Lymphopenia at diagnosis is highly prevalent in myelodysplastic syndromes and has an independent negative prognostic value in IPSS-R-low-risk patients

Lymphopenia is associated with an increased mortality in several medical conditions. Its prognostic impact in myelodysplastic syndromes (MDS) is less well studied. Hence, we analyzed 1023 patients from the Düsseldorf MDS-registry with regard to the absolute lymphocyte count (ALC) at diagnosis. An ALC below the median of the population (1.2 × 109/l) was associated with lower counts of neutrophils (median 1.35 vs. 1.92 × 109/l, p < 0.001) and platelets (median 100 vs. 138 × 109/l, p < 0.001) and with a significant lower overall survival in univariate analysis (whole cohort: median 36 vs. 46 months, p = 0.016; 721 patients without hematopoietic stem cell transplantation or induction chemotherapy: median 36 vs. 56 months, p = 0.001). For low-risk MDS according to IPSS-R, an ALC < 1.2 × 109/l was of additional prognostic value in a multivariate Cox regression model together with age (< or ≥65 years) and LDH (< or ≥normal value of 240 U/l; HR 1.46, 95% CI: 1.03–2.08, p = 0.033). These data support the hypothesis of subtle but clinical relevant changes of the adaptive immune system in MDS. Further studies are necessary to identify the ALC cut-off best suitable for prognostication and the mechanisms responsible for the impairment of lymphoid homeostasis in MDS.


Introduction
The myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematological stem cell disorders arising primarily in the elderly population. Genetic and epigenetic changes in hematopoietic stem cells and alterations in the hematopoietic niche are the main factors that render hematopoiesis ineffective and increase the risk of leukemic transformation due to increased genetic instability.
An accurate risk stratification regarding the latter event is essential for newly diagnosed MDS patients. The most frequently used score for risk stratification, the international prognostic scoring system (IPSS) 1 and its revised version 2 take the degree of cytopenias, the bone marrow blast count and presence and type of cytogenetic changes determined by conventional metaphase cytogenetics into account. Higher-risk MDS patients are candidates for allogeneic hematopoietic stem cell transplantation (HSCT) because of the risk of leukemic transformation and a very short overall survival (OS) even without transformation. For lower-risk MDS patients best supportive care and improvement of cytopenias are the cornerstones of therapy. Additional risk factors such as elevated lactate-dehydrogenase (LDH), bone marrow fibrosis, and recently more and more recognized the mutational profile as determined by next generation sequencing (NGS) are important additional factors that can be considered for clinical decision making, especially in intermediate risk patients according to the IPSS-R 3 .
A low-lymphocyte count is a prognostic factor in solid tumors and lymphoid malignancies 4 . The prevalence and prognostic impact of lymphopenia in MDS patients has been studied less systematically. We, therefore, conducted this study to provide additional evidence with a focus on MDS subtypes according to the WHO 2016 classification, the IPSS-R and the presence of comorbidities.

Material and methods
We screened the Düsseldorf MDS-registry for patients with information about the absolute lymphocyte count (ALC) at diagnosis and the IPSS-R category. The Düsseldorf MDS-registry covers virtually all newly diagnosed MDS-cases in the Greater Düsseldorf Area since 1982. Structure of the registry and the diagnostic criteria for inclusion of cases have been described earlier in detail 5 . The local ethics committee (University Hospital, Heinrich Heine University, Düsseldorf) approved the study, which followed the 2000 revision of the Declaration of Helsinki. All patients gave their written informed consent before being included into the Düsseldorf MDS registry.
Patients with an ALC > 5.0 × 10 9 /l were excluded from the dataset, since they could suffer from a concomitant lymphoproliferative disorder, as were cases with a white blood cell count > 13.0 × 10 9 /l, since they could represent myelodys plastic/myeloproliferative neoplasms. For stratification of patients into lymphopenic and nonlymphopenic, a cut-off of <1.0 × 10 9 /l lymphocytes was used, according to the normal range of our institutional laboratory (1.0-4.4 × 10 9 /l). For survival analyses, an ALC of 1.2 × 10 9 /l was used as an additional discriminator, since this value was of prognostic relevance in earlier studies [6][7][8] and represented the median of our cohort. Data for the MDS comorbidity index (MDS-CI) were retrieved from the dataset of a study published previously 9 .
Categorical variables were analyzed by frequency tables and compared by the χ 2 -test, continuous variables were described by median (range) and compared between different groups by the Mann-Whitney (comparison of two groups) or the Kruskal-Wallis test (comparison of ≥3 groups), since data did not follow a normal distribution. Overall and leukemia-free survival were calculated in months from the date of diagnosis to the respective event date. Time-to-event-curves were calculated by the Kaplan-Meier method and the log-rank test was used for univariate comparison. Cox proportional hazard regression model was applied for multivariate analysis. All statistical tests were two-sided and a p value < 0.05 was considered statistically significant. All analyses were performed with IBM SPSS Statistics, Version 25.
Totally, 133 patients (13%) underwent HSCT and 81 (8%) were treated with at least one course of an intensive induction chemotherapy. The remaining patients received other disease modifying treatments like immunomodulatory drugs (Lenalidomide or Thalidomide, n = 62) or hypomethylating agents (Azacytidine, n = 76, Decitabine n = 12), were treated with lowintensity treatments like erythropoiesis stimulating agents or immunosuppression (cyclosporine ± anti-thymocyte globulin) or received best supportive care, including transfusion and iron chelation.
Distribution of MDS subtypes according to the WHO 2016 classification, cytogenetic risk groups according to the IPSS-R and the IPSS-Risk categories, as well as peripheral blood values, blast counts in bone marrow and peripheral blood and additional parameters (LDH, presence or absence of bone marrow fibrosis, transfusion dependency) at the time point of diagnosis are listed in detail in Table 1.

