Abstract
Day 15 absolute lymphocyte count (ALC-15) after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) has been reported to be a significant predictor for survival in multiple hematological malignancies. Limitations of previous reports included their retrospective nature and the lack of ALC-15 lymphocyte subset analysis. To address these limitations, from 2/2002 until 2/2007, 50 non-Hodgkin lymphoma (NHL) patients were enrolled in a prospective study. The primary end point of the study was to confirm prospectively the ALC-15 survival role after APHSCT in NHL. The secondary end point was to identify the ALC-15 lymphocyte subset affecting survival after APHSCT. With a median follow-up of 22.2 months (range: 6-63.7 months), patients with an ALC-15 ≥ 500 cells/μl (n = 35) experienced superior overall survival (OS) and progression-free survival (PFS) compared with those who did not (median OS : not reached vs 5.4 months, 3 years OS rates of 80% (95% CI: 55-95%) vs 37% (95% CI: 15-65%), p < 0.0001; and median PFS: not reached vs 3.3 months, 3 years PFS rates of 63% (95% CI: 40-85%) vs 13% (95%CI: 4-40%), p < 0.0001, respectively). Univariately, CD16+/56+/CD3- natural killer (NK) cell was the only ALC-15 lymphocyte subset identified as a predictor for survival. Patients with an NK cell ≥ 80 cells/μl (n = 38) experienced superior OS and PFS compared with those who did not (median OS : not reached vs 5 months, 3 years OS rates of 76% (95%CI: 57-92%) vs 36% (95%CI: 11-62%), p < 0.0001; and median PFS: not reached vs 3 months, 3 years PFS rates of 57% (95%CI: 38-85%) vs 9 % (95%CI: 1-41%), p < 0.0001, respectively). Multivariate analysis showed that NK cells are independent predictor for survival. This is the first study confirming the ALC-15 survival role prospectively and identifying NK cells as the key ALC-15 lymphocyte subset affecting survival after APHSCT.
Keywords: Absolute lymphocyte count, Autologous stem cell transplantation, Natural killer cells, non-Hodgkin lymphoma, survival
Introduction
The clinical efficacy of autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) in treating malignancies has been solely attributed on the high-dose chemotherapy to eradicate tumor cells [1]. However, despite high-dose chemotherapy, APHSCT is still not curative for a number of patients with malignant diseases [1]. One hypothesis for the high relapse rate observed in APHSCT is the presumptive lack of graft-versus-tumor (GVT) effect present in allogeneic stem cell transplantation (Allo-SCT), where it is associated with a significant therapeutic impact on clinical outcomes [2]. Recent reports have shown that early post-transplant lymphocyte recovery has a direct impact on survival in patients undergoing Allo-SCT [3-4], suggesting that the early lymphocyte recovery post-transplant can be used as a surrogate maker for GVT.
Likewise, early lymphocyte recovery defined as an absolute lymphocyte count at day 15 (ALC-15) after APHSCT has been reported as significant, independent prognostic factor for survival in acute myelogenous leukemia [5], breast cancer [6-7], Hodgkin’s lymphoma [8-9], non-Hodgkin’s lymphoma [9-13], multiple myeloma [10, 13-14], and primary systemic amyloidosis [15], suggesting the possibility of an autologous GVT similar to the GVT seen in the Allo-SCT. Limitations of these previous publications included their retrospective nature and the lack of ALC-15 lymphocyte subset analysis. To address these limitations, we prospectively enrolled non-Hodgkin’s lymphoma (NHL) patients in a study to determine the role of ALC-15 on survival and to identify the ALC- 15 lymphocyte subset affecting survival after APHSCT.
Materials and methods
To participate in the study, patients were required to be candidates for APHSCT, chemo-sensitive disease prior to APHSCT, mobilize stem cells using growth factor only (granulocyte-colony stimulating-factor [G-CSF]), signed informed consent for participation and were willing to have research blood collected at day 15 post-APHSCT. Patients were excluded if they refused to provide consent, failed to mobilize stem cells by G-CSF and required bone marrow harvest, were infused with both peripheral blood and bone marrow harvest derived stem cells, or participated in clinical trials No patients were lost to follow-up. From 2/2002 and 2/2007, 50 NHL patients were enrolled in the study. All patients signed written, informed consent to participate in the study. Approval of the study was obtained from the Mayo Clinic Institutional Review Board and was in accordance with US federal regulations and the Declaration of Helsinki.
End points
The primary end point of the study was to assess prospectively the impact of ALC-15 on overall survival (OS) and progression-free survival (PFS) from the time of APHSCT. The second end point was to determine the ALC-15 lymphocyte subset affecting survival after APHSCT. The third end point was to confirm prospectively the role of the autograft absolute lymphocyte count (A-ALC) with the ALC-15 recovery post-APHSCT.
