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Early lymphocyte recovery is a predictive factor for prolonged survival after autologous hematopoietic stem cell transplantation for acute myelogenous leukemia

Abstract

Absolute lymphocyte count (ALC) recovery correlates with survival after autologous hematopoietic stem cell transplantation (AHSCT) for patients with multiple myeloma, non-Hodgkin's lymphoma, and metastatic breast cancer. The role of ALC recovery in relationship to clinical outcome after AHSCT in patients with acute myelogenous leukemia is unknown. We analyzed 45 patients who underwent AHSCT at Mayo Clinic, Rochester, Minnesota between 1990 and 2000. The ALC threshold was selected at 500 cells/μl on day 15 post-AHSCT based on our previous studies. Thirty-two females and 13 males were included in the study with a median age of 45 years (range 12–75). The median follow-up was 14 months with a maximum of 129 months. The median overall and leukemia-free survival were significantly better for the 23 patients with ALC at day 15 500 cells/μl compared with 22 patients with ALC <500 cells/μl (not yet reached vs 10 months, P < 0.0009; 105 vs 9 months, P < 0.0008, respectively). In conclusion, ALC 500 cells/μl on day 15 post-AHSCT is associated with better survival in acute myelogenous leukemia and requires further studies.

Introduction

Absolute lymphocyte count (ALC) correlates with survival in allogeneic stem cell transplantation.1,2 We have recently reported a correlation between ALC recovery on day 15 post-autologous hematopoietic stem cell transplantation (AHSCT) and prolonged survival in patients with multiple myeloma (MM),3 non-Hodgkin's lymphoma (NHL),3 and metastatic breast cancer.4 ALC is a powerful, independent prognostic factor for overall survival (OS) and progression-free survival (PFS) in patients with MM and NHL. To assess whether ALC recovery is associated with survival in patients with acute myelogenous leukemia (AML) post-AHSCT, we analyzed ALC recovery on day 15 post-AHSCT in AML.

Materials and methods

Patient population

Forty-five consecutive patients underwent AHSCT for AML at Mayo Clinic Rochester, MN, between January 1990 and December 2000. One patient had two autologous hematopoietic stem cell transplantations. Data were only used from the first transplant for this patient in the study. Data from transplant recipients were collected prospectively and entered into a computerized database. Response to therapy, relapse, and survival data were updated continuously. No patients were lost to follow-up. All patients gave written, informed consent allowing utilization of their medical records for medical research. Approval for the retrospective review of these records was obtained from the Mayo Clinic Institutional Review Board and was in accordance with US federal regulations and the Declaration of Helsinki.

Eligibility criteria

The eligibility criteria for patients to undergo AHSCT included adequate hepatic, pulmonary, cardiac and renal function; ECOG performance status of 0, 1, or 2; and no active infection. Patients were excluded if they did not meet the eligibility criteria or had any other concomitant illness that would preclude AHSCT.

Prognostic factors

The prognostic factors used were selected from multiple publications5,6,7,8 which have identified various prognostic factors for AML at diagnosis, pre-transplantation and post-transplantation. The prognostic factors at diagnosis included: cytogenetic abnormalities (favorable, intermediate, unfavorable), French–American–British (FAB) classification (M3 vs other), gender, and white blood cell (WBC) count (>10.5 × 109/l). The prognostic factors before transplantation included: age (>45 years old), clinical status (complete remission, partial remission, or relapse untreated), complete remission (CR) status alone, the number of consolidation regimens after achieving CR, duration of complete remission after induction chemotherapy (12 months), interval from CR1 or CR2 to transplantation (>4 months), and performance status prior to transplantation (ECOG 1). The prognostic factors after transplantation included: ALC at day 15 post-AHSCT, absolute neutrophil count (ANC) at day 15 post-AHSCT, conditioning regimens (total body irradiation (TBI) + chemotherapy vs chemotherapy alone), growth factor (G-CSF vs GM-CSF), platelet count at day 15 post-AHSCT and stem cell source (bone marrow vs peripheral blood). Favorable cytogenetic abnormalities included t(8;12), t(15;17) and inv(16)/t(16;16). Cytogenetic abnormalities of intermediate prognosis included normal karyotype and missing Y chromosome; all others as well as complex chromosomal changes were classified as unfavorable.6