Lymphocyte counts in different MDS subgroups
Despite considerable inter-individual variations of the ALC, significant differences were detected between specific MDS subgroups, with a moderate extent in terms of absolute numbers (see Table 2).
For patients suffering from secondary MDS or having received HSCT or induction chemotherapy OS was not influenced by lymphopenia (median OS 26 vs. 21 months, p = 0.89 and 46 vs. 31 months, p = 0.14, respectively).
Due to these findings, further survival analyses were restricted to patients suffering from primary MDS without HSCT or induction chemotherapy (n = 721). The influence on survival of an ALC < 1.2 × 10 9 /l in this group was highly significant (mean OS 36 vs. 56 months, p = 0.001; Fig. 1b). It remained an independent prognostic factor in a multivariate cox regression model together with the cytogenetic risk groups according to the IPSS-R. However, after additional inclusion of bone marrow blast count (< or ≥5%) and level of cytopenias (hemoglobin < or ≥10 g/dl, absolute neutrophil count < or ≥1.8 × 10 9 /l and platelets < or ≥100 × 10 9 /l) into the model, an ALC < 1.2 × 10 9 /l lost its additional prognostic value (p = 0.086, details are shown in Table 3).

Discussion
Analyzing a large cohort of 1023 uniformly characterized patients, we found evidence for a significant impairment of lymphopoiesis in MDS. Totally, 38% of patients were lymphopenic (ALC < 1.0 × 10 9 /l) and the median lymphocyte count of 1.22 × 10 9 /l observed in our cohort was conssiderably lower than the median ALC of about 2.0 × 10 9 /l, published for a large cohort of healthy, nonhispanic caucasian US Americans in the corresponding age groups 10 . In addition, an ALC < 1.2 × 10 9 /l was associated with a worse OS in univariate analysis.
These results are consistent with previously published data on lymphopenia in MDS and its impact on survival. A prognostic role for an ALC < 1.2 × 10 9 /l in MDS with del(5q), including patients with an increased blast count and additional cytogenetic abnormalities, was first reported by Holtan et al. 6 . Subsequently, a lymphopenia defined by this cut-off was considered to be an independent prognostic factor in addition to IPSS and WPSS in MDS without del(5q) as well 7 . Within an unselected cohort of primary MDS, an ALC < 1.2 × 10 9 /l was associated with an inferior OS in univariate analysis but failed to reach statistical significance, when including the more refined cytogenetic risk categories according to IPSS-R into a multivariate Cox regression model 8 .
In addition to the confirmation of these previous observations in an independent cohort, our findings add some new aspects to the role of lymphopenia in MDS. First, the additional prognostic value of a low-lymphocyte count seems to be restricted to low-risk patients, if the IPSS-R is used for risk stratification. However, for this subgroup it can be considered as a relative strong additional prognostic marker, since it retained its independent prognostic value in a multivariate Cox regression model together with age and an elevated LDH, two additional risk factors well-known to affect prognosis in MDS 2,11 . Table 3 Factors affecting prognosis in MDS patients without hematopoietic stem cell transplantation or induction chemotherapy (n = 721) in univariate analysis and two multivariate models (multivariate I: absolute lymphocyte count < 1.2 × 10 9 /l together with the cytogenetic risk groups according to IPSS-R; multivariate II: absolute lymphocyte count < 1.2 × 10 9 /l together with the cytogenetic risk groups according to IPSS-R, bone marrow blast count and the degree of cytopenias) The lack of an additional prognostic value for the IPSS-R very low-risk category in our cohort may be due to the very low patient number in this cohort (n = 51). Biology and clinical course of high-risk and very high-risk MDS are primarily characterized by genomic instability and AML evolution. In this setting, an additional impairment  of the adaptive immune system does not seem to have additional prognostic value. Alterations of both the innate and the adaptive immune system have long been recognized as a feature in MDS 12,13 . The T-cell compartment has been most extensively studied, with a focus on its involvement in the pathophysiology of MDS. To date, it is unknown, whether lymphopenic MDS patients differ from MDS patients with a normal lymphocyte count with regard to the distribution or functional capacity of different lymphocyte subsets. Whether lymphopenia in MDS is a direct consequence of underlying hematopoietic stem cell defect (s) or arises from immune-modulating stimuli related to the disease or to other host conditions remains to be elucidated as well.