A-ALC, ALC-15 and ALC-15 lymphocyte subset analysis
Autograft absolute lymphocyte count (A-ALC) was determined as previously reported: {% collection lymphocytes × absolute autograft white blood cell count)/kilogram (kg). ALC-15 was determined from the differential white blood cell count obtained at day 15 post-APHSCT [16]. ALC-15 lymphocyte subset was analyzed as followed: mononuclear cells from peripheral blood samples at day 15 post-APHSCT were collected and concentrated to 1 × 106 cells/ml. According to manufactures’ recommendations, immunophenotyping was performed for T-cell panel (CD3/CD4/CD8), B-cell (CD19) and Natural killer (NK) cells (CD16+/CD56+/CD3-). Stained cells are then analyzed using flow cytometry (FACScallibur, Becton-Dickson, CA). Cells were analyzed for the percentage of cells expressing said antigens as well as the average quantity of antigen expression.
Prognostic factors
Prognostic factors for post-APHSCT OS and PFS evaluated in this study included the International prognostic index (IPI) {age≥ 60, extra-nodal sties ≥ 2, lactate dehydrogenase (LDH), performance status (PS) ≥ 2, and stage ≥ 3}[17]. In addition, disease status prior to transplant (complete remission [CR] vs partial response [PR]), the infused CD34+ cell dose, ALC-15, A-ALC, and ALC-15 lymphocyte subsets :{day 15 CD3 (CD3-15), day 15 CD4 (CD4-15), day 15 CD8 (CD8-15), day 15 CD19 (CD19-15), and day 15 CD16+/56+/3- ([CD16+/56+/3-]-15)}, and the number of prior treatments before APHSCT ≥ 2 were also evaluated.
Peripheral blood stem cell collection
All patients received G-CSF (10 μg/kg) daily for 5-7 consecutive days by subcutaneous injection. Once their peripheral blood CD34+ cell count was ≥ 10 cells/μL, patients began daily apheresis until they achieved a target of 5 × 106 CD34+ cells/kg. A minimum target of 2 × 106 CD34+ cells/kg was required for the patient to be considered for transplantation. Patients were assigned to either the Baxter Amicus (Baxter, Healthcare Corp, Deerfield, IL, USA), Fenwal CS 3000 Plus (Baxter, Healthcare Corp, Deerfield, IL, USA), or COBE Spectra (Gambro BCT, Lakewood, CO, USA) based on instrument availability on day of collection. Instrument settings used for the collection procedures have previously been described [18]. The number of apheresis collection was dependent upon collection of an adequate number of CD34+ cells to achieve hematologic engraftment post-APHSCT.
Conditioning regimen
All patients received Carmustine (BCNU) 300 mg/m2 on day -6; etoposide 100mg/m2 twice a day on days -5,-4,-3, and-2; cytarabine 100mg/m2 twice a day on days -5,-4,-3, and-2; and melphalan 140 mg/m2 on day -1 (BEAM). All patients received growth factor support starting on day +6 with sargramostim 500 mcg daily until evidence of neutrophilic engraftment defined as an absolute neutrophil count ≥ 500 cells/μl on three consecutive days.
Response and survival
Response criteria were based on the guidelines from the NHL international Workshop [19]. Complete response (CR) was defined as complete regression of all measurable or evaluable disease including radiologically demonstrable disease, bone marrow involvement, or peripheral blood involvement. Partial response (PR) was defined as a 50% or greater reduction in the sum of the products of the longest diameter and perpendicular diameters of measurable lesion(s), with a 50% or greater decrease in hepatomegaly or splenomegaly (measured from the costal margin) in the presence of previously known liver or spleen involvement. Stable disease was defined as less than PR with no progressive disease. Disease progression was defined as a 50% or more increase in the sum of the products of the longest diameter and the perpendicular diameter of measurable lesion(s) from the pre-study measurement, the appearance of new lesions, or a 2-cm increase in spleen or liver size due to lymphoma. Relapsed disease was defined as the appearance of any new lesion or increase by 50% or more in the size of previously involved sites.
OS was measured from the date of transplant to the date of death or last followup. PFS was defined as the time from transplant to the time of progression, relapse, death, or last follow-up.
