Non‐tumour bone marrow lymphocytes correlate with improved overall survival in childhood acute lymphoblastic leukaemia

Abstract Composition of tumour immune cell infiltrates correlates with response to treatment and overall survival (OS) in several cancer settings. We retrospectively examined immune cells present in diagnostic bone marrow aspirates from paediatric patients with B‐cell acute lymphoblastic leukaemia. Our analysis identified a sub‐group (∼30% of patients) with >2.37% CD20 and >6.05% CD7 expression, which had 100% OS, and a sub‐group (∼30% of patients) with ≤2.37% CD20 and ≤6.05% CD7 expression at increased risk of treatment failure (66.7% OS, P < 0.05). Immune cell infiltrate at diagnosis may predict treatment response and could provide a means to enhance immediate treatment risk stratification.


INTRODUCTION
Conventional chemotherapeutic regimens cure a significant proportion of paediatric patients with B-cell acute lymphoblastic leukaemia (B-ALL). Improvements in survival rates observed over recent decades can be attributed to the introduction of efficient testing for minimal residual disease during treatment and new combinations of chemotherapeutics. However, considerable adverse effects are associated with regimens used to treat paediatric B-ALL. Strategies to identify patients in whom lower dose treatment would be clinically effective or treatment intensification would enhance survival, represent attractive avenues to reduce long-term cytotoxicity and/or enhance overall survival (OS).
Recent advances in our understanding of tumour immunology suggest that an individual's immune response, pre-therapy against their own tumour, significantly influences disease progression. 1  in solid tumours have been shown to correlate with prognosis, and in some instances, to predict patient survival more accurately than any other parameter. 2,3 In B-ALL, two recent reports examining the immunological composition of bone marrow (BM) at diagnosis demonstrate correlation between CD4 + (where CD is cluster of differentiation) T lymphocytes and favourable early response in paediatric patients 4 and CD8 + T lymphocytes and improved OS in adult patients. 5 In this study, we examined the composition at diagnosis of non-malignant lymphocytes in the BM of paediatric patients with B-ALL. We aimed to identify whether non-malignant lymphocytes routinely measured in BM aspirate by flow cytometry are associated with OS.

METHODS AND RESULTS
We reviewed the medical records of 153 children diagnosed with ALL at Alder Hey Children's Hospital, Liverpool, between 2002 and We assessed the measured relative frequency values of CD markers in diagnostic BM aspirate between survivors and nonsurvivors. Tumour lymphocytes (CD19 + , CD10 + ) and non-malignant B-lymphocytes (CD19 + , CD10 − ) were equivalent between study groups; however, an increased relative frequency of the mature B-lymphocyte marker, CD20, was observed in survivors (P = 0.0295; Fig. 1A). T lineage cells (CD2 + ) were equivalent between study groups; however, an increased relative frequency of the mature T-lymphocyte and natural killer (NK) cell marker, CD7, was observed in survivors (P = 0.0447; Fig. 1B). Following associated receiver operating characteristic analysis, cut-offs of 2.37% CD20 expression (sensitivity 88%, specificity 56%) and 6.05% CD7 expression (sensitivity 88%, specificity 53%) were selected to differentiate between patients with high and low expression.
Comparing patients with high CD20 expression (>2.37%; n = with CD4 + T cells. 8,9 Lymphocytes are also immune effector cells, and may mediate tumour cell death through granule exocytosis or death receptor signalling, cytotoxic T cells and NKs are key effectors in these mechanisms. Interaction between B and T cells serves to augment adaptive immune responses through cross-priming of T cells, reciprocal enhancement of activation signalling in each cell type and the F I G U R E 1 CD20 + lymphocytes were increased in survivors compared to non-survivors (A), CD7 + lymphocytes were increased in survivors compared to non-survivors (B). Enhanced OS (C) and LFS (D) was observed in paediatric patients with B-ALL having >2.37% CD20 + lymphocytes. Enhanced OS (E) but not LFS (F) was observed in paediatric patients with B-ALL having >6.05% CD7 + lymphocytes. 100% OS was observed in patients having >2.37% CD20 + and >6.05% CD7 + lymphocytes (G). 87.5% LFS was observed in patients having >2.37% CD20 + and >6.05% CD7 + lymphocytes compared to 61.1% LFS in patients having ≤2.37% CD20 and ≤6.05% CD7 expression (H).
production of immunostimulatory cytokines. 9 in this study, three had no clinical high risk features and were treated according to Regimen A. All three of these children had low CD20 levels at diagnosis, and two of these three also had low CD7 levels. Reliable identification of very good risk and very high risk patients at diagnosis potentially offers the opportunity to reduce or intensify therapy from induction onward. Our data clearly define two important patient sub-groups; firstly, one for which the chemotherapeutic treatment regimen was 100% successful, and secondly, one in which patients were at significantly increased risk of treatment failure. Notwithstanding the limitations of this single centre retrospective study of small numbers, it seems relevant that the tumour immune contexture in paediatric ALL receives future attention, both to confirm our observations and to investigate mechanistically the benefit to patients of mature immune cell BM infiltrate at treatment commencement.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.