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Lymphocyte expansion after unrelated cord blood allogeneic stem cell transplantation in adults

Abstract

Limited information is available regarding the incidence and features of lymphocyte expansions after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Large granular lymphocytes (LGL) expansions have been reported after bone marrow or peripheral blood, but not after unrelated cord blood (UCB) allo-HSCT, associated with indolent clinical courses and favorable outcomes. Here, we considered 85 recipients of UCB allo-HSCT to more broadly define the impact of lymphocytosis, not limited to LGL. Sustained lymphocytosis was observed in 21 (25%) patients at a median onset of 12.6 months and with a median duration of 12 months. Immunophenotypic analysis showed predominantly CD8+ T and/or polyclonal B-cell expansions. Three patients only had monoclonal T-cell expansion. CMV reactivation was significantly more frequent in the group of patients with lymphocytosis (76% vs 28%, P=0.0001), but was not associated with survival. Conversely, 2-year disease-free survival and overall survival were significantly higher for lymphocytosis patients (85% vs 55%, P=0.01 and 85% vs 63%, P=0.03, respectively). In conclusion, expansion of T or B lymphocytes after UCB allo-HSCT in adults is not a rare event. Although occurring relatively late after transplant, this feature is predictive of a better outcome for the patients.

Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a good option for the treatment of hematological malignancies. Leukemia eradication is obtained by the direct cytotoxic effect of the conditioning regimen, but also through the immune recognition of malignant cells by donor lymphocytes, referred to as the GvL effect.1 A better understanding of the interplay, after allo-HSCT, between immune recovery and the GvL effect is however still needed. Peripheral lymphocytosis, encountered after myeloablative or reduced-intensity conditioning is an ill-defined feature with no prognostic impact described. Most reports in the literature deal with large granular lymphocyte (LGL) expansions and only seldom report B-cell increases.2 With an incidence of 3–18%, LGL proliferations occur generally late after allo-HSCT with a median onset of 9–16 months.3, 4, 5 Such expansions can be polyclonal, oligoclonal or monoclonal, arising from either CD3+ T cells or CD3 natural killer cells or both. LGL expansion has been frequently linked to CMV reactivation, an indolent clinical course and a usually favorable outcome. Most available data were described in the setting of allo-HSCT using bone marrow or peripheral blood (PB) as the stem cell source. Here, we report data regarding the incidence and features of more broadly defined lymphocyte expansions, not limited to LGL, after unrelated cord blood (UCB) transplantation in adults.

Subjects and methods

Ninety-eight UCB allo-HSCT were performed in adults (18 years) between October 2005 and October 2014 at Nantes University Hospital. The 85 UCB allo-HSCT patients alive at 3 months post transplant were considered for final analyses as lymphocyte expansion is generally a late event after transplant. Most patients received double CB units (n=83) and a reduced-intensity conditioning regimen (n=72), for various hematological diseases. A few patients received myeloablative (n=9) or sequential (n=4) conditioning. All patients benefited from GvHD prophylaxis with cyclosporine and mycophenolate mofetil, whereas only eight patients received anti-thymocyte globulin (Thymoglobulin, Sanofi-Aventis, Gentilly, France). All patients also received G-CSF from day +1 until neutrophil recovery. All patients also had regular complete blood counts, which were reviewed to assess lymphocytosis. Whenever detected, the date of onset and termination of PB lymphocyte expansions were collected. Such lymphocyte expansions were defined as 4 × 109/L.6 PB immunophenotyping allowed to discriminate expansions of cytotoxic T cells (CD3+CD8+CD56+), natural killer (NK) cells (CD3CD16+/CD56+) and B cells (CD19+). Of note, in one patient, lymphocytosis was due to the lymphoma relapse: this patient was not considered in the lymphocytosis cohort.

Engraftment was monitored by quantitative PCR of donor- and recipient-specific single nucleotide polymorphisms.7 Both whole PB and sorted CD3+ T cells were used to assess chimerism. As DNA was thus available from these T cells, it was possible to retrospectively investigate the clonality of TCR-γ/β gene rearrangements in samples collected during sustained lymphocytosis. These clonality assays used the Biomed-2 PCR method.8

Lymphocyte expansion data were also analyzed with respect to viral reactivation episodes, acute or chronic GvHD, relapse and survival.

Statistics were performed using the Medcalc software (Ostend, Belgium). For continuous variables, data were tested for normality, and reported as medians or means. Categorical variables were reported as frequencies (%). Disease-free survival (DFS) was considered as the time from UCB allo-HSCT to either relapse, death or last follow-up if censored. Overall survival (OS) was considered as the time from UCB allo-HSCT to death or last follow-up if censored. These tests were also performed considering only patients alive 1 year after UCB allo-HSCT (median time of lymphocytosis expansion onset in our study), taking this date as the starting point. OS and DFS were evaluated using Kaplan–Meier survival curves and log-rank tests. Cumulative incidences were evaluated using the R solution.