Statistical analysis
OS, PFS, and actuarial risk of relapse were analyzed using the approach of Kaplan and Meier [20]. Differences between survival curves were tested for statistical significance using the two-tailed log-rank test. The Cox proportional hazards model [21] was used to evaluate ALC-15 and ALC-15 lymphocyte subsets as a prognostic factor for post-APHSCT OS and PFS times as well as to assess and adjust for other known prognostic factors. Multivariate analysis done using Cox regression models tested all variables with a p ≤ 0.2 in the univariate analysis. CD3-15, CD4-15, CD8-15, and CD19- 15 were used as continuous variables. Our data did not show any correlation between a specific cut-point and clinical outcomes for these variables. A cut-off of ≥ 80 [CD16+/56+/3-]-15 cells/μl was supported by the data as it yielded the greatest differential in survival based on χ2 values analyzed at different cut-points (between the 25th and 75th quartiles) from log-rank tests.
Prediction of ALC-15 was explored further in logistic regression models, univariately assessing continuous and dichotomized values of ALC-15 lymphocyte subsets. χ2-tests were used to determine relationships between categorical variables. The Wilcoxon-rank test was used to determine associations between continuous variable and categories, and Spearman correlation coefficients were used to evaluate associations for continuous variables. All p-values represented were two-sided and statistical significance was declared at p < 0.05.
Results
Patient characteristics
The median age at the time of transplant for this cohort of 50 patients was 57.7 years (range: 23-73). Distributions of additional baseline characteristics for these patients are presented in Table 1 and are summarized based on whether patients achieved and ALC-15 ≥ 500 cells/μl vs < 500 cells/μl. The only differences between the groups were the infused A-ALC, CD8-15, and [CD16+/56+/3-]-15. None of the patients received purged or CD34+ selected stem cells.
Table 1.
Baseline characteristics of patients according to ALC-15 (≥ 500 vs < 500 cells/μl)
| Characteristics | ALC-15 ≥ 500 cells/μl (N=35) | ALC-15 < 500 cells/μl (N=15) | p-value |
|---|---|---|---|
| Age, years, median (range) | 56 (23-71) | 60 (46-73) | 0.18 |
| Gender | 0.53 | ||
| Female | 13 | 4 | |
| Male | 22 | 11 | |
| Lymphoma type | 0.35 | ||
| DLBCL | 23 | 11 | |
| Mantle cell | 8 | 2 | |
| T-cell | 4 | 1 | |
| Follicular | 0 | 1 | |
| Prognostic factors at diagnosis | |||
| Extra nodal sites | 0.41 | ||
| 0 | 21 | 11 | |
| 1 | 11 | 4 | |
| 2 | 2 | 0 | |
| LDH (U/L), median (range) | 200 (150-1515) | 225 (152-358) | 0.47 |
| Performance status | 0.47 | ||
| 0 | 21 | 10 | |
| 1 | 12 | 5 | |
| 2 | 2 | 0 | |
| Stage | 0.45 | ||
| I | 5 | 1 | |
| II | 2 | 3 | |
| III | 6 | 2 | |
| IV | 22 | 9 | |
| IPI factors | |||
| Age, years | 0.55 | ||
| ≥ 60 | 15 | 8 | |
| < 60 | 20 | 7 | |
| LDH | 0.36 | ||
| Abnormal | 13 | 8 | |
| Normal | 22 | 7 | |
| Extra nodal sites | 0.22 | ||
| ≥ 2 | 2 | 0 | |
| < 2 | 33 | 15 | |
| Performance status | 0.23 | ||
| ≥ 2 | 2 | 0 | |
| < 2 | 33 | 15 | |
| Stage | 0.71 | ||
| I/II | 7 | 4 | |
| III/IV | 28 | 11 | |
| IPI score | 0.4 | ||
| 0 | 4 | 1 | |
| 1 | 11 | 4 | |
| 2 | 15 | 7 | |
| 3 | 5 | 3 | |
| IPI index | 0.75 | ||
| ≥ 2 | 20 | 10 | |
| < 2 | 15 | 5 | |
| Apheresis machine | 0.28 | ||
| Baxter Amicus | 23 | 9 | |
| Fenwal CS 3000 | 2 | 3 | |
| COBE Spectra | 10 | 3 | |
| Number of prior treatments before APHSCT | 0.14 | ||
| 1 | 9 | 1 | |
| 2 | 24 | 11 | |
| 3 | 2 | 2 | |
| 4 | 0 | 1 | |
| Disease status prior to salvage chemotherapy | 0.09 | ||
| CR | 7 | 0 | |
| Relapse | 28 | 15 | |
| Disease status prior to APHSCT | 0.18 | ||
| CR | 13 | 2 | |
| PR | 22 | 13 | |
| Number of apheresis collection, median (range) | 4 (1-7) | 3 (2-7) | 0.29 |
| Infused CD34+ cells × 106/kg, median (range) | 4.76 (2.03-8.56) | 3.93 (2.26-8.03) | 0.81 |
| Infused A-ALC lymphocytes × 109/kg, median (range) | 0.609 (0.187-1.655) | 0.183 (0.02-1.263) | <0.003 |
| CD3-15 cells/μl, median (range) | 421 (8-2069) | 165 (0.3-1413) | 0.09 |
| CD4-15 cells/μl, median (range) | 171.5 (3-1091) | 123 (19.7-793) | 0.27 |
| CD8-15 cells/μl, median (range) | 151.5 (3-813) | 63 (13-570) | <0.02 |
| CD19-15 cells/μl, median (range) | 2 (0-227) | 1 (0-10) | 0.58 |
| [CD16+/56+/3-]-15 cells/μl, median (range) | 134 (66-999) | 91 (10-231) | <0.0001 |
Abbreviations: A-ALC= autograft, absolute lymphocyte count; APHSCT= autologous hematopoietic stem cell transplantation; CR = complete remission; DLBCL = diffuse large B-cell lymphoma; IPI = International Prognostic index; LDH = lactate dehydrogenase; PR = partial response.
