Minimal Residual Disease

PCR detection of residual Bcl-2/IgH-positive cells after high-dose therapy with autologous stem cell transplantation is a prognostic factor for event-free survival in patients with low-grade follicular non-Hodgkin's lymphoma

Summary:

This study was designed to evaluate the results of high-dose therapy followed by purged autologous stem cell transplantation (ASCT) for patients with low-grade follicular non Hodgkin's lymphoma (LGFL), and the prognostic significance of PCR detection of residual Bcl-2/IgH-positive cells after ASCT. Between 1992 and 1998, 49 patients with LGFL received total body irradiation and high-dose cyclophosphamide followed by purged ASCT. PCR amplification of the Bcl-2/IgH rearrangement was performed at diagnosis, on stem cell collections before and after purging and on bone marrow and blood samples after ASCT. With a median follow-up of 76 months (37–103) 34 patients remain alive and event-free. A total of 20 patients had disease recurrence, three patients developed secondary myelodysplastic syndrome (MDS). In all, 11 patients died; 10 deaths were because of recurrent disease, one because of MDS. Kaplan–Meier estimates of event-free survival (EFS) and overall survival (OS) at 5 years were 65% (±7%) and 77% (±6%), respectively. Patients who achieved a sustained molecular complete response (CR) had a lower risk of disease recurrence and experienced significantly longer EFS (93% (±6%) vs 11% (±7%) P=0.0008) and OS (100 vs 55% (±12%) P=0.0057). In conclusion, myeloablative therapy followed by purged ASCT may induce long EFS in patients with LGFL. The achievement of sustained molecular CR after ASCT improves EFS and OS.

Main

Low-grade follicular non-Hodgkin's lymphomas (LGFL) is an incurable disease with current conventional chemotherapy regimens: the median survival is between 5 and 10 years without a plateau. All patients relapse with a disease-free survival of 25% at 5 years and of less than 10% at 10 years.1,2,3,4 An increasing number of reports on the use of intensive myeloablative therapy with autologous stem cell support in patients with LGFL show encouraging results in comparison to conventional chemotherapy.5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 However, since there is evidence of tumor contamination of most of the bone marrow (BM) or peripheral blood stem cell (PBSC) grafts,20 autografting carries the risk of reinfusing abnormal cells, which may contribute to disease recurrence.21,22 Attempts have therefore been made to reduce or remove lymphoma cells from the grafts.11,12,13,14,15,16,17,18,19 LGFL bears a characteristic chromosomal abnormality involving the Bcl-2 gene on chromosome 18 and the Ig heavy chain (IgH) locus on chromosome 14. Bcl-2/IgH rearrangement is present in about 50–70% of LGFL.23 Minimal residual disease (MRD) can be detected by PCR amplification of Bcl-2/IgH.24 This technique allows the evaluation of the graft purging quality as well as the monitoring of MRD after autologous stem cell transplantation (ASCT).

We have reviewed our single-center results of purged ASCT for patients with LGFL. The aims of this study were to evaluate (1) the ability of three purging techniques to reduce tumor-cell contamination of the graft, (2) the efficacy of high-dose therapy, (3) the effect of the infusion of a PCR-negative graft on the probability of remaining in complete remission after ASCT, and (4) the prognostic significance of PCR detection of residual cells after ASCT.

Patients and methods

Patients

Between 1992 and 1998, 49 patients with LGFL underwent treatment with myeloablative radiochemotherapy followed with purged ASCT. There were 18 women and 31 men. Median age was 46 years (range 28–60), and median duration of disease before ASCT 8 months (range 6 months–12 years). The Ann Arbor disease stage at diagnosis was III in 15 patients and IV in 34; all stage IVs, were because of BM involvement. At the time of ASCT, 32 patients were in first response (seven complete and 25 partial) and 17 patients in second or third response (four complete and 13 partial). Clinical characteristics of the patients are listed in Table 1.