Results

Characteristics of the cohort

Sustained non-relapse lymphocytosis was observed in 21 (25%) of the 85 cases of UCB allo-HSCT studied (Table 1). There were 10 females and 11 males, with a median age of 59 years old (range 32–71). Most of these 21 patients had received UCB allo-HSCT for a myeloid-lineage related disease (67%) and 67% were in CR at the time of UCB allo-HSCT. This was not significantly different in the group without lymphocytosis where, respectively, 50% of the patients had an initial myeloid-related disease and 75% were in CR at the time of UCB allo-HSCT. Of note, however, lymphocytosis was observed in none of the 10 patients with ALL nor in the seven patients with Hodgkin lymphoma.

Table 1 Patient characteristics and outcome: comparison between the groups of patients with or without lymphocytosis after UCB allo-HSCT

A majority of lymphocyte expansions occurred in patients who had received reduced-intensity conditioning, whereas only one case was observed following myeloablative conditioning and one after a sequential regimen. No difference in the occurrence of lymphocytosis was related to anti-thymocyte globulin administration, number of nuclear cells or CD34+ cells infused. Lymphocytosis was also unrelated to the number or type of HLA mismatches between recipients and CB units. Only a trend was seen with double HLA A and B mismatch (P=0,055), more frequent in patients with lymphocytosis. Sixty-four patients were alive 1 year post transplant, 19 in the group with lymphocytosis and 45 in the other group.

Lymphocytosis after UCB allo-HSCT is of T or B immunophenotype

The median onset of lymphocyte expansion after UCB-HSCT was 12.6 months (range 1.4–49) (Figure 1). The median initial lymphocyte count was 4.76 × 109/L (range: 4–9.22) at the time of expansion diagnosis. The median duration of expansion was 12 months (range 1–52).

Figure 1
figure1

Cumulative incidence of the occurrence of lymphocyte expansion after UCB allo-SCT.

Immunophenotypic analysis was possible for 20 of the 21 patients with lymphocytosis. This showed nine CD8+ T and one NK LGL expansions. Seven cases of polyclonal B-cell expansions were also documented, whereas three patients presented both T CD8+ and B-cell expansions. Of note, all B-cell expansions tested were moderately but undoubtedly CD5+. Sorted material from chimerism studies was available for seven of the nine patients with T-cell expansion. Two patients had polyclonal profiles, whereas oligoclonality was observed in two patients and monoclonality in three. For these three patients, a control of clonality performed a few months later demonstrated persistence of a similar profile (Figure 2). As for one of them no engraftment was obtained, this clone was of recipient origin. Moreover, among the three patients with a T-cell clone, none presented any sign of LGL leukemia. As full engraftment was demonstrated by chimerism studies, lymphocyte expansions were of UCB origin for 19/21 patients, whereas the last two had autologous reconstitution.

Figure 2
figure2

Persistence of Dβ1-Jβ TCR (UPN4 and UPN16) and Dβ2-Jβ TCR (UPN9) rearrangement profile at the time of lymphocytosis (2–8 months post UCB allo-HSCT) and 5–18 months post UCB allo-HSCT. A full colour version of this figure is available at the Bone Marrow Transplantation journal online.

Acute and chronic GvHD developed, respectively, in 37% and 58% of lymphocytosis patients, and in 52% and 47% of the 64 patients without lymphocyte expansion (P=NS). Lymphocytosis and acute or chronic GvHD were seen in 9 of 10 patients with CD8+ T-cell expansion, but only in one patient with B-cell expansion (P=0,003). Seven patients had hypogammaglobulinemia at the time of diagnosis of lymphocyte expansion and all of them presented with bacterial, mostly bronchial, infections.

Lymphocytosis after UCB allo-HSCT is associated with viral infections

Viral reactivation rates, as assessed by specific PCR, were similar in both groups of patients for human herpesvirus-6 (86% in the lymphocytosis group vs 73%, P=NS) and EBV (29% and 36%, respectively, P=NS). CMV reactivation was conversely significantly more frequent in the group of patients with lymphocytosis (76% vs 28%, P=0.0001). For half of them, CMV reactivation occurred early, before day +30 after UCB allo-HSCT. There were also more patients seropositive for CMV before transplantation in this group (68.4% vs 37,9%, P=0.002). Interestingly, CMV reactivation was significantly higher in the 10 patients of the T or NK group compared with the 7 patients with B-cell expansion (100% vs 57%, P=0.05). Nine of the 16 cases of CMV reactivation required foscarnet or ganciclovir therapy. The median time between CMV infection and lymphocytosis onset was 9 months (range, 20 days–25 months). This does not include one patient who had CMV reactivation together with a BK virus cystitis, treated by IVIgs in whom CMV reactivation appeared 6 months after the end of lymphocytosis. Interestingly, it was the only NK lymphocytosis case of our series.

