abnormalities in the lymphoma clone is further supported by the identification of clones that are immunophenotypically identical to B-cell chronic lymphocytic leukemia (CLL) in healthy individuals from CLL kindreds (14–18%) as well as in the general population (3.5% in age bracket >65 years).²⁴ Also of note is that the majority (20/23 or 87%) of recipients in this study received TCD grafts possibly resulting in a greater degree of immunosuppression than non-TCD grafts.

The results of the current study further support the notion that monitoring of MRD should be performed by quantitative type of PCR assays, preferably RQ-PCR, but not qualitative type of PCR assays.^{16, 18, 19, 20, 25, 26, 27, 28} Only a subset (3/8 or 37.5%, Tables 1 and 2) of recipients in our study with detectable t(14;18) post transplant demonstrated evidence of FL relapse. Notably, the quantity of t(14;18)-positive cells appears to be the determining factor for clinical outcome. All recipients (n=5) with a low quantity (<0.01%) of detectable t(14;18)-positive cells remained in long-term clinical remission, similar to recipients with undetectable t(14;18) cells post transplant. These results further strengthen our previous observation that a negative or low quantity of t(14;18)-positive cells (<0.01%) after SCT may correlate with clinical remission.¹⁶ Similar to our observations, increasing quantitative levels of cells carrying t(14;18) seen with serial determinations has been shown to correlate with FL recurrence after autologous bone marrow transplant.¹⁹

Our findings further denote that it may be essential to screen for the donor's t(14;18) status before using t(14;18) as the target for monitoring minimal residual disease (MRD) by RQ-PCR in FL patients after alloSCT to determine if there is a confounding donor's t(14;18) clone. We demonstrated that the significance of post transplant presence of t(14;18) in the recipients might be affected by whether the recipients had received grafts from donors carrying t(14;18) cells or not. Highlighting this is our experience in which two patients that were t(14;18) negative pre-transplant became t(14;18) positive at levels of <0.01% post transplant after receiving grafts from donors carrying t(14;18) cells. Additionally, another two patients that were t(14;18) positive pre-transplant showed low levels (<0.01 to 0.04%) of t(14;18) cells after transplant. These four patients were free of FL with follow-up ranging from 43 to 157 months, suggesting that these t(14;18) cells may be of donor origin and do not indicate pending relapse. However, the possibilities that these t(14;18) cells represent molecular relapse or random events that occur in 'normal' people cannot be completely excluded. Additional testing such as sequencing of the t(14;18) breakpoint region of donor and recipient samples would be needed to verify that the t(14;18) clones from the recipient and donor are truly the same clone. This determination could not be performed in the current study due to the limited availability of donor and recipient samples.

In summary, our results indicate that transplanting FL patients with grafts from donors carrying t(14;18) cells is not an uncommon event. The lack of an association of t(14;18) detected in donors with adverse clinical outcome documented in this study suggests that these donors are suitable for alloSCT. The current study is, however, limited by its retrospective nature and small sample size. Prospective studies with a larger sample size are indicated to validate our findings. Moreover, It may be necessary to screen for the donor's t(14;18) status before using t(14;18) for monitoring MRD by RQ-PCR to exclude the possibility of confounding donor's t(14;18) clone. Prospective studies evaluating this matter by sequencing in order to check for identical or different clones are warranted.

References

References