No patients were lost to follow-up. The median follow-up for the cohort was 22.2 months (range: 6-63.7 months). By the time of this analysis, 21 patients (42%) had evidence of relapse and 12 patients (24%) had died. The day 100 transplant-related mortality for the cohort was only 2% (1/50). One patient died of an intracranial bleed, the rest due to progressive lymphoma.
Post-transplant survival and ALC-15
The median post-transplant OS (Figure 1a) and PFS (Figure 1b) times were significantly better for patients that achieved an ALC-15 ≥ 500 cells/μl compared with patients with an ALC-15 < 500 cells/μl (not reached vs 5.4 months, 3 year OS rates of 80% (95%CI: 55-95%) vs 37% (95%CI: 15-65%), p < 0.0001; not reached vs 3.3 months, 3 year PFS rates of 63% (95%CI: 40-85%) vs 13% (95%CI: 4-40%), p < 0.0001, respectively).
Figure 1.
a. Overall survival (OS) of patients with an absolute lymphocyte count at day 15 (ALC-15) ≥ 500 cells/μl vs patients with an ALC-15 < 500 cells/μl. The median OS was not reached in the group of patients with an ALC-15 ≥ 500 cells/μl and 5.4 months with the group of patients with an ALC-15 < 500 cells/μl. The OS rates at three years were 80% and 37%, respectively (p < 0.0001).
b. Progression-free survival (PFS) of patients with an absolute lymphocyte count at day 15 (ALC-15) ≥ 500 cells/μl vs patients with an ALC-15 < 500 cells/μl. The median PFS was not reached in the group of patients with an ALC-15 ≥ 500 cells/μl and 3.3 months with the group of patients with an ALC-15 < 500 cells/μl. The PFS rates at three years were 63% and 13%, respectively (p < 0.0001).
Day 15 post-APHSCT lymphocyte subsets and ALC-15
By day 15 post-APHSCT, 30% (15/50) achieved normal CD3 count (normal range: 582-1992 cells/μl), 18% (9/50) normal CD4 count (normal range: 401-1532 cells/μl), 38 % (19/50) normal CD8 count (normal range: 152-838 cells/μl), 4% (2/50) normal CD19 count (normal range: 71-567 cells/μl), and 76% (38/50) normal CD16+/56+/3- count {natural killer (NK) cells} (normal range: 80-597 cells/μl). By univariate analysis, we identified a correlation between ALC-15 and CD3-15 (rs = 0.4, p < 0.006), CD4-15 (rs = 0.36, p < 0.01), CD8-15 (rs = 0.41, p < 0.004), and [CD16+/56+/3-]-15 (rs = 0.62, p < 0.0001). No correlation was identified between ALC-15 and CD19-15 (rs = 0.04, p = 0.72) or ALC-15 and CD34 (rs = 0.11, p = 0.5). When CD3-15, CD4-15, CD8-15, and [CD16+/56+/3-]-15 were accounted in a multivariate logistic regression model, [CD16+/56+/3-]-15 was the only remaining significant correlate for ALC-15 (p < 0.0001).
ALC-15 lymphocyte subset and relapse
Twenty-one patients relapsed at a median of 5.1 months (range: 1.2-28.4 months) with a 3 year actuarial risk of relapse for NK cells of 21% (95% CI: 12-45%); CD3-15 of 45% (95% CI: 30-70%); CD8-15 of 48% (95% CI: 35-75%); CD4-15 of 54% (95%CI: 32-73%); and CD19-15 of 60% (95% CI: 30-80%) (figure 2).
Figure 2.
Actuarial risk of relapse for ALC-15 lymphocyte subsets.
