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BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3 -mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3 -mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P <0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P <0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P =0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches., (Copyright© 2020 Ferrata Storti Foundation.)

Biologists and clinicians agree that the B-cell receptor influences the behavior of chronic lymphocytic leukemia, and promising new drugs are aimed at receptor-associated kinases. Engagement of surface immunoglobulin by antigen is a key driver of malignant cells with outcome influenced by the nature of the cell, the level of stimulation and the microenvironment. Analysis of surface immunoglobulin-mediated signaling in the two major disease subsets defined by IGHV mutational status reveals bifurcation of responses toward proliferation or anergy. Mutated chronic lymphocytic leukemia, generally of relatively good prognosis, is mainly, but not exclusively, driven towards anergy in vivo. In contrast, unmutated chronic lymphocytic leukemia shows less evidence for anergy in vivo retaining more responsiveness to surface immunoglobulin M-mediated signaling, possibly explaining increased tumor progression. Expression and function of surface immunoglobulin M in unmutated chronic lymphocytic leukemia appear rather homogeneous, but mutated chronic lymphocytic leukemia exhibits a highly heterogeneous profile that may relate to further variable clinical behavior within this subset. Anergy should increase susceptibility to apoptosis but, in leukemic cells, this may be countered by overexpression of the B-cell lymphoma-2 survival protein. Maintained anergy spreads to chemokines and adhesion molecules, restraining homing and migration. However, anergy is not necessarily completely benign, being able to reverse and regenerate surface immunoglobulin M-mediated responses. A two-pronged attack on proliferative and anti-apoptotic pathways may succeed. Increased understanding of how chronic lymphocytic leukemia cells are driven to anergy or proliferation should reveal predictive biomarkers of progression and of likely response to kinase inhibitors, which could assist therapeutic decisions., (Copyright© Ferrata Storti Foundation.)

This work investigates the possibility that HLA-G, a molecule modulating innate and adaptive immunity, is part of an immune escape strategy of chronic lymphocytic leukemia cells. A 14 base pair insertion/deletion polymorphism (rs66554220) in the 3'-untranslated region of HLA-G influences mRNA stability and protein expression. The analysis of a cohort of patients with chronic lymphocytic leukemia confirmed that del/del individuals are characterized by higher levels of surface and soluble HLA-G than subjects with the other two genotypes. In line with its role in immunomodulation, the percentage of regulatory T lymphocytes is higher in del/del patients than in patients with the other genotypes and correlates with the amounts of surface or soluble HLA-G. Furthermore, addition of sHLA-G-rich plasma from patients with chronic lymphocytic leukemia induces natural killer cell apoptosis and impairs natural killer cell lysis, with effects proportional to the amount of soluble HLA-G added. Lastly, the presence of an HLA-G 14 base pair polymorphism is of prognostic value, with del/del patients showing reduced overall survival, as compared to those with other genotypes. These results suggest that: (i) the HLA-G 14 base pair polymorphism influences the levels of surface and soluble HLA-G expression, and (ii) the over-expression of HLA-G molecules contributes to creating tolerogenic conditions.

Background: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive., Design and Methods: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters., Results: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets., Conclusions: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

Antigen stimulation may be important for splenic marginal zone lymphoma pathogenesis. To address this hypothesis, the occurrence of stereotyped B-cell receptors was investigated in 133 SMZL (26 HCV+) compared with 4,414 HCDR3 sequences from public databases. Sixteen SMZL (12%) showed stereotyped BCR; 7 of 86 (8%) SMZL sequences retrieved from public databases also belonged to stereotyped HCDR3 subsets. Three categories of subsets were identified: i) "SMZL-specific subsets" (n=5), composed only of 12 SMZL (9 HCV-from our series); ii) "Non-Hodgkin's lymphoma-like subsets" (n=5), comprising 5 SMZL (4 from our series) clustering with other indolent lymphomas; iii) "CLL-like subsets" (n=6), comprising 6 SMZL (3 from our series) that belonged to known CLL subsets (n=4) or clustered with public CLL sequences. Immunoglobulin 3D modeling of 3 subsets revealed similarities in antigen binding regions not limited to HCDR3. Overall, data suggest that the pathogenesis of splenic marginal zone lymphoma may involve also HCV-unrelated epitopes or an antigenic trigger common to other indolent lymphomas.

Background: We previously reported ongoing mutational and isotype switch events in the immunoglobulin (Ig) heavy chain (H) locus in hairy cell leukemia. Those analyses raised questions on the incidence and type of selective influences occurring on the tumor B-cell receptor of hairy cell leukemia., Design and Methods: To further investigate this issue, we examined the full IGH and kappa and lambda light chains (IGkappa and IGlambda) variable and constant region transcripts expressed in a large cohort of patients with hairy cell leukemia (n=88)., Results: Multiple IgH isotypes were expressed in 46/56 (82%) cases of hairy cell leukemia. Comparison of tumor with normal B-cell repertoires revealed preferential usage of IGHV3-21, IGHV3-30 and IGHV3-33 in hairy cell leukemia (p=0.001, p=0.003 and p=0.001, respectively). Light chain analysis demonstrated preferential Igl use with an inverted IGk:IGl ratio (0.7:1) and universal usage of IGLJ3. Analysis of LCDR3 junctions revealed highly homologous motifs in 40% of IGL. Parallel analysis of IGH and IGL showed selective pairing of IGHV3-21/30/33 segments to specific LCDR3-J3 subsets (p=0.008). Of 40 cases of hairy cell leukemia, 38 had mutated IGHV and/or IGK/LV, with variations in 13/13 cloned cases, while two had 100% unmutated IGHV and IGK/LV., Conclusions: Overall, biased IGV usage, preference for Iglambda with universal IGLJ3 usage and a high incidence of LCDR3 homologous motifs suggest selective influences on the B-cell receptor of hairy cell leukemia. Ongoing mutations and isotype switching suggest that influences occur on the tumor B-cell receptor at ectopic sites.