Treatments and outcome

At the time of analysis, the median follow-up for the 21 lymphocytosis patients was 26 months (range, 3–83). None of the lymphocytosis patients required treatment for LGL proliferation. However, six patients had recurrent upper respiratory tract infections that required antibiotics or IVIg for two of them. In one case, there was a severe thrombocytopenia post UCB allo-HSCT. None of the 21 patients had autoimmune manifestations. At the time of UCB allo-HSCT, 14 and 3 patients were in CR or PR, respectively (81%), and none of them relapsed. Four patients had active or refractory disease at the time of UCB allo-HSCT. One of them never achieved remission and another relapsed 78 days after UCB allo-HSCT. At the time of analysis, one patient had relapsed and four had died, the causes of death being disease in one case and transplant-related mortality in three. These events were significantly lower than in the group of patients without lymphocytosis (P=0.003 for relapses and P=0.04 for death). As a result, 2-year DFS (Figure 3a) and OS (Figure 3b) were significantly different at, respectively, 85% (lymphocytosis group) vs 55% (non-lymphocytosis group) (P=0.01), and 85% vs 63% (P=0.03). Significantly better DFS (Figure 3c; P=0,03), but only a trend to better OS (Figure 3d) (P=0,1), were also observed in lymphocytosis patients alive 1 year post UCB allo-HSCT, compared with the other group. OS and DFS were not significantly different between patients with or without CMV reactivation.

Figure 3
figure3

Comparison of disease-free and overall survival between patients with lymphocytosis (dotted line) or not (solid line) considering the whole cohort (n=85, a and b), and patients alive at 1 year post transplant (n=64, c and d), respectively. DFS, disease-free survival.

Discussion

The aim of this study was to retrospectively examine the features of peripheral lymphocytosis (defined as 4 × 109/L) occurring in some UCB allo-HSCT patients. We observed here that 25% of the patients alive 3 months post UCB allo-HSCT developed lymphocytosis at a median time of 1 year post transplant. This is more frequent than the 3% reported by Mohty et al.3 after bone marrow/PBSC allo-HSCT, yet the six patients described by these authors had milder expansions, all clearly defined as CD8+CD57+ LGL. Similarly, lymphocytosis followed predominantly reduced-intensity conditioning in this study.3 Lymphocytosis was often transient with nonetheless a median duration of 12 months (1–52). This is lower than the 31 months reported by Nann-Rütti et al.,4 but comparable to the 10 months reported by Mohty et al.3 As described, post UCB allo-HSCT, lymphocytosis was also indolent with no sign of LGL leukemia.4, 9, 10 Only two patients had a persistent indolent clonal T-cell expansion up to 18 months after UCB allo-HSCT. At variance with previous reports,11 GvHD was not more frequent in lymphocytosis patients, nor HLA mismatched patients. However, post UCB allo-HSCT lymphocytosis patients with chronic GvHD had significantly more frequently a CD8+ expansion. This suggests a role for these cells in GvHD, yet these expansions of cytotoxic T cells could also exert a GvL function. Conversely, B-cell lymphocytosis was more frequently seen in patients without GvHD. Lymphocytosis after UCB allo-HSCT involved equally CD8+ and B cells in contrast with bone marrow/PBSC allo-HSCT, which has been reported to be more often associated with CD8+ expansions.3

CMV but not human herpesvirus-6 nor EBV reactivation was significantly associated with post UCB allo-HSCT lymphocytosis. CMV reactivation may be considered as an expression of chronic lymphocyte stimulation as previously proposed.4 It explains perhaps that CMV reactivation was more associated with CD8+ than B-cell expansions. Because these cells were of donor origin, it can be postulated that they represent primo-activation upon encounter with CMV. B-cell expansions were polyclonal and curiously often associated with CD5 intermediate expression suggesting a proliferation of innate B1 cells from the UCB allo-HSCT.12

Although the antileukemic effect of CMV infection was first suggested by Lönnqvist et al.,13 and similarly reported in adult14, 15, 16 and pediatric17 cohorts, here, CMV reactivation did not influence DFS nor OS. However, we observed significantly fewer relapses or disease-related deaths in the group of post UCB allo-HSCT lymphocytosis patients, where a significantly higher rate of CMV reactivation was observed (76%). The absence of effect of CMV reactivation on DFS and OS suggests possibly a bystander GvL effect of lymphocyte expansions.18 Interestingly, two lymphocytosis patients did not engraft and did not relapse, suggesting an antitumor effect of allo-HSCT even in cases without sustained donor engraftment.19 This absence of relapse associated also to lymphocytosis in a context of CMV reactivation maybe a witness of the bystander effect described above.

Late relapse may occur after UCB allo-SCT and death owing to the procedure can be also documented at distance from the graft,20, 21 probably related to persistent immune deficiency. Here, lymphocyte expansion was found to be associated with a favorable outcome, overall as well as when considering patients alive 12 months post UCB allo-HSCT, the median time of occurrence of this event. This better outcome could be related to a positive anti-GvL effect of UCB allografted cells when they proliferate.

In conclusion, expansion of T or B lymphocytes after UCB allo-HSCT in adults is not a rare event. Although occurring relatively late after transplant, this event is predictive of a positive outcome.

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Correspondence to Y Le Bris.

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Le Bris, Y., Guillaume, T., Ménard, A. et al. Lymphocyte expansion after unrelated cord blood allogeneic stem cell transplantation in adults. Bone Marrow Transplant 52, 854–858 (2017). https://doi.org/10.1038/bmt.2016.364

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