Post-transplant survival and natural killer (NK) cells
NK -15[CD 16+/56+/3-]-15, as a continuous variable, was the only ALC-15 lymphocyte subset associated with OS and PFS after APHSCT (Table 2). The median post-transplant OS (Figure 3a) and PFS (Figure 3b) times were significantly better for patients that achieved a NK cell at day 15 (NK-15) ≥ 80 cells/μl compared with patients with an NK-15 < 80 cells/μl (not reached vs 4.9 months 3 year OS rates of 76% (95%CI: 57-92%) vs 36 (95%CI: 11-62%), p < 0.0001; not reached vs 3.0 months 3 year PFS rates of 57% (95%CI: 38-85%) vs 9%(95%CI:1-41%), p < 0.0001, respectively). Patients with an ALC-15 < 500 cells/μl (N = 15), but with a NK-15 ≥ 80 cells/μl (N = 4), experienced superior OS and PFS post-APHSCT compared with those who did not (not reached vs 4.6 months, 3 year OS rates of 67% (95%CI: 35-95%) vs 27% (95%CI: 9-55%), p < 0.04; 17 vs 2.8 months, 3 years PFS rates of 25% (95%CI: 10-70%) vs 9% (95%CI: 1-40%), p < 0.05, respectively).
Table 2.
Univariate analysis for overall and progression-free survival
| Prognostic Factors | Overall Survival | Progression-Free Survival | ||||
|---|---|---|---|---|---|---|
| RR | 95%CI | p-value | RR | 95%CI | p-value | |
| Age ≥ 60 years | 1.042 | 0.985-1.116 | 0.16 | 1.046 | 1.000-1.101 | 0.06 |
| ALC-15 ≥ 500 cells/μl | 0.342 | 0.161-0.626 | <0.0004 | 0.403 | 0.253-0.623 | <0.0001 |
| A-ALC | 0.262 | 0.030-0.629 | <0.01 | 0.165 | 0.017-0.518 | <0.004 |
| Extra nodal sites > 1 | 1.520 | 0.352-3.561 | 0.46 | 1.094 | 0.257-2.436 | 0.87 |
| LDH abnormal | 1.342 | 0.757-2.470 | 0.31 | 0.998 | 0.997-1.011 | 0.20 |
| PS > 1 | 1.231 | 0.681-1.386 | 0.24 | 1.125 | 0.512-4.771 | 0.81 |
| Stage III/IV | 1.757 | 0.774-7.513 | 0.20 | 1.371 | 0.797-2.832 | 0.28 |
| Disease status at transplant: CR | 0.495 | 0.116-1.129 | 0.11 | 0.627 | 0.303-1.080 | 0.10 |
| Infused CD34 (continuous variable) | 1.174 | 0.855-1.600 | 0.30 | 1.033 | 0.798-1.311 | 0.80 |
| Apheresis machine S vs A/F | 0.274 | 0.181-1.017 | 0.06 | 0.247 | 0.161-0.967 | <0.05 |
| CD3-15 | 1.000 | 0.499-1.001 | 0.67 | 0.999 | 0.998-1.000 | 0.71 |
| CD4-15 | 1.000 | 0.998-1.002 | 0.68 | 0.999 | 0.997-1.001 | 0.81 |
| CD8-15 | 1.000 | 0.997-1.003 | 0.81 | 0.999 | 0.996-1.002 | 0.58 |
| CD19-15 | 0.991 | 0.926-1.008 | 0.49 | 0.984 | 0.919-1.003 | 0.17 |
| [CD16+/56+/3-]-15 | 0.671 | 0.531-0.831 | <0.01 | 0.796 | 0.682-0.917 | <0.03 |
| [CD16+/56+/3-]-15 ≥ 80 cells/μl | 0.328 | 0.169-0.587 | <0.0002 | 0.382 | 0.245-0.592 | <0.0001 |
| Number of prior treatments before APHSCT ≥ 2 | 1.878 | 0.532-2.171 | 0.4 | 0.934 | 0.319-1.699 | 0.9 |
Abbreviations: A = Amicus machine; ALC = absolute lymphocyte count; A-ALC = autograft absolute lymphocyte count; F = Fenwal CS 3000 machine; LDH = Lactate dehydrogenase; PS = performance status; and S = Spectra machine.