Conditioning regimens

The conditioning regimens included: cyclophosphamide (60 mg/m2 on days −5, −4) and TBI (220 rads, twice a day on days −3, −2 and −1; 1320 cGy total dose) 33 patients; busulfan (1mg/kg on day −7 to day −4) and cyclophosphamide (60 mg/kg on days −3 and −2) eight patients. Four patients received busulfan (1 mg/kg on day −7 to day −4) and cyclophosphamide (50 mg/kg on day −5 to −2). All patients had stem cells reinfusion after high-dose therapy.

Stem cell source

The stem cell source for the AHSCT was either bone marrow (BM) or peripheral blood stem cell (PBSC). Twenty-five patients received unmobilized BM stem cells, while 20 patients received PBSC mobilized with Ara-C and granulocyte colony-stimulating factor (G-CSF). During the early years of the program, bone marrow was the preferred source of stem cells, whereas in the later years peripheral blood collection was the primary stem cell source. Patients unable to mobilize adequate peripheral CD34 cells (2 × 106/kg) underwent bone marrow harvest.

Supportive care

Post-transplantation growth factors have been in use since 1991. Three patients did not receive post-transplant growth factors. Of the 42 patients who received growth factors post-transplantation, 30 patients received G-CSF and 12 patients received granulocyte–macrophage colony-stimulating factor (GM-CSF). Different prophylactic antibiotics, antifungal, antiviral medications and transfusion requirements were used as per bone marrow transplant supportive care guidelines during the transplant phase.

Hematological engraftment

Neutrophil engraftment was defined at the first day of ANC 500 cells/μl × 3 consecutive days. Platelet engraftment was defined a platelet count of 20 × 109/l independent of transfusion support. ALC threshold (immunologic engraftment) was determined at 500 cells/μl at day 15 after transplantation based on our previous studies.

Response and survival

Complete remission (CR) was defined as normal bone marrow morphology with 20% cellularity and fewer than 5% blasts, resolution of previously abnormal cytogenetics, no evidence of extramedullary leukemia, ANC 1500/μl and platelets 100000/μl for at least 4 weeks. Patients with regenerated peripheral blood values but more than 5% and less than 25% myeloblasts were considered to be in partial remission (PR), as were patients fulfilling the bone marrow criteria of CR but without full recovery of peripheral blood platelet and/or white blood cell counts.

Overall survival (OS) time was measured from the date of transplantation to the date of death or last follow-up. Leukemia-free survival (LFS) was defined as time from transplantation to disease progression, relapse, or death.

Statistical analysis

OS and LFS times were analyzed using the method described by Kaplan and Meier.9 Univariate analysis was performed using the log-rank test, to identify patient characteristics that were predictive of outcome after AHSCT. The Cox proportional hazards model10 was used to assess ALC at day 15 as a prognostic factor for post-transplant OS and LFS rates as well as to adjust for other known prognostic factors. The prognostic factors included in the analysis are outlined under the prognostic factors in the Materials and methods section. We did not have records available at diagnosis for cytogenetics in seven patients.

The cut-off of ALC 500 cells/μl was based on our previous study.6 This choice of cut-point was supported by the data, as it yielded the greatest differential in survival based on χ2 values from log-rank tests. The χ2 and Fisher's exact tests were used to determine relationships between nominal variables; nonparametric tests were used for continuous variables. All P values represented are two-sided and statistical significance was declared at P < 0.05.

