Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Genes selection using deep learning and explainable artificial intelligence for chronic lymphocytic leukemia predicting the need and time to therapy.

Analyzing gene expression profiles (GEP) through artificial intelligence provides meaningful insight into cancer disease. This study introduces DeepSHAP Autoencoder Filter for Genes Selection (DSAF-GS), a novel deep learning and explainable artificial intelligence-based approach for feature selection in genomics-scale data. DSAF-GS exploits the autoencoder's reconstruction capabilities without changing the original feature space, enhancing the interpretation of the results. Explainable artificial intelligence is then used to select the informative genes for chronic lymphocytic leukemia prognosis of 217 cases from a GEP database comprising roughly 20,000 genes. The model for prognosis prediction achieved an accuracy of 86.4%, a sensitivity of 85.0%, and a specificity of 87.5%. According to the proposed approach, predictions were strongly influenced by CEACAM19 and PIGP, moderately influenced by MKL1 and GNE, and poorly influenced by other genes. The 10 most influential genes were selected for further analysis. Among them, FADD, FIBP, FIBP, GNE, IGF1R, MKL1, PIGP, and SLC39A6 were identified in the Reactome pathway database as involved in signal transduction, transcription, protein metabolism, immune system, cell cycle, and apoptosis. Moreover, according to the network model of the 3D protein-protein interaction (PPI) explored using the NetworkAnalyst tool, FADD, FIBP, IGF1R, QTRT1, GNE, SLC39A6, and MKL1 appear coupled into a complex network. Finally, all 10 selected genes showed a predictive power on time to first treatment (TTFT) in univariate analyses on a basic prognostic model including IGHV mutational status, del(11q) and del(17p), NOTCH1 mutations, ß2-microglobulin, Rai stage, and B-lymphocytosis known to predict TTFT in CLL. However, only IGF1R [hazard ratio (HR) 1.41, 95% CI 1.08-1.84, P=0.013), COL28A1 (HR 0.32, 95% CI 0.10-0.97, P=0.045), and QTRT1 (HR 7.73, 95% CI 2.48-24.04, P<0.001) genes were significantly associated with TTFT in multivariable analyses when combined with the prognostic factors of the basic model, ultimately increasing the Harrell's c-index and the explained variation to 78.6% (versus 76.5% of the basic prognostic model) and 52.6% (versus 42.2% of the basic prognostic model), respectively. Also, the goodness of model fit was enhanced (χ2 = 20.1, P=0.002), indicating its improved performance above the basic prognostic model. In conclusion, DSAF-GS identified a group of significant genes for CLL prognosis, suggesting future directions for bio-molecular research.

Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rß1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rß1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rß1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rß1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rß1-positive CLL cells in the spleen. Our findings indicate that IL-12Rß1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

Lymphocyte Doubling Time As A Key Prognostic Factor To Predict Time To First Treatment In Early-Stage Chronic Lymphocytic Leukemia.

The prognostic role of lymphocyte doubling time (LDT) in chronic lymphocytic leukemia (CLL) was recognized more than three decades ago when the neoplastic clone's biology was almost unknown. LDT was defined as the time needed for the peripheral blood lymphocyte count to double the of the initial observed value. Herein, the LDT prognostic value for time to first treatment (TTFT) was explored in our prospective O-CLL cohort and validated in in two additional CLL cohorts. Specifically, newly diagnosed Binet stage A CLL patients from 40 Italian Institutions, representative of the whole country, were prospectively enrolled into the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). Two independent cohorts of newly diagnosed CLL patients recruited respectively at the Division of Hematology in Novara, Italy, and at the Hospital Clinic in Barcelona, Spain, were utilized as validation cohorts. In the training cohort, TTFT of patients with LDT >12 months was significantly longer related to those with a shorter LDT. At Cox multivariate regression model, LDT ≤ 12 months maintained a significant independent relationship with shorter TTFT along with IGHV unmutated (IGHVunmut) status, 11q and 17p deletions, elevated ß2M, Rai stage I-II, and NOTCH1 mutations. Based on these statistics, two regression models were constructed including the same prognostic factors with or without the LDT. The model with the LTD provided a significantly better data fitting (χ2 = 8.25, P=0.0041). The risk prediction developed including LDT had better prognostic accuracy than those without LDT. Moreover, the Harrell'C index