ALBERT

Description: Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD 〈 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 798 Interleukin-7 (IL-7) belongs to the hematopoietic family of cytokines, being essential for T cell development in the thymus and maintenance of peripheral T cells. IL-7 signals through IL-7R, which consists of the gamma common chain as well as an alpha-chain. The alpha-chain, is also used by the receptor of thymic stromal–derived lymphopoitin (TSLP), a cytokine with complex effects on cytokine profiles, including induction of TNF production by dendritic cells. We have sequenced IL-7Ralpha and identified the existence of 4 single nucleotide polymorphisms (+510C/T, +1237 A/G, +2087T/C and +3110A/G), that all give rise to amino acid substitutions. In a previously published study, we found that the G allele of the +1237 SNP of the donor was associated with increased overall mortality and treatment related mortality in patients having matched unrelated donors (MUD) SCT. The aim of the present investigation was to validate these findings in a study of a British-French cohort of SCT patients. Patients and methods: IL-7Ralpha polymorphisms were determined by a SSP-PCR system in genomic DNA from 119 transplants with either matched sibling donors (n=93) or MUD (n=26). The indication for SCT included AML (n=32), CML (n=27), ALL (n=18), other malignancies (n=29) and benignant haematological disorders (n=11). Results: Donor homozygozity for the T allele at position +510 was associated with increased overall mortality (HR:1.99 (95%CI:1.03-3.84) p=0.04) as well as treatment related mortality (TRM) (HR: 1.38 (95% CI:1.38-6.03) p=0.0054), as evaluated by multivariate testing using Cox regression analysis. In contrast, this SNP was unrelated to the risk of relapse. A similar pattern was found for the +1237 SNP, with increased TRM associated with homozygozity for the G allele in the donor (HR: 2.28 (95%CI:1.09-4.77) p=0.03) , although the association between this genotype and overall mortality was not significant (HR: 1.72 (95%CI:0.90-3.26) p=0.10). In line with this, the frequency of grade 3-4 acute GvHD was related to the +510 genotype of the donor: (TT (50%), CT (8%), and CC (15%) (OR=7.70 (95%CI:2.47-24), p= 0.0004) as was the +1237 SNP: GG (43%), AG (6%) and AA (16%) (OR=6.26 (95%CI:2.06-19), p=0.001) (multivariate analysis). There was no clear association between the recipient genotype of these IL-7Ralpha SNPs or with the +2087 or +3110 SNPs. Conclusion: The presented data confirms our previous finding that the +1237 genotype of the donor is related to TRM and acute GvHD after SCT. Moreover, the data suggests a similar pattern for the +510 SNP of the donor, in line with the fact that +510 and +1237 SNPs are in close linkage disequlibrium. These findings suggest that IL-7 and/or TSLP pathways may be of pathogenetic importance for transplant related mortality and acute GvHD, and suggest that modulation of these pathways may have an impact on the outcome in SCT. Finally, the study suggests that selection of donors based on IL-7Ralpha typing, in addition to HLA typing, may help to improve the outcome in SCT. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Monocytes and neutrophilic granulocytes are critical for innate immunity, through recognition and phagocytosis of pathogens, and mediation of acute inflammation through secretion of pro-inflammatory cytokines. Furthermore, monocytes constitute an important link between innate and adaptive immunity through antigen presentation to lymphocytes. Patients with chronic lymphocytic leukemia (CLL) have an increased risk of infection, despite often displaying normal neutrophil and monocyte counts. CLL-cells depend on interactions with the immune microenvironment (IME) for proliferation and survival, while inducing changes in surrounding immune cells. However, changes in myeloid cell function in patients with CLL remain sparsely documented. Since the btk-inhibitor ibrutinib impairs B-cell receptor signaling and disrupts CLL-IME interactions (Niemann et al, CCR, 2016), we investigated the impact of ibrutinib and venetoclax on monocyte- and neutrophil phenotype and function in CLL patients. Methods: Nine patients treated with ibrutinib 420 mg daily for 8 weeks followed by addition of venetoclax with an initial 5-week ramp up period were included. Blood samples were taken at baseline, after 8 weeks ibrutinib monotherapy, and after another 8 weeks of ibrutinib and venetoclax combination therapy. Immune phenotype was assessed in whole blood by an 8-tube, 10 color flow cytometry panel with custom designed lyophilized antibodies (Duraclone). Immune function was characterized using TruCulture, a whole blood-ligand stimulation assay applying the toll like receptor (TLR) ligands heat killed candida albicans (TLR 2,4,6), lipopolysaccharide (LPS; TLR4), resiquimod (single-stranded RNA-virus analog, TLR7,8), and Poly:IC (double-stranded RNA-virus analog, TLR3), after which the cytokine response was measured. Informed consent from patients and approval from the ethics committee was obtained. Results: Monocyte and neutrophil counts, as wells as distribution of mature and immature neutrophils, were within normal range at baseline (n=9) and remained unchanged throughout treatment. At baseline, expression of HLA-DR on monocytes was suppressed, but increased significantly upon combination treatment with ibrutinib and venetoclax (p=0.04). HLA-DR expression on neutrophils was high at baseline, remained unchanged upon ibrutinib treatment (n=8), but declined after addition of venetoclax (n=7) (p

Description: Introduction: The role of T cell receptor (TCR) γδ cells in allogeneic hematopoietic stem cell transplantation (HSCT) is becoming of increasing interest1,2. In contrast to conventional alloreactive TCR αβ cells, TCR γδ cells are believed to have anti-tumor effects without causing graft-versus-host disease (GVHD). We conducted a single-center, prospective study to assess the impact of early TCR γδ cell immune reconstitution on overall survival, relapse and acute GVHD after HSCT. Methods: From October 2015 to March 2017, 108 consecutive patients transplanted for malignant diseases at the Bone Marrow Transplant Unit, Department of Hematology, Copenhagen University Hospital, Rigshospitalet, were included in the study, table 1. Fresh blood samples days 28, 56, 91, 180 and 360 after transplantation were analyzed for absolute concentrations of CD3-, CD4- and CD8 positive T cells together with a multi-color flow cytometry panel with staining for TCRαβ, TCRγδ, Vδ1, Vδ2, CD3, CD4, CD8, HLA-DR, CD196, CD45RO, CD45RA, CD16, CD56, CD314 (NKG2D) and CD337 (NKp30) for immune phenotyping. Results: After a median of 673 (386-913) days, 28 (26%) patients had died from relapse (n=14) and from transplant-related-mortality(n=14), respectively. A total of 24 (22%) patients experienced relapse during the observation time with median time to relapse of 177 (56-778) days. Acute GVHD grade 2-4 was diagnosed in 38 (35%) of patients. Patients were divided into two groups by dichotomization at the median value of TCR γδ cell concentrations for Kaplan-Meier analyses of overall survival (OS), relapse-free survival (RFS) and cumulative incidence analyses (Gray's test for competing risks) of relapse and acute GVHD. Patients with high concentrations of TCR γδ cells 56 days after transplantation had significantly higher OS and RFS compared with patients with low concentrations, p

Description: Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity. Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients). Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts. Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations. Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (〉20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments. Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells. The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. Disclosures No relevant conflicts of interest to declare.