Results

Forty-five patients with AML who underwent AHSCT were identified for the study with a median age of 45 years (range 12–75) at transplantation. Patient characteristics prior to stem cell collection and at stem cell transplantation are described in Tables 1 and 2, respectively. Twenty-two patients developed AML de novo and three patients secondary AML from myelodysplastic syndrome. Two patients had central nervous system involvement and four patients presented with extramedullary disease (gum swelling, one patient and lymph nodes, three patients). Patients received a median number of two chemotherapy regimens prior to AHSCT (range 1–4) and a median number of treatment cycles of four (range 1–9). The median duration of complete remission after induction chemotherapy prior to transplantation was 9 months (1–88 months). The median time from diagnosis to undergo autologous stem cell transplantation was 14 months (range 3–88 months). Prior to transplantation 13 patients were in first CR, 20 patients in second CR, one patient in first PR, one patient in second PR, and 10 patients in first-relapsed untreated. The median duration from the time the patients were classified in complete remission (CR1 or CR2) to AHSCT was 5 months (range 0.5–37 months). Five patients received a purged stem cell product and none of the patients received CD34-selected stem cells. The median ANC and platelet count at day 15 post-AHSCT were 0.6 × 109/l (range 0.05–5.4 × 109/l) and 20 × 109/l (range 3–64 × 109/l), respectively. Twenty-nine patients achieved an ANC 500 cells/μl at day 15 and 24 patients achieved untransfused platelet count 20 × 109/l at day 15 post-AHSCT. Twenty-three patients had ALC 500 cells/μl on day 15 with a median ALC of 760 cells/μl (range 520–1980 cells/μl) and 22 patients had ALC <500 cells/μl with a median ALC of 125 cells/μl (range 10–410 cells/μl). There were no associations between ALC recovery at day 15 post-AHSCT and the type of conditioning regimen (P = 0.92), the number of pre-transplant chemotherapy regimens (P = 0.74), the number of pre-transplant treatment cycles (P = 0.50), ANC at day 15 post-AHSCT (P = 0.54), platelet count at day 15 post-AHSCT (P = 0.77), or stem cell source (BM or PBSC) (P = 0.14). In the 20 patients in whom CD34 counts were available (19 PBSC and one BM), the median number of CD34-positive cells infused was 4.7 × 106/kg (range 2.1–6.2 × 106/kg). The mononuclear cell count (MNC), available in 25 patients (one PBSC and 24 BM), had a median of 2.0 × 108/kg (range 0.5–9.2 × 108/kg). CD34 cell counts (n = 20, P = 0.33), and MNC (n = 25, P = 0.47) values were not significant predictors of ALC recovery.

Table 1 Patients’ characteristics prior to stem cell collection
Table 2 Stem cell transplantation patients’ characteristics

Overall survival and progression-free survival

By March 2001, 20 deaths had occurred among the 45 patients in the study. Eighteen patients died due to recurrent or progressive disease. The transplant-related mortality was 4%. One patient died of diffuse alveolar hemorrhage and the other of acute respiratory distress syndrome. Of the transplant-related mortality, one patient had ALC 500 cells/μl and one patient with ALC <500 cells/μl. There were six deaths in the 23 patients with ALC 500 cells/μl and 14 deaths in the 22 patients with ALC <500 cells/μl. Three patients had relapsed of leukemia but remain alive (one patient with ALC 500 cells/μl and two patients with ALC <500 cells/μl). Twenty-two patients remain alive without relapse (16 patients with ALC 500 cells/μl and six patients with ALC <500 cells/μl). None of the patients developed clinically evident autologous graft-versus-host disease. All patients were followed with a median follow-up of 14 months and a maximum of 129 months.

The median OS (Figure 1) and LFS (Figure 2) were significantly greater for patients with ALC 500 cells/μl vs ALC <500 cells/μl (not reached vs 10 months, P < 0.0009; 105 vs 9 months, P< 0.0008, respectively). The estimated 5-year overall survival for patients with ALC 500 cells/μl is 68% (standard error = 11.0%) vs ALC <500 cells/μl of 19% (standard error = 11.0%) and for the leukemia-free survival for patients with ALC 500 cells/μl of 66% (standard error = 24.0%) vs ALC <500 cells/μl of 8% (standard error = 7.0%).

Figure 1
figure1

Overall survival according to absolute lymphocyte recovery (ALC).

Figure 2
figure2

Leukemia-free survival according to absolute lymphocyte recovery (ALC).

