Introduction

High-dose chemotherapy and autologous SCT (ASCT) is associated with long-term remission in approximately 40% of patients with relapsed non-Hodgkin's lymphoma (NHL).¹ Predictors of post transplant outcome include performance status, time from diagnosis to relapse or transplant, and chemosensitivity to salvage therapy.² Early lymphocyte recovery measured by an absolute lymphocyte count (ALC) >500/l obtained on day 15 post transplant is independently associated with favorable outcomes after autografting for NHL,^{3, 4, 5} multiple myeloma,³ Hodgkin's disease,^{5, 6} acute myeloid leukemia⁷ and metastatic breast cancer.^{8, 9}

Myeloid growth factors are often administered after hematopoietic cell transplantation to accelerate neutrophil recovery.^{10, 11, 12} In addition to the myeloid effects, GM-CSF affects monocyte and lymphocyte recovery after standard¹³ or high-dose chemotherapy;¹⁴ GM-CSF may therefore affect the prognostic significance of early lymphocyte recovery post autografting. We analyzed the outcomes of 268 consecutive NHL autograft recipients who received post transplant GM-CSF to hasten neutrophil recovery, after stratification according to the ALC obtained on day +15 post transplant.

Patients and methods

Patients with chemosensitive-relapsed NHL autografted at Washington University between January 1996 and May 2003 were divided into two groups according to their ALC measured on post transplant day +15 (ALC<500 vs ALC500). Patient's basic demographic data (age and gender), disease-related information (stage, grade, histology, time of diagnosis and pre-transplant therapy), stem cell mobilization, conditioning regimens, post transplant complications, engraftment, relapse and survival information were extracted from a comprehensive bone marrow transplant computerized database. ALC was calculated by multiplying the percentage of lymphocytes by the total number of WBCs obtained on a complete blood count (CBC) drawn on day +15 post transplant. This retrospective study was approved by the Washington University Institutional Review Board.

As part of an institutional policy, ASCT was offered to patients with chemosensitive-relapsed NHL, while patients with refractory disease were preferentially offered either allogeneic transplantation or experimental therapies. Salvage regimens used to assess chemosensitivity included platinum-based regimens (ESHAP (etoposide, solumedrol, cytosine arabinoside, platinum) or DHAP (dexamethasone, cytosine arabinoside, platinum)); and ifosfamide-based regimens (MINE (mesna, ifosfamide, mitoxantrone, etoposide) or ICE (ifosfamide, carboplatin, etoposide)), administered with or without rituximab (R). Involvement of the marrow by NHL excluded patients from ASCT at our center. Peripheral blood stem cells (PBSC) were the major source of stem cell support, with only four receiving bone marrow and one patient receiving both. Stem cells were mobilized with G-CSF (10 g/kg/day for 4 days, 88%) or with other hematopoeitic growth factors (GM-CSF 10 g/kg/day or experimental agents, 12%); collected through large volume (20 l) apheresis, mixed with DMSO, and cryopreserved in liquid nitrogen at -190°C. In all, 111 patients (41%) underwent fractionated total-body irradiation (TBI), while the rest (59%) received other preparative regimen including BEAM (BCNU, etoposide, ara-C, melphalan) and BEAC (BCNU, etoposide, ara-C, CY). All patients were nursed in laminar airflow rooms equipped with high-efficiency particular air filters for protective isolation, and received routinely GM-CSF (Leukine, Berlex, Richmond, CA, USA) subcutaneously at the dose of 250 g/m² starting on day 0 and continued until the absolute neutrophil count exceeded 500 /mm³ on three consecutive days. Patients were discharged upon neutrophil engraftment, in the absence of fevers, and if they were able to maintain adequate oral intake.

The ² or student t-test (Mann–Whitney test if normality assumption violated) were used to determine the comparability of the two groups (ALC<500 vs ALC500) in terms of baseline characteristics including age, gender, tumor grade, time from initial diagnosis to transplant, and pre-transplant therapies. For the binary outcomes (such as complications, days +30 and +100 survivals) the differences between the two groups were compared by ² or Fisher's exact test. The distributions of time-to-event outcomes, including time-to-engraftment, overall survival (OS) and disease-free survival (DFS), were summarized with Kaplan–Meier product limit estimators and compared by log-rank test. The analyses of OS and DFS were performed separately for patients with low-grade (LG) and intermediate-grade (IG)/high-grade (HG) NHL. A multivariate Cox model was also fitted on the pooled data while stratifying the potential effects of NHL types.

