Donor chimerism does not predict response to donor lymphocyte infusion for relapsed chronic myelogenous leukemia after allogeneic hematopoietic cell transplantation

Article Outline

• Abstract
• Introduction
• Materials and methods
  • Patients
  • Donor lymphocyte infusions
  • Definitions of outcomes
  • Statistical analysis
• Results
  • All patients
  • Patients with CML
    • Effect of chimerism on remission and survival of CML patients
    • Effect of chimerism on GVHD and aplasia
  • Patients with non-CML malignancies
• Discussion
• Acknowledgements
• References
• Copyright

Abstract 

We studied the effect of donor chimerism level on the outcome of donor lymphocyte infusion (DLI) therapy in 42 patients with persistent or relapsed hematologic malignancies after non-T cell-depleted allogeneic hematopoietic cell transplantation. Seventy-five percent of chronic myelogenous leukemia (CML) and 39% of non-CML patients entered remission after DLI therapy. Remission and survival rates were similar for CML patients irrespective of their pre-DLI donor chimerism levels; however, remission occurred sooner in patients with ≥10% pre-DLI donor chimerism. None of the non-CML patients with <10% pre-DLI donor chimerism and 47% of those with ≥10% pre-DLI donor chimerism attained remission. The 2-year survival rates after DLI were 75% for CML and 17% for non-CML patients. We conclude that a low level of donor marrow chimerism is not an adverse prognostic factor for response to DLI in CML patients, but for non-CML patients it may confer worse outcomes. Better methods to augment the response to DLI for patients with hematologic malignancies other than CML that recur after allogeneic hematopoietic cell transplantation are needed, whereas for relapsed CML patients, combination therapies including imatinib mesylate or other promising antileukemic agents may provide outcomes superior to those with DLI alone.

Keywords:  Donor lymphocyte infusion, Chimerism, Hematopoietic cell transplantation

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Introduction 

Donor lymphocyte infusions (DLI) can produce lasting remissions in patients with relapsed chronic myelogenous leukemia (CML) after allogeneic hematopoietic cell transplantation (HCT), but they work less well for other malignancies [1], [2]. This form of adoptive immunotherapy induces a graft-versus-leukemia effect (GVL) mediated by graft-versus-host alloreactive donor T cells [1], [3]. Persistent or recurring disease after allogeneic HCT reflects a state of mixed chimerism in which infusion of donor lymphocytes could overcome the persistence of recipient cells and mediate a leukemia-specific or an allospecific GVL response [4]. DLI therapy can also cause complications such as severe graft-versus-host disease (GVHD) and myelosuppression, which occur in up to 40% of patients, and the mortality attributed to DLI therapy may be as high as 18% at 2 years after DLI [1], [2].

Variables known to have a positive influence on survival after DLI therapy include underlying CML (rather than other hematologic malignancies) and achievement of complete remission (CR) [1], [5]. The presence of donor T lymphocytes may correlate with achievement of remission after DLI for patients who relapse after lymphocyte-depleted bone marrow transplantation [6]. In this study, we describe the effect of the pre-DLI donor chimerism level on remission, survival, and toxicities after DLI therapy for relapsed hematologic malignancies in recipients of T cell-replete allogeneic HCT.

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Materials and methods 

Patients 

Forty-two patients received DLI at our institution between July 1993 and December 1999. Written, informed consent was obtained from all patients and donors, and the study was approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. Twenty-four patients had CML, 10 had acute myelogenous leukemia, 6 had myelodysplastic syndrome, 1 had acute lymphocytic leukemia, and 1 had juvenile myelomonocytic leukemia. Patients had received T cell-replete HCT from HLA-matched siblings (n = 34), a 1 antigen-mismatch sibling (n = 1), or HLA-matched (n = 7) unrelated donors. Among 18 non-CML patients, 11 received chemotherapy a median of 75 days (range, 30-144 days) before DLI, and 4 patients (36%) attained CR attributable to chemotherapy before DLI. Three CML patients (12%) and 3 non-CML patients (17%) had received multiple courses of DLI (median, 2) for persistent or progressive disease. Two (67%) of the 3 CML patients and none of the 3 non-CML patients attained CR. If a patient received multiple courses of DLI, the date of the last DLI before CR was attained was considered in evaluating outcomes. If a patient had not attained CR, the date of the first administered DLI course was considered in evaluating outcomes. For the 2 CML patients who attained CR, the initial DLI course was 4 and 8 months before the successful DLI, respectively, and both patients attained CR 5 months later. Patient characteristics are shown in Table 1. The date of the last follow-up was June 2002 or the date of last contact with a patient.