Univariate and multivariate analysis

Of the 38 patients with available data at diagnosis for cytogenetics, subset analysis shows that intermediate and unfavorable cytogenetics were significant predictors for OS in the univariate analysis. ALC at day 15 and complete remission status before transplantation were also significant predictors for OS and LFS in the univariate analysis. In the multivariate analysis, ALC at day 15 post-AHSCT was found to be the only significant prognostic factor for both overall survival and leukemia-free survival (OS, RR = 0.46, P < 0.0047 and LFS, RR = 0.53, P < 0.0076). Table 3 summarizes the significant prognostic factors for the univariate and multivariate analyses. Neither for OS and LFS were ANC count at day 15 post-AHSCT (P = 0.81 and P = 0.44, respectively), CFU-GM/CD34 counts of the graft (P = 0.27 and P = 0.42, respectively), or PBSC vs BM (P = 0.37 and P = 0.60, respectively) found to be statistically significant prognostic factors in the univariate analysis.

Table 3 Univariate analysis and multivariate analysis: overall and LFS rates

Discussion

Our data show that ALC (500 cells/μl on day 15) post-AHSCT has a significant association with better survival in patients with AML. This is the first study describing the association of prolonged survival and ALC post-AHSCT in patients with AML. Multivariate analysis in our study showed that ALC at day 15 post-AHSCT is the most powerful, independent prognostic indicator for overall and leukemia-free survival for patients with AML. However, larger studies are required to confirm the impact of ALC recovery on survival post-AHSCT in patients with AML.

A potential explanation for the survival advantage associated with ALC recovery post-AHSCT in AML patients is the possibility that early immune reconstitution may have a protective effect against disease progression. This is analogous to the graft-versus-leukemia (GVL) effect in allogeneic bone marrow transplant recipients reported in patients with AML where the donor immune system is considered to be responsible for eradication of residual disease in the host.11 While reappearance of neutrophils and platelets is often considered the endpoint of hematologic recovery after AHSCT, immunological reconstitution is a gradual process that may not be completed until months to years post-transplantation.12,13 Delayed quantitative and qualitative T and B cells are observed from months to years post-AHSCT.12,13

Post-AHSCT immune-reconstitution studies have shown that natural killer (NK) cells recover normal absolute and relative numbers within 1 month after transplant.14,15 Our own data demonstrate normal absolute NK cell counts and function at 2 weeks post-AHSCT in patients with MM and NHL.16 Early NK cell recovery has been reported by others after standard chemotherapy,17 autologous stem cell transplantation14 and allogeneic bone marrow transplantation.18 Maturation of NK cells can occur in the absence of a functional thymus in mice and humans, perhaps allowing for prompt NK cell recovery in adults.19,20 Lowdell et al21 demonstrated 100% leukemia-free survival at 100 months compared with 20% leukemic-free survival for patients post- AHSCT that maintained in vitro a higher lymphocyte cytotoxic activity against their own leukemic blasts. NK cells were the cells mediating the lymphocyte cytotoxic activity. Based on these data, it is reasonable to hypothesize that NK cells may play an important immuno-surveillance role early after AHSCT for AML patients.

Due to the retrospective nature of this study, we do not know the lymphocyte subsets involved in the ALC recovery and their relationship to clinical outcome. The lack of correlation between CD34 counts or MNC and day 15 ALC recovery suggests the possibility that the repopulating lymphocytes post-AHSCT may be derived from sources other than transplanted stem cells, for example, mature lymphocytes present in the reinfused auto-graft. This is the subject of an ongoing prospective study.

In conclusion, ALC 500 cells/μl on day 15 post-AHSCT was the strongest predictor of clinical outcome in patients with AML in this study. The observed clinical benefit of early ALC, as a reflection of early immune reconstitution, may be analogous to the GVL effect seen in the allogeneic transplant setting. This study further supports our prior work underscoring the importance of lymphocyte recovery post-AHSCT in patients with malignancies, as higher survival rates have now been observed in patients with NHL,3 MM3 and metastatic breast cancer,4 who reach an ALC 500 cells/μl on day 15 post-AHSCT.

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Porrata, L., Litzow, M., Tefferi, A. et al. Early lymphocyte recovery is a predictive factor for prolonged survival after autologous hematopoietic stem cell transplantation for acute myelogenous leukemia. Leukemia 16, 1311–1318 (2002). https://doi.org/10.1038/sj.leu.2402503

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Keywords

  • acute myelogenous leukemia
  • absolute lymphocyte count
  • autologous hematopoietic stem cell transplantation

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