Figure 3.
a. Overall survival (OS) of patients with a natural killer cells at day 15 (NK-15) ≥ 80 cells/μl vs patients with an NK-15 < 80 cells/μl. The median OS was not reached in the group of patients with an NK-15 ≥ 80 cells/μl and 5 months with the group of patients with an NK-15 < 500 cells/μl. The OS rates at three years were 76% and 36%, respectively (p < 0.0001).
b. Progression-free survival (PFS) of patients with a natural killer cells at day 15 (NK-15) ≥ 80 cells/μl vs patients with an NK-15 < 80 cells/μl. The median PFS was not reached in the group of patients with an NK-15 ≥ 80 cells/μl and 3 months with the group of patients with an NK-15 < 500 cells/μl. The OS rates at three years were 57% and 9%, respectively (p < 0.0001).
NK-15, disease status and prior treatment to APHSCT
NK-15 was able to discriminate among patients that were heavy (≥ 2 prior treatments) or less (1 treatment) pre-treated and patients that presented in CR or PR prior to APHSCT. In the less pre-treated group (N =10), the median post-transplant OS and PFS (times were significantly better for patients that achieved a NK cell at day 15 (NK- 15) ≥ 80 cells/μl (N = 7) compared with patients with an NK-15 < 80 cells/μl (N =3) (not reached vs 5 months 3 year OS rates 67% (95%CI: 23-91%) vs 0%, p < 0.0001; not reached vs 2.6 months 3 year PFS rates of 65% (95%CI:27-92%) vs 0%, p < 0.003, respectively). In the heavy pre-treated group (N=40), the median post-transplant OS and PFS times were significantly better for patients that achieved a NK cell at day 15 (NK- 15) ≥ 80 cells/μl (N=32) compared with patients with an NK-15 < 80 cells/μl (N=8) (not reached vs 3 months 3 year OS rates of 85% (95%CI: 58-98%) vs 36% (95%CI: 18- 65%), p < 0.003; not reached vs 3.0 months 3 year PFS rates of 63% (95%CI: 40-87%) vs 9% (95%CI: 1-43%), p < 0.0001, respectively). In the CR group (N = 15) prior to APHSCT, the median post-transplant OS and PFS times were significantly better for patients that achieved a NK cell at day 15 (NK-15) ≥ 80 cells/μl (N = 13) compared with patients with an NK-15 < 80 cells/μl (N =2) (not reached vs 10 months, 3 year OS rates of 100% vs 50% (95%CI: 20-80%), p < 0.01; not reached vs 3.0 months, 3 year PFS rates of 90% (95%CI: 60-100%) vs 0%), p < 0.0001, respectively). In the PR group (N=35), the median post-transplant OS and PFS times were significantly better for patients that achieved a NK cell at day 15 (NK-15) ≥ 80 cells/μl (N =25) compared with patients with an NK-15 < 80 cells/μl (N =10) (not reached vs 5 months, 3 year OS rates of 72% (95%CI: 48-93%) vs 30% (95%CI: 10-61%), p < 0.0005; not reached vs 3.3 months, 3 year PFS rates of 52% (95%CI: 30-77%) vs 10% (95%CI: 1-43%), p < 0.0004, respectively).
Source of ALC-15 and ALC-15 lymphocyte subsets
To confirm prospectively our previous retrospective reports demonstrating that AALC correlated with ALC-15, we analyzed if A-ALC or infused CD34+ cells not only correlated with ALC-15, but also with ALC-15 lymphocyte subsets. First we identified no association between the collected CD34+ cells and A-ALC (rs = 0.001, p = 0.95). There was no association between CD34+ cells and ALC-15 (rs = 0.11, p =0.5), CD3-15 (rs = 0.16, p = 0.3), CD4-15 (rs = 0.16, p = 0.3), CD8-15 (rs = 0.21, p =0.1), CD19-15 (rs = 0.04, p =0.8), and [CD16+/56+/3-]-15 (rs = 0.11, p =0.4). On the contrary, we identified an association between A-ALC and ALC-15 (rs = 0.55, p < 0.0001), CD3-15 (rs = 0.29, p < 0.04), CD4-15 (rs = 0.28, p < 0.05), CD8-15 (rs = 0.32, p < 0.02), and [CD16+/56+/3-]-15 (rs = 0.38, p < 0.007). No association was observed between A-ALC and CD19-15 (rs = 0.14, p = 0.32). Patients that achieved an ALC-15 ≥ 500 cells/μl were infused with a higher A-ALC vs those who achieved an ALC-15 < 500 cells/μl (0.609 (range: 0.187-1.655) vs 0.183 (range: 0.02-1.263) × 109 lymphocytes/kg, p < 0.0027, respectively). Despite setting the specific apheresis instrument parameters to collect CD34+ cells, the COBE Spectra collected more A-ALC per collection compared with the Baxter Amicus and the Fenwal CS3000 Plus (median A-ALC lymphocytes × 109/kg: 0.2 (range: 0.034-0.793) vs 0.146 (range: 0.01-0.88) vs 0.105 (range: 0.043-0.265), p < 0.04, respectively).