Table 1. Patient Characteristics

Donor lymphocyte infusions 

Donor lymphocytes from the original donor were obtained by lymphapheresis. Before 1996, CML and non-CML patients received 3 lymphapheresis products, irrespective of the cell dose. Starting in February 1996, all CML patients received a fixed dose of 1 × 10⁸ CD3⁺ T cells per kilogram (n = 8), whereas non-CML patients received 3 lymphapheresis products. No patients were receiving immunosuppressive medications at the time of DLI.

Definitions of outcomes 

CR of leukemia was defined as the normalization of blood counts and marrow cellularity, the absence of any morphologic evidence of leukemia on peripheral blood and marrow evaluation, and the absence of cytogenetic abnormalities. For patients with CML, CR was defined as complete cytogenetic remission (0% Ph⁺ cells and no other cytogenetic abnormalities) and molecular remission (no BCR/ABL messenger RNA detected by reverse transcription-polymerase chain reaction [PCR] analysis). All the CML and non-CML patients deemed in CR also had evidence of 100% donor chimerism.

Chimerism was evaluated by quantitative PCR of informative polymorphic variable number tandem repeat regions [7]. Posttransplantation genomic DNA isolated from recipient bone marrow mononuclear cells was amplified with fluorescent PCR primers for variable number tandem repeat regions found to distinguish donor from recipient alleles. The fluorescent PCR products were separated by gel electrophoresis on an Applied Biosystems 373 Sequencer (Applied Biosystems, Foster City, CA). The GeneScan software (Applied Biosystems) package was used to correlate allele peak areas with the percentage of donor or recipient DNA. The chimerism assay can detect a minimum of 5% donor-derived DNA on a recipient DNA background.

Complete donor chimerism was defined as the presence of 100% donor-derived hematopoietic cells, and mixed chimerism was defined as the presence of a mixture of donor and recipient hematopoietic cells on bone marrow analysis. We chose a cutoff of 10% for the donor chimerism analysis to compare patients who had only minor donor chimerism with those who had a larger fraction of donor cells present at the time of the DLI infusion. The cutoff of 10% is a value close to the median pre-DLI donor chimerism level for the CML patients (8.45%) and therefore thought to be relevant for analysis. Chimerism analyses of peripheral blood were not considered for categorization in this study.

The clinical manifestations of acute GVHD were graded I to IV according to criteria described by Thomas et al.[8], and chronic GVHD was classified as limited or extensive (requiring treatment), as described by Shulman et al.[9]. GVHD that occurred ≤3 months after HCT or DLI was defined as acute, and beyond 3 months it was defined as chronic.

Statistical analysis 

The estimation of survival and relapse-free survival was calculated by the Kaplan-Meier method. Survival was measured from the time of the last DLI until death or last follow-up [10]. Relapse-free survival was measured from the time of confirmed CR until relapse, death, or last follow-up, whichever came first. The cumulative incidence of CR was calculated by treating deaths without attaining remission as a competing risk [11]. The statistical comparisons among factors were completed by the log-rank test. For comparisons among factors with n <5 in a group, we used the Fisher exact test. Univariate analyses were performed for the factors of pre-DLI donor chimerism, DLI dose, age, time from transplantation to relapse and from relapse to DLI, acute and chronic GVHD after DLI, CR after DLI, and relapse phase at DLI (for CML patients). Acute and chronic GVHD were tested as time-dependent factors in a Cox regression model [12].

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Results 

All patients 

Twenty five (60%) of 42 patients achieved CR after DLI. Eighteen (72%) of the 25 patients had CML, and 7 patients (28%) had non-CML diseases (6 patients with acute myelogenous leukemia and 1 patient with myelodysplastic syndrome). As previous studies have shown, patients treated for relapsed CML have higher survival rates compared with those treated for relapsed non-CML diseases (Figure 1). Because of marked differences in survival, all subsequent analyses are based on the 2 groups of patients: those with CML and non-CML diseases.

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• Figure 1. 

  Survival after DLI for CML and non-CML patients.

Patients with CML 

Eighteen (75%) of 24 CML patients (95% confidence interval, 50%–100%) achieved molecular remission after DLI; this was confirmed by conversion to negative BCR/ABL status by PCR analysis. The median survival was 54 months (range, 14–79 months) for patients who achieved CR (n = 18), versus 6 months (range, 2.6–35 months) for those who did not obtain CR after DLI (n = 6; P <.01). The median time to CR was 104 days (range, 35–279 days) after DLI. Two patients (11%) relapsed 3.5 and 65 months after CR was documented. The first patient died of respiratory failure secondary to radiation pneumonitis 5 months after relapse, and the other was treated with another course of DLI and is still alive with chronic-phase CML 7 months after relapse. Sixteen patients (67%) remain alive in CR after a median follow-up of 59 months (range, 30–79 months). Five years after DLI, CML patients had an overall probability of survival of 75% (range, 58%–92%), and for those who attained CR, the relapse-free survival was 94% (range, 82%–100%).