Here, some of our observations point towards a direct relationship between MDS pathophysiology and low lymphocyte counts. In our cohort, neither age nor comorbidities were associated with a lower ALC and significant differences with regard to the ALC were noted between different MDS subtypes. Notably, ALCs were lower in subtypes with a worse prognosis, like higher-risk MDS according to the IPSS-R, cases of therapy related MDS and in patients being transfusion dependent at diagnosis. In addition, lymphopenia was associated with lower peripheral blood counts as markers of disease severity, a finding that has been described previously 8 .
Some of the key features of T-cell alterations seen in aging individuals 14 have also been described as features of T-cell lymphopoiesis in MDS, as for example a skewed Tcell receptor repertoire 15 and a contracted pool of CD8+ T cells 16 . Hence, the overlapping features of the physiology of aging hematopoiesis and the pathophysiology of MDS, which have come into focus recently 17 , may provide some explanations for the lymphopenia occurring in MDS-patients.
Many changes of the adoptive immune system in the elderly, often referred to as immunosenescence, can be directly traced back to changes in the stem cell compartment, which typically shows a myeloid-skewing with a diminished capacity for lymphoid differentiation 18 . Subclinical inflammatory changes in the bone marrow microenvironment are a further hallmark of the aging hematopoietic stem cell niche 19 . Recently, microinflammation in the bone marrow mediated by the NLRP3 inflammasome has been identified as one driver of the MDS phenotype 20 and activation of the NLRP3inflammasome within the bone marrow is known to inhibit B-cell lymphopoiesis at least in vitro 21 . Additional alterations in the bone marrow niche linking impairment of lymphopoiesis and inflammatory changes likely exist and may not only cause ineffective hematopoiesis, but also compromise homeostasis of both B and T cells.
Furthermore, lymphocytes can be part of the MDSclone. For MDS with del(20q) 22 , Monosomy 7 (ref. 23 ), trisomy 8 (ref. 24 ) and del(5q) 23 , the respective cytogenetic anomalies have been described in B-and/or T-cell lymphocytes, leading to the hypothesis that the MDS clones arise in a common myeloid and lymphoid progenitor at least in some cases. This was demonstrated in a study applying NGS to bone marrow cell populations sorted according to their differentiation stage in MDS-RS-SLD 24 . The initiating SF3B1 mutation was found in a lymphomyeloid progenitor and is consistently detectable at the pro-B-cell progenitor stage. Since the mutation was rarely observed in mature B cells, it can be concluded that SF3B1-mutations are likely to impair B-cell development.
Concerning T-cell involvement in myeloid neoplasms, mutations in the epigenetic regulator DNMT3A are detectable in AML patients in both leukemic blasts and in T cells at diagnosis 25 , and may persist in first complete remission in both compartments 26 . Since DNMT3A has recently been shown to be involved in T-cell development 27 , DNMT3A mutations may affect T-cell biology and due to the many pathways shared between MDS and AML, MDS could be derived from lympho-myeloid clonal hematopoiesis at least in some subtypes. However, the clonal involvement of T cells in MDS remains to be studied systematically.
Both Lenalidomide 28,29 , and hypomethylating agents 30 alter lymphocyte biology during treatment. Hence, these treatments could possibly be able to reverse the negative prognostic impact of a lymphopenia at diagnosis. Only a limited number of patients in our cohort were treated with these compounds, so we are not able to address the question whether a low ALC remains a significant prognostic factor in patients treated with Lenalidomide and hypomethylating agents. Further studies addressing this question are needed. Since the ALC is readily available in clinical routine this question can be addressed in clinical trials or register studies. Furthermore, the additional prognostic value of the ALC should be investigated after refinement of the IPSS-R by the inclusion of adverse molecular features revealed by NGS 31 .
Taken together, our data support the hypothesis of subtle but clinically relevant changes of the adaptive immune system in MDS. Further studies are necessary to define the ALC cut-off most suitable for prognostication and to identify the mechanisms underlying the impairment of lymphoid homeostasis in MDS.