Univariate and multivariate analysis
Age, A-ALC, ALC-15, [CD16+/56+/3-]-15, disease status at transplant, apheresis machine, and stage were predictor for OS; and age, A-ALC, ALC-15, [CD16+/56+/3-]- 15, disease status at transplant, LDH, and apheresis machine were predictor for PFS as each of these variables reached a p ≤ 0.2 in the univariate analysis (Table 2). In the multivariate analysis. [CD16+/56+/3-]-15 remained an independent predictor for OS and PFS post-APHSCT and truncated ALC-15 and A-ALC (Table 3).
Table 3.
Multivariate analysis for overall and progression-free survival
| Prognostic Factors | Overall Survival | Progression-Free Survival | ||||
|---|---|---|---|---|---|---|
| RR | 95%CI | p-value | RR | 95%CI | p-value | |
| Age ≥ 60 years | 1.098 | 1.012-1.204 | <0.02 | 1.124 | 1.050-1.215 | <0.0004 |
| ALC-15 ≥ 500 cells/μl | 0.266 | 0.117-1.143 | 0.8 | 0.471 | 0.254-1.743 | 0.15 |
| A-ALC | 0.326 | 0.139-1.358 | 0.8 | 0.320 | 0.156-1.477 | 0.15 |
| [CD16+/56+/3-]-15 ≥ 80 cells/μl | 0.521 | 0.212-0.774 | <0.0002 | 0.420 | 0.241-0.617 | <0.0001 |
| Disease status at transplant: CR | 0.676 | 0.357-0.855 | <0.04 | 0.576 | 0.481-0.927 | <0.03 |
| LDH | 1.000 | 0.966-1.003 | 0.63 | |||
| Apheresis machine S vs A/F | 0.366 | 0.204-1.731 | 0.6 | 0.504 | 0.313-1.111 | 0.12 |
| Stage III/IV | 1.521 | 0.852-2.113 | 0.15 | |||
Abbreviations: A = Amicus machine; ALC = absolute lymphocyte count; A-ALC = autograft absolute lymphocyte count; F = Fenwal CS 3000 machine; LDH = Lactate dehydrogenase; PS = performance status; and S = Spectra machine.
Discussion
Patients achieving an ALC-15 ≥ 500 cells/μl after APHSCT have a superior clinical outcome compared with those who do not [5-15]. Major limitations of previous reports included their retrospective nature and no ALC-15 lymphocyte subsets analysis to identify the key ALC-15 lymphocyte subset conveying the superior survival observed with a higher ALC-15 recovery. Therefore, we set out to address these limitations in a prospective fashion by analyzing not only ALC-15, but also ALC-15 lymphocyte subsets in NHL patients undergoing APHSCT.
The stratification of our cohort group using ALC-15 ≥ 500 cells/μl was based on previous publications [5-6,8,10,15]. Both groups were balanced for all patient characteristics and prognostic factors except for A-ALC, CD8-15, and NK cells at day 15 defined as [CD16+/56+/3-]-15. Our study confirmed prospectively that patients achieving an ALC-15 ≥ 500 cells/μl experienced superior survival compared with those who did not. No association between the ALC-15 and B-cells was identified. A possible explanation for this observation is the fact that all patients in the study received prior rituximab therapy depleting the B-cell compartment. However, T-cells and NK cells were associated with ALC-15 recovery. By multivariate analysis, NK cells were the major lymphocyte subset contributing the ALC-15 recovery. This finding is in accordance with previous publications regarding immune-reconstitution after transplant, where NK cells recovered early post- allogeneic or autologous stem cell transplantation [22-25]. NK cells recovered not only quantitatively, but also qualitatively when compared with the other lymphoid compartments (i.e., T or B-cells) at day 15 after APHSCT [26]. The only ALC-15 lymphocyte subset associated with clinical outcome was NK cells. NK-15 was associated with a lower actuarial risk of relapse compared with the other ALC-15 lymphocyte subsets. NK cells as a continuous or dichotomized variable was associated with post-APHSCT OS and PFS. Moreover, NK-15 was an independent prognostic factor in the multivariate analysis compared to A-ALC and ALC-15, suggesting that NK-15 is a more specific marker of the autologous GVT observed in APSHCT.