Because the donor chimerism level may predict the response to DLI [6], we compared remission and survival rates for CML patients with <10% or ≥10% donor chimerism at the time of DLI (pre-DLI donor chimerism). Survival rates at 5 years after DLI were similar for CML patients with <10% or ≥10% pre-DLI donor chimerism, as shown in Figure 2. CR was attained by 10 (77%) of 13 CML patients with <10% and by 8 (73%) of 11 patients with ≥10% pre-DLI donor chimerism. Patients with <10% chimerism achieved CR later (median, 5.6 months; range, 1.6–9.2 months) than those with ≥10% pre-DLI donor chimerism (median, 3 months; range, 1–5.8 months; P = .05). Although chimerism level may reflect leukemia cell burden, a more detailed analysis showed that high CR rates were obtained for all CML patients irrespective of the chimerism subgroups (Figure 3). Among the patients who achieved CR, the survival rates and risk of relapse were similar irrespective of the pre-DLI donor chimerism. The median pre-DLI donor chimerism for the CML patients who attained CR was 5%, compared with 10% for those who did not achieve CR.

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• Figure 2. 

  Overall survival after DLI for CML patients based on the pre-DLI donor chimerism.

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• Figure 3. 

  Complete response rates based on the pre-DLI donor chimerism levels (CML patients: n = 4 for 0%, n = 9 for 1%–9%, n = 5 for 10%–50%, and n = 6 for 51%–100% pre-DLI donor chimerism; non-CML patients: n = 1 for 0%, n = 2 for 1%–9%, n = 2 for 10%–50%, and n = 13 for 51%–100% pre-DLI donor chimerism).

Overall, GVHD developed in 17 (71%) of 24 CML patients after HCT and in 12 patients (50%) after DLI. Among the 17 patients who had acute and/or chronic GVHD after HCT, 13 (76%) achieved CR after DLI, compared with 5 (71%) of 7 patients without GVHD after HCT (P = not significant). CR rates were similar for the patients in whom GVHD did or did not develop after HCT or DLI or both (data not shown; P = not significant).

Acute GVHD (grade II to IV) occurred in 2 (15%) of 13 CML patients with <10% pre-DLI donor chimerism and in 5 (45%) of 11 patients with ≥10% pre-DLI donor chimerism (P = .11). Chronic GVHD necessitating treatment developed in 5 (38%) of 13 CML patients with <10% and in 5 (45%) of 11 patients with ≥10% pre-DLI donor chimerism (P = not significant). Remission (Table 2) and 5-year survival rates (Table 3) were similar among patients with or without acute or chronic GVHD.

Table 2. Factors Influencing Remission after DLI

NA indicates not applicable; NS, P >.20; CI, confidence interval; NC, nucleated cells.

Table 3. Factors Influencing Survival after DLI

NA indicates not applicable; NS, P >.20; CI, confidence interval; NC, nucleated cells.

None of the patients with ≥10% pre-DLI donor chimerism, but 2 of 13 CML patients (15%), both with 5% pre-DLI donor chimerism levels, developed bone marrow aplasia 1 and 4 months after the last course of DLI, without any associated GVHD. Both patients had received 2 courses (3 doses each) of DLI before pancytopenia developed, with total corresponding doses of 12 × 10⁸ and 7 × 10⁸ nucleated cells per kilogram. Both patients were in CR at the time of pancytopenia, received marrow and peripheral blood stem cell support with antithymocyte globulin preinfusion conditioning, and remain alive in CR at 27 and 71 months after DLI. These data are concordant with prior observations that residual donor hematopoiesis in relapsed patients may protect from DLI-induced bone marrow aplasia [13].

Patients with non-CML malignancies 

The overall probability of survival for non-CML patients was 17% (95% confidence interval, 0%–34%) at 2 years after DLI (Figure 1). Seven (39%) of 18 non-CML patients achieved CR. Among them, 4 (36%) of 11 patients pretreated with chemotherapy achieved CR before DLI and had a median survival of 20 months (range, 2.6–97 months). Three (43%) of 7 patients treated with DLI without prior induction chemotherapy attained CR at a median of 29 days (range, 27–70 days) after DLI and had a median survival of 5 months (range, 2.6–36 months). Three non-CML patients were treated with multiple courses of DLI; 2 never attained CR, whereas 1 patient was in CR from chemotherapy before DLI.