Table 1 Patient characteristics

Treatment programme

For the 32 patients transplanted in first response, tumor reduction before stem cell harvesting was achieved with three cycles of VCAP regimen (cyclophosphamide 1500 mg/m2 day 1, doxorubicine 80 mg/m2 day 1, vincristine 2 mg day 1, prednisone 60 mg/m2 days 1–5). A total of 17 patients were transplanted with more advanced disease. Before graft harvesting they received either the VCAP regimen (11 patients) or the CHOP regimen (two patients) or platinum containing chemotherapy (three patients). One patient did not receive any chemotherapy. For these 17 patients, first-line therapy had consisted of chlorambucil (one patient) or mini-CHVP (10 patients) or other CHOP-like regimens (five patients) or radiotherapy alone (one patient). The conditioning regimen consisted of total body irradiation (TBI), with six 2 Gy fractions over 3 days with lung shielding at 8 Gy followed by 60 mg/kg of cyclophosphamide on two consecutive days. The graft was reinfused within 48 h after the last dose of cyclophosphamide. Most of the patients received G-CSF (Neupogen, Amgen) intravenously at a daily dose of 300 μg given from day 7 after ASCT until granulocyte engraftment. Irradiated, leukocyte poor platelets and red blood cells were transfused when platelet counts were <20 × 109/l and when the hemoglobin was <8 g/dl, respectively. Episodes of febrile neutropenia were first treated with broad-spectrum antibiotics. Intravenous amphotericin B was used when fever persisted 48 h after initiation of antibacterial antibiotics. Patients were housed in laminar air flow rooms until neutrophil recovery.

Stem cell collection and processing

Bone marrow was harvested from 28 patients under general anesthesia. In 23 patients, the mononuclear cell fraction was isolated on Ficoll–hypaque and processed with three rounds of two anti-B-cell monoclonal antibodies (anti-CD20, anti-CD19) and baby rabbit complement, as previously described.6 In five patients, immunomagnetic purging was performed and the mononuclear cell fraction was incubated with anti-B-cell antibodies. The sensitized cells were mixed with magnetic sheep anti-mouse SAM-coated beads to allow rosette formation. Rosettes and unbound beads were removed by passage through the Maxsep™ Magnetic Cell Separator (Baxter Biotech Group). In 21 patients peripheral PBSCs were collected with three leukaphereses following G-CSF mobilization. The first two leukapheresis products were pooled and subjected to positive selection of CD34+ cells over a computer-driven avidin immunoaffinity column device (CEPRATE stem cell concentrator system: Cell Pro Inc. Bothell, WA, USA) as previously described.25 The product of the third leukapheresis was kept unmanipulated as a rescue in case of nonengraftment.

Clonogenic assay and flow cytometry analysis for hematopoietic progenitor cells

In 28 BM collections performed between 1992 and 1995, only clonogenic assays were carried out, formation of colony-forming unit granulocyte-macrophage was scored as previously described.25 For the 21 PBSCs collected from 1994, quantitation of CD34+ cells was performed by flow cytometry analysis as described elsewhere.25

Bcl-2/IgH rearrangement analysis

To determine the presence of Bcl-2/IgH rearrangement, fresh samples of lymph node, BM, and blood were obtained from patients at diagnosis. Samples from stem cell collections were analyzed before and after purging. Following ASCT, patients were monitored regularly, with BM and/or blood samples for PCR analysis taken at 3, 6, 12 months and yearly thereafter. Nested oligonucleotide amplification was performed at the major breakpoint of the Bcl-2/IgH hybrid gene as previously described.25 The limit of sensitivity of the technique, determined by serial dilutions of the Karpas 422 cell line, was two tumor cells in 105 normal cells.

Evaluation and statistical methods

Survival was calculated from the day of ASCT to the date of death from any cause or the date of last follow-up. An event was defined as relapse of disease, or histological transformation, or occurrence of myelodysplastic syndrome. Event-free survival (EFS) was calculated from the day of ASCT to the date of an event or the date that the patient was known to be alive and event-free. Overall survival (OS) and EFS curves were estimated by the method of Kaplan and Meier26 and compared by the log rank test.27 Comparison of patient characterstics who were PCR positive or PCR negative was performed with the χ2 test. The criterion for statistical significance was P<0.05. Comparison of non parametric values used the Wilcoxon rank sum test. The statistical software was SPSS Version 9.0.