Results

Patients

Between January 1996 and May 2003, 273 consecutive patients with chemosensitive NHL were autografted at Washington University. Complete information and day +15 ALC value were available for 268 patients, all of whom are included in this analysis. Patients were divided into two groups based on day +15 ALC: ALC500 (n=151, 56%) and ALC<500 (n=117, 44%). Patient characteristics were well-balanced between the two groups with regard to sex, grade of NHL, pre-transplant exposure to rituximab, preparative regimen and time from diagnosis to transplant within each grade (Table 1). The number of patients with residual radiological abnormalities at the time of pre-transplant staging was comparable in both the two groups (data not shown).

Table 1 - Patients characteristics.

Full table

Post transplant complications

Early (pre-engraftment) post transplant complications were evenly distributed in the two groups (Table 2), with the exception of neutropenic colitis that was observed more commonly in patients with ALC <500 (20 vs 10%, P=0.02). Neutropenic fevers occurred in 76 vs 81% (P=0.36), neutropenic colitis in 20 vs 10% (P=0.02), grades 2–3 mucositis in 55 vs 50% (P=0.46), and arrhythmias in 12 and 12% (P=0.98) for ALC<500 and ALC500 patients, respectively.

Table 2 - Effects of day +15 ALC on post transplant complications.

Full table

Engraftment

Patients with a post transplant day +15 ALC<500 were transplanted with a stem cell product containing a median CD34 count of 3.04 10⁶ cells/kg and patients with an ALC>500 received a median CD34 count of 3.13 10⁶ cells/kg (P=0.88). Median time-to-neutrophil engraftment (absolute neutrophil counts >500 /mm³ for three consecutive days) was 12 days (95% CI 11–12) for ALC500 and 13 days (95% CI 12–14) for the ALC<500 group (P=0.003). A delay in platelet recovery was observed in the ALC<500 group. Median time-to-platelet engraftment (untransfused platelet counts >50 000 /mm³) was 36 days (95% CI: 30–48) for the ALC<500 vs 29 days (95% CI: 25–33) for the ALC>500 group (P=0.0003, Figure 1). Delayed platelet engraftment was independent from age, time from diagnosis to transplantation, number of prior chemotherapy cycles and number of CD34+ cells infused/kg. We have previously reported a delay in platelet recovery among patients with pre-transplant exposure to rituximab,¹⁵ we, therefore, analyzed the outcomes of those patients according to the ALC on day 15. In this group of patients, time-to-untransfused platelet count >50 000 /mm³ was 44 days (95% CI: 35–59 days) for the ALC<500 vs 33 days (95% CI: 29–42 days) for the ALC>500 group (P=0.1). Median duration of hospitalization was comparable in the ALC<500 and ALC500 groups (15 vs 14 days, P=0.71), and the two groups had equivalent days +30 (97 vs 97%, P=0.99) and +100 (90 vs 93%, P=0.35) survivals.

Figure 1.

Probabilities of platelet engraftment (untransfused platelet counts >50 000/mm³) in days for ALC<500 (n=117) and ALC500 (n=151) patients.

Full figure and legend (40K)

Survival

DFSand OS were analyzed separately for patients with LG (ALC<500, n=48; ALC 500, n=61) and IG/HG (ALC<500, n=63; ALC 500, n=84) NHL. The median follow-up was 22 months. For patients with LG NHL, there was no significant difference in DFS or OS according to their ALC recovery (P=0.88 and P=0.97, respectively). The 3-year DFS was 51 and 54%, and 3-year OS was 71 and 66% for the ALC<500 and ALC500 group, respectively (Figure 2). For patients with IG/HG NHL, the associations between ALC and DFS/OS were not statistically different (with P=0.35 and P=0.20, respectively). The 3-year DFS was 45 and 57%, and 3-year OS was 59 and 63% for the ALC<500 and ALC 500 groups, respectively (Figure 3). Multivariate Cox models based on the pooled data showed that the associations between early ALC recovery and survivals remained insignificant (P=0.60 for DFS and P=0.36 for OS) after adjusting for the effect of NHL grades and prior use of rituximab.

Figure 2.

Disease-free survival (DFS) and overall survival (OS) in patients with low-grade NHL based on day +15 ALC.

Full figure and legend (51K)

Figure 3.

Disease-free survival (DFS) and overall survival (OS) in patients with intermediate-grade NHL based on day +15 ALC.