None of the 3 patients with <10% pre-DLI donor chimerism and 7 (47%) of 15 patients with ≥10% donor chimerism attained CR (P = .25). Moreover, none of the patients with <50% and 54% of those with ≥50% pre-DLI donor chimerism attained CR (Figure 3), but the difference was not statistically significant (P = .10). Survival rates were also similar for non-CML patients with <10% or ≥10% pre-DLI donor chimerism (Table 3). The median chimerism for the non-CML patients was 94% (range, 0%-100%), with 97% (range, 75%-100%) for those who attained CR and 65% (range, 0%-100%) for those who did not attain CR (P = .08).

Five (28%) of 18 non-CML patients developed GVHD after HCT, and 8 patients (44%) developed GVHD after DLI. None of the 5 patients who had GVHD and 7 (54%) of 13 patients without GVHD after HCT achieved CR. Acute (grade II to IV) and chronic GVHD occurred with similar incidence in patients with <10% or ≥10% pre-DLI donor chimerism (33% acute GVHD for both groups and 0% and 22% chronic GVHD for the patients with <10% or ≥10% donor chimerism, respectively). Notably, for non-CML patients, both the CR rates and the 2-year survival rates were higher for patients who developed chronic GVHD Table 1, Table 2.

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Discussion 

DLI can provide a direct GVL effect and offer an effective therapy for relapsed hematologic malignancies after hematopoietic stem cell transplantation [14]. Because DLIs are particularly effective in CML, deciding between the use of DLI and alternative therapies, such as the tyrosine kinase inhibitor imatinib mesylate, for relapsed CML patients may be influenced by certain prognostic factors. Relapsed non-CML diseases have a poor prognosis; only a minority of patients have a sustained response to DLI. Alternatively, a second transplantation can have transplant-related mortality of up to 69% [15], and 3-year leukemia-free survival rates vary between 11% and 52%, depending mainly on the interval between the first transplantation and relapse [15], [16], [17]. In an analysis of non-CML patients only, the best survival after second HCT was observed in patients who were in CR before receiving the second HCT and who relapsed beyond 292 days after the first HCT [17]. Prior studies have emphasized that early disease stage [5] and chronic GVHD are correlated with favorable outcomes after DLI [18], [19]. The role of the pre-DLI donor chimerism in predicting outcomes after DLI is more controversial. In patients who relapsed after T cell-depleted HCT, inferior remission rates (15% versus 77%) were seen for those with ≤40% as compared with >40% donor T lymphocytes at the time of DLI [6]. Other authors have not observed any chimerism influence on attaining CR after DLI [20] or in general outcomes for patients with multiple myeloma [21].

In this study, we found a small number of relapses and high remission and survival rates after DLI in CML patients, irrespective of the pre-DLI donor chimerism levels. In addition, low pre-DLI donor chimerism did not increase the risk of relapse in CML patients. Although a low donor chimerism may merely be a marker of disease relapse and therefore of worse outcomes, this was not the case for the CML patients—outcomes were as good as or better for those with lower pre-DLI donor chimerism levels. In contrast, for non-CML patients, pre-DLI donor chimerism levels were nonsignificantly lower (65% versus 97%; P = .08) for the patients who did not attain CR.

The use of DLI as the favored first-line therapy in relapsed CML patients after allogeneic HCT has been recently challenged by the specific inhibitor of the BCR/ABL tyrosine kinase imatinib mesylate (Gleevec; Novartis, Atlanta, GA). Recent reports showed encouraging results, with overall response rates as high as 79% and complete cytogenetic responses of 35% [22], [23]. In the study of Kantarjian et al.[22], imatinib mesylate was used in 28 patients, of whom 13 (46%) received DLI a median of 4 months (range, 2-39 months) before imatinib mesylate. Although it is likely that the responses noted in those patients (11 of 13 responded to imatinib mesylate) were due to imatinib mesylate itself, a DLI influence cannot be discarded, because it is well described that responses after DLI may be protracted and occur as late as 6 to 12 months after therapy [2], [4]. As we and others report durable molecular CR after DLI for relapsed CML after allogeneic HCT, no long-term series have yet been described using imatinib mesylate. For non-CML malignancies, DLI has more limited efficacy, and therefore its use in conjunction with induction chemotherapy or other novel postremission therapies should be explored.

In summary, we report that pre-DLI donor chimerism levels do not predict either response or survival and should not be considered a prognostic determinant factoring into the decision-making process of using DLI for treating relapsed CML after allogeneic HCT. Given the long-term excellent outcomes with DLI in CML (as shown here), the unknown durability of responses to imatinib mesylate, and the established differences in toxicity, future strategies need to address how to prioritize the use of these treatments.

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Acknowledgements 

We thank Roby Nicklow for assistance with patient follow-up. This work was supported by National Institutes of Health grant nos. PO1-CA-65493, P30-CA-77598, and R01-CA-72669.

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