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Background Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. Patients and methods Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. Results Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). Conclusion Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.

The expression and role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in B cells are not yet explored in contrast to myeloid cells, where these β2-integrin type receptors are known to participate in various cellular functions, including phagocytosis, adherence and migration. Here we aimed to reveal the expression and role of CR3 and CR4 in human B cells. In B cells of healthy donors CR3 and CR4 are scarcely expressed. However, two patients with chronic lymphocytic leukemia (CLL) characterized by a peculiar immune-phenotype containing both CD5-positive and CD5-negative B cell populations made possible to study these molecules in distinct B cell subsets. We found that CD11b and CD11c were expressed on both CD5-positive and CD5-negative B cells, albeit to different extents. Our data suggest that these receptors are involved in spreading, since this activity of CpG-activated B cells on fibrinogen could be partially blocked by monoclonal antibodies specific for CD11b or CD11c. CpG-stimulation lead to proliferation of both CD5-positive and CD5-negative B cells of the patients with a less pronounced effect on the CD5-positive cells. In contrast to normal B cells, CLL B cells of both patients reacted to CpG-stimulation with robust IL-10 production. The concomitant, suboptimal stimulus via the BCR and TLR9 exerted either a synergistic enhancing effect or resulted in inhibition of proliferation and IL-10 production of patients' B cells. Our data obtained studying B cells of leukemic patients point to the role of CR3 and probably CR4 in the interaction of tumor cells with the microenvironment and suggest the involvement of IL-10 producing B cells in the pathologic process.

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy. Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies. Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. Disclosures No relevant conflicts of interest to declare.