Results

Results of in vitro purging procedures

A Bcl-2/IgH rearrangement was detected in 28 of 49 patients (57%) at diagnosis in fresh samples of lymph node and/or bone marrow, and/or blood. In all, 27 stem cell collections were tested for Bcl-2/IgH rearrangement before and after purging (one was not tested). Four (15%) were found to be PCR negative before in vitro purging (three BM and one PBSC). In vivo purging occurred in three patients in the first partial response and in one patient in the second partial response, all of whom had received the VCAP regimen. Within 23 PCR-positive harvests, B-cell depletion or CD34+ cell selection resulted in complete tumor-cell depletion of the graft in six cases (26%): three out of 15 B-cell-depleted grafts and three out of 13 CD34 positively selected grafts. The small numbers of patients preclude a meaningful comparison of the effectiveness of each purging technique. The six successfully purged were achieved in grafts from patients in clinical CR without histological BM infiltration in two cases, and from patients in clinical PR in four cases, including two patients with BM infiltration.

Results of high-dose therapy with purged ASCT

A total of 21 patients received a CD34 positively selected graft (containing a median number of 2.7 × 106 CD34+ cells/kg (range 0.97–40)) and 28 received a B-cell-depleted bone marrow graft (for which the number of CD34+ cells was not available). All patients but one engrafted. The median time to reach a granulocyte count >0.5 × 109/l and an unsupported platelet count > 20 × 109/l was 14 days (range 7–31) and 16 days (range 4–64), respectively. One patient experienced early graft failure and received the rescue graft. Granulocytes and platelets engrafted faster in patients who had received a CD34+ selected PBSC transplant than in those transplanted with purged BM: 11 days (range 7–14) vs 17 days (range 10–31) (P=0.009) and 11 days (range 4–49) vs 20 days (range 7–64) (P<0.0001) for granulocyte and platelet engraftment, respectively. The treatment was well tolerated. No early adverse events were observed except for neutropenic fevers. No toxic deaths were recorded. Clinical complete remission (CR) was achieved in the 49 patients. After a median follow-up time of 76 months (range 37–107 months) 38 of 49 patients are still alive. In all, 11 patients died at a median of 16 months (range 3–84) after ASCT. A total of 10 deaths were because of recurrent disease with histological transformation, and one death was because of myelodysplastic syndrome (MDS). As shown in Figure 1a, the estimated 5-year OS is 77% (±6%). A total of 20 patients had lymphoma recurrence after a median of 12 months (range 3–98). Three patients have developed MDS 16, 16 and 36 months after ASCT, one of whom died; 19 continue in remission, one with MDS. Therefore, the 5-year EFS is 65% (±7%) (Figure 1b).

Figure1
figure1

(a, b) Kaplan–Meier's estimate of probability of OS (a) and EFS (b) from ASCT for the 49 patients.

Prognostic factors for OS and EFS

We analyzed the prognostic significance of clinical characteristics and of results of PCR analysis performed at diagnosis, in the grafts and during the follow-up period. The 5-year OS and EFS of the patients with Bcl-2-negative lymphoma at diagnosis were no different from those of patients with Bcl-2-positive lymphoma at diagnosis (P=0.9).

Clinical prognostic factors for OS and EFS

There was no correlation between OS or EFS and the number of previous lines of chemotherapy, or disease status at the time of ASCT.

Prognostic impact of complete graft purging

Given the small size of the two groups of patients, we found no significant differences in term of OS or EFS between the 10 patients transplanted with a PCR-negative graft (OS: 89% (±10%), EFS: 78% (±14%)) and the 17 patients who had received a PCR-positive graft (OS: 68% (±15%), EFS: 64% (±12%)).

Prognostic significance of PCR detection of Bcl-2/IgH rearrangement during the follow-up period

PCR-based monitoring was performed during the follow-up period in 24 of the 28 relevant patients (Figure 2). Out of the 24 patients, 10 remained PCR negative from ASCT to the last follow-up, five became PCR negative 12–48 months (mediane 12 months) after ASCT without further treatment and then remained PCR negative over time. Only one of these 15 patients relapsed 17 months after ASCT. Seven patients remained PCR positive from ASCT to the last follow-up, and two converted to PCR positivity, 12 and 24 months after ASCT, and remained PCR positive. In this group of nine patients, eight have relapsed 11–98 months after ASCT (median 22 months). Hence, the persistence or the durable reappearence of PCR-positive cells is associated with a higher risk of relapse (8/9 vs 1/15 P=0.001). As shown in Figure 3, the 5-year OS and EFS for the 15 patients who achieved a sustained molecular CR (100 and 93 (±6%), respectively) were significantly better than OS and EFS for the nine patients who never achieved a durable molecular CR (55% (±12%) (P=0.0057) and 11% (±7%) (P=0.0008), respectively).