Identification of ways to enhance ALC-15 and NK cells-15 recovery will provide new immunologic therapeutic interventions to improve clinical outcomes post-APHSCT. We previously demonstrated no association between infused CD34+ cells and ALC-15 and this finding is confirmed by the current study. In prior reports we showed that the infused A-ALC directly impacted ALC-15 recovery in NHL [27] and multiple myeloma [28] patients post-APHSCT. Our findings regarding the association between A-ALC and ALC-15 have been recently confirmed retrospectively in multiple myeloma [29-30]. Our study shows a strong positive correlation not only between infused A-ALC and ALC-15, but also between A-ALC and T and NK- cells, but not B-cells. This observation is important because stem cell mobilization and collection in APHSCT should not be viewed only as a means to achieve hematopoietic engraftment, but also as a means to achieve immunologic engraftment with direct consequences to clinical outcome [31]. Due to the strong correlation between A-ALC and ALC-15 and the fact that A-ALC is known pre-APSHCT, a low A-ALC pre-APSHCT may translate into slow ALC-15 recovery leading to poor clinical outcomes. To overcome the lack of a immune response due to a low A-ALC, clinicians may intensify conditioning hoping to improve clinical outcomes post-APSHCT. Thus, therapeutic maneuvers to enhance A-ALC collection should translate into better clinical outcomes after APHSCT. This is confirmed by the fact that patients that achieved an ALC-15 ≥ 500 cells/μl were infused with higher numbers of A-ALC. To enhance A-ALC collection, we previously published that the apheresis instruments can affect A-ALC collection [18]. This is confirmed in this study as the settings used at our institution with the COBE Spectra collected more A-ALC than the settings used at our institution with the Amicus or the Fenwal CS3000 Plus, even though the parameters for each apheresis machine were set to maximize CD34+ cell collection. However, even though the COBE Spectra collected more A-ALC and was associated with better survival, in the multivariate analysis, the apheresis machine statistical significance was canceled out by the NK-15, suggesting that it not the apheresis machine affecting survival, instead, it is the amount of A-ALC collected by the apheresis machine translating into a higher ALC-15/NK-15 recovery affecting survival post- APHSCT. Because of this finding, we have an opened Phase III randomized double-blind trial to assess if modified apheresis instrument settings compared with the standard settings can improve survival through higher A-ALC collection translating into faster immune recovery post-APHSCT.
A limitation of the study is the heterogenous group of non-Hodgkin’s lymphoma histologies included in the study. However, ALC-15 has been reported to be an independent prognostic factor for survival in T and B-cell NHL, as well as, mantle cell lymphoma post-APHSCT [9-13]. Another limitation of the study is that even though we identified prospectively an association between ALC-15 and NK-15 subset and survival post-APHSCT, it does not imply causation. We hope that our Phase III study will directly answer that the alleged survival advantage observed with early lymphocyte recovery post-APHSCT is caused by the ALC-15 and the NK-15 subset. In addition, ALC-15/NK-15 might be possible a surrogate marker found in less heavily pretreated patients that would have better clinical outcomes for this reason rather than higher lymphocyte count recovery. Nevertheless, we identified that patients with a higher NK-15 experienced superior survival regardless if they were heavy pretreated or not, or if there were in CR or PR prior to APHSCT. However, larger prospective studies are warranted to confirm our findings with a more balance cohort of patients, as younger patients, higher CR rates pre-APHSCT, and higher number of patient with mantle cell lymphoma that would require longer follow-up to assess progression of disease were observed in the ALC-15 ≥ 500 cells/μl group. To our knowledge, one negative study has been published showing no association between ALC-15 and survival post- APHSCT in NHL by Tiwari et al [32]. An explanation for the negative result reported by Tiwair et al could be answered by the current study as the main limitation of Tiwari et al study was the lack of ALC-15 lymphocyte subset analysis. In the current study, even patients with an ALC-15 < 500 cells/μl, but with a NK-15 ≥ 80 cells/μl experienced superior survival compared with those who did not, as well as NK-15 was independent prognostic factor compared to ALC-15 in the multivariate analysis. This finding suggests that NK-15 could be a more powerful, discriminatory prognostic factor for survival than ALC-15 post-APHSCT.
In summary, this is the first study confirming prospectively the prognostic significance of ALC-15 and identifying NK cells as the key ALC-15 lymphocyte subsets conveying the superior survival after APHSCT. In addition, this study also confirms prospectively that A-ALC is the main source of ALC-15 and ALC-15 lymphocytes subsets. We eagerly await the results an ongoing Phase III to enhance A-ALC collection to maximize ALC-15 recovery with the hope of improving clinical outcomes post- APHSCT. Further studies to understand the underlying mechanism of how NK cells eradicate tumor cells post-APHSCT are warranted [33].
Acknowledgments
This study was supported in part by the Grant CA90628-04A from the National Institute of Health, and the University of Iowa/Mayo Clinic Lymphoma Spore CA97274.
Footnotes
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