Full figure and legend (55K)

Discussion

The favorable prognostic significance of early lymphocyte recovery on the outcomes of autografting was independently demonstrated in various hematological^{3, 4, 5, 6, 7} and non-hematological malignancies.^{8, 9} It is unclear why early lymphocyte recovery is associated with better DFS and OS, and mechanisms are far from being clearly understood. It is hypothesized that early lymphocyte recovery reflects early post transplant immune reconstitution which confers a protective effect against disease progression.⁷ Alternatively, it is possible that the early lymphocyte recovery may identify a group of good risk patients who have an inheritently functional immune system despite the presence of malignancy and its treatment. Indeed, a recent report has shown an association between lymphocyte graft content and post transplant lymphocyte recovery.¹⁶ In a series of 190 patients autografted for relapsed NHL, higher stem cell lymphocyte content was observed in patients with an ALC>500 compared to those with an ALC<500 on day +15; and a stem cell ALC>0.5 10⁹/kg was independently associated with longer OS and progression-free survival.

The effects of myeloid growth factors on hematopoietic reconstitution following autografting are well-established with either bone marrow, or to a lesser extent, peripheral blood stem cells.¹⁰ In most studies, administration of post transplant growth factors was found to be cost effective through a reduction of the duration of neutropenia, neutropenic fevers and hospitalization. Those growth factors act to varying degrees on progenitor cells at the early stages of differentiation, resulting in self-replication and/or differentiation along specific pathways (granulocytes, monocyte/macrophages, lymphocytes and/or fibroblasts, stromal or endothelial cells). Although acceleration of myeloid recovery is the major effect associated with myeloid growth factors given post chemotherapy, effects on lymphocyte recovery have also been described. Gore et al.¹³ compared lymphocyte recovery from 19 AML patients receiving GM-CSF following induction chemotherapy to a historical control of 25 patients treated with an identical regimen in the absence of cytokine. A transient, but marked lymphocytosis preceded myeloid recovery in patients treated with GM-CSF. These lymphocytes expressed memory T-cell phenotype and dominant clonal rearrangements. Interestingly, patients who developed peak lymphocyte counts 1000/l were more likely to achieve remission than those with a lower peak. In a randomized trial comparing the effects of post transplant G-CSF and GM-CSF in breast cancer patients receiving high-dose chemotherapy and autografting, a faster granulocyte and lymphocyte (CD3+, CD8+ and CD56+), recovery was observed in patients receiving G-CSF, while a higher HLA-DR expression by CD3+ and a faster lymphocyte proliferative capacity occurred in the GM-CSF group. GM-CSF seemed to exert an earlier effect on all T-lymphocyte subsets, preventing a complete drop during the post-chemotherapy nadir.¹⁴ Information on administration of post transplant myeloid growth factors in trials analyzing the prognostic significance of early post transplant lymphocyte recovery was either not reported,^{3, 4, 5, 16} or if reported, the impact of those growth factors on lymphocyte recovery was not analyzed.⁷

Given the effects of GM-CSF on lymphocytes, we hypothesized that routine administration of GM-CSF post transplant alters the prognostic significance of early lymphocyte recovery. In our study, patients with early lymphocyte recovery had similar characteristics and early complications as compared to those with delayed recovery and despite a statistically (but not clinically) and significantly faster neutrophils and platelets recovery. Additionally, neither DFSs nor OSs were different when patients were stratified by LG and IG/HG NHL. This study is limited by a relatively short follow-up (median 22 months). However, in Porrata's NHL analysis, patients with late lymphocyte recovery relapsed relatively early post transplant. Indeed for those patients, and in comparison to early lymphocyte recovery patients, median OSs and DFSs were 16 vs 55 months (P=0.0063) and 9 vs 49 months, (P=0.0067), respectively in the diffuse large cell group; and 9 months vs not reached (P=0.0001) and 7 vs 108 months, (P=0.0001), respectively in the follicular group.^{4, 5} Recent data suggest that absolute NK cell subsets measured on day +15 post transplant and the kinetic of lymphocyte recovery, rather than its extent, are better prognostic indicators than the ALC measured on day 15.^{4, 17} Given the retrospective nature of this analysis, and the fact that peripheral blood lymphocyte subsets were not prospectively measured in this patient population, analysis of these factors could not be done.

In conclusion, this study failed to reproduce the favorable outcomes associated with early lymphocyte recovery post autografting for NHL. We hypothesize that post transplant GM-CSF may have contributed to this apparent loss of prognostic significance.

References

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Acknowledgements

We thank Dr Luis Porrata for reviewing the manuscript and insightful comments, Angela Smith and Larisa Duffey for providing data from the cryopreservation laboratory.