Figure2
figure2

PCR analysis of samples of stem cell collection before and after purging, and of BM or blood samples during the follow-up period after ASCT in 28 patients with BcL-2/IgH rearrangement. +: represents PCR-positive results; −: represents PCR-negative results; R: relapses.

Figure3
figure3

(a, b) OS (a) and EFS (b) curves according to molecular status after ASCT: sustained molecular CR in 15 patients (solid line) or no molecular CR in nine patients (dotted line).

Clinical factors for achievement of a molecular CR

The achievement of a molecular CR after ASCT was not associated with disease status at ASCT, since, of the 24 patients monitored after ASCT, a durable molecular response was achieved in 11/17 transplanted in first response vs 4/7 transplanted with more advanced disease. A durable molecular CR was achieved in 4/4 patients transplanted in clinical CR at the time of ASCT, vs 11/20 patients transplanted in clinical PR.

Impact of PBSC vs BM transplantation on the achievement of molecular CR

A total of 15 patients received BM grafts; three BM harvests were PCR negative before in vitro purging, and three became PCR negative after B-cell depletion. After ASCT six patients who had received BM grafts remained or became PCR negative and one of them relapsed, seven remained or became PCR positive, of whom all relapsed, two were not evaluated and both relapsed. In all, 13 patients received PBSC grafts, of whom one was PCR negative before in vitro purging and three became PCR negative after CD34+ cell selection. After ASCT, nine patients remained or became PCR negative and none relapsed, two became PCR positive, of whom one relapsed, and three were not evaluated, of whom two relapsed. Finally, 13 out of 16 evaluated patients who had received BM grafts, vs nine out of 10 evaluated patients who had received PBSC graft achieved a molecular CR (P=NS). The OS and EFS of the BM group and the PBSC group were 73 (±12%) and 46 (±13%) (P=0.1), and 85 (±10%) and 84 (±10%) (P=0.1), respectively.

Impact of complete graft purging on the achievement of a molecular CR

We found no association between the reinfusion of a PCR-negative graft and the achievement of a durable molecular CR after ASCT: 10 of the 27 Bcl-2-positive patients evaluated (37%) were transplanted with a PCR-negative graft of whom 7/9 remained PCR negative 12–83 months after ASCT (one relapsed at 18 months), 2/9 became PCR positive at 3 and 36 months (one relapsed at 12 months), one patient was not monitored and he did not relapse. A total of 17 patients were transplanted with a PCR-positive graft: 6/14 remained PCR positive, three of whom have relapsed at 19, 36 and 51 months, 8/14 became PCR negative; the PCR conversion occurring between 3 months and 4 years (median 12 months) after ASCT and persisting until the last follow-up, none of whom relapsed. Three of the patients who had received a PCR-positive graft were not monitored after ASCT; these three patients relapsed at 3 and 6 months from the ASCT (Figure 2). Thus, based on a small number of patients, PCR negativity of the graft is not associated with a better chance of achieving sustained PCR negativity after ASCT.

Discussion

In this report, we present the results of 49 purged autotransplant in patients with LGFL and show that the achievement of molecular CR after ASCT improved OS and EFS. High-dose therapy with TBI and high-dose cyclophosphamide followed by purged ASCT proved to be effective regarding tumor reduction, since every patient achieved clinical CR, and 15 of the 24 (62%) patients evaluable for Bcl-2/IgH rearrangement achieved a molecular CR. The 5-years EFS of 65% (±7%) observed in this study compares favorably with the results of conventional chemotherapy1,2,3,4 and is comparable to those previously observed after high-dose therapy with ASCT.5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 The only factor found to be associated with an improvement in EFS is the achievement of a durable molecular CR after ASCT, but no parameter was identified as predictive of the probability of achieving a molecular CR. The probability of EFS was not found to be related to disease status at the time of ASCT, differing from most of the studies previously reported.5,8,9,10,11,12,16,18 A possible explanation might be that in our series ASCT was performed only in patients with chemosensitive disease and without histological transformation. We found a significant difference in term of recurrence risk, OS and EFS between the nine patients in whom Bcl-2-positive cells persisted or reappeared after ASCT and the 15 patients who remained, or became durably Bcl-2 negative. These results are in accordance with previous studies.14,15,16,17,18,19,28,29,30,31 Therefore, the achievement of a molecular CR should be attempted in the therapeutic program. Achieving molecular CR should combine adequate tumor reduction before the graft harvesting, an effective method of in vitro graft purging, an effective conditioning regimen, and methods that could eradicate residual disease in patients remaining PCR positive after ASCT. In our study, the VCAP regimen produced 18% of clinical CRs and allowed the collection of PCR-negative stem cells in 15% of the 27 PCR-positive patients. This compares favorably with conventional chemotherapy that is usually unable to eradicate Bcl-2-positive cells;24 however, better results have been reported with sequential high-dose therapy that allowed the achievement of PCR negativity in 28–68% of PBSC or BM harvests.7,29 The question of whether the presence of lymphoma cells within the reinfused graft contributes to recurrence remains contentious: the prognostic value of the ability to purge residual lymphoma cells from grafts emerged from three previous studies,17,29,30 but was not found in three other studies.13,14,18 Here, six out of 23 (26%) PCR-positive stem cell collections were successfully purged. These results are similar to most of those previously reported.13,14,15,17,18,29 In addition, four stem cell harvests were PCR negative before in vitro purging. Thus, 10 (37%) of the evaluable grafts were PCR negative, and we observed no improvement of EFS in patients transplanted with PCR-negative graft as compared to those transplanted with grafts that contained PCR-detectable cells, but the number of patients in each group is small. In addition, the sensitivity of our PCR assay was relatively low (2/105) compared to others. Thus, it is possible that the residual tumor burden in the PCR-negative and -positive grafts was similar. Only quantitative evaluation would allow correct measurement of this parameter. It is not possible to ascertain whether PCR-positive cells in the autograft contributed to disease recurrence or signified a greater tumor burden associated with a higher risk of treatment failure, since within the 17 PCR-positive grafts, 14 originated from patients not in clinical CR at time of ASCT, in whom the higher risk of relapse may have been be related to reduced chemoradiosensitivity of the residual tumor cells. Regarding the conditioning regimen, reports on conventional radiotherapy32 suggest that TBI may play an essential role because of the particular radiosensitivity of LGFL, and in most studies TBI is included in the conditioning regimen. Three out of the 49 patients developed MDS, and this is of particular concern in patients who may experience prolonged survival after ASCT. The three patients had been transplanted with first-line therapy after three cycles of a CHOP-like regimen, had relapsed after ASCT and had achieved a second response with CHOP-like chemotherapy, before the occurrence of MDS. The risk of MDS is well described after high-dose therapy with ASCT,15,16,17 and may also relate to prior chemotherapy and/or to chemotherapy administered at the time of relapse post-transplant. Finally, additional therapeutic interventions in patients who remain PCR positive after ASCT should be considered: ongoing studies with IFNα,33 monoclonal antibodies, either conjugated to toxins or unconjugated, such as Rituximab34 are attractive treatments for minimal residual disease, and do not involve an increased risk of MSD. The use of a nonmyeloablative conditioning regimen reduces the risk of allogeneic transplantation-related toxicity, thus making a second transplant feasible.35 This strategy could also be proposed for the treatment of residual disease persisting or reappearing after ASCT. In conclusion, patients with LGFL would be more likely to be cured if Bcl-2-positive cells could be eliminated, and therefore attainment of molecular CR should be attempted. Nevertheless, it is difficult to ignore the evidence that PCR amplification has detected residual lymphoma cells in patients with LGFL who have had very long-term CRs.36 Only prospective studies can determine the benefit of high-dose therapy supported with purged stem cells.

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Acknowledgements

We gratefully acknowledge the excellent technical assistance of A Cassidanius, S Bercegeay, and C Even.

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Mahé, B., Milpied, N., Mellerin, M. et al. PCR detection of residual Bcl-2/IgH-positive cells after high-dose therapy with autologous stem cell transplantation is a prognostic factor for event-free survival in patients with low-grade follicular non-Hodgkin's lymphoma. Bone Marrow Transplant 31, 467–473 (2003). https://doi.org/10.1038/sj.bmt.1703829

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Keywords

  • follicular non-Hodgkin's lymphoma
  • autologous stem cell transplantation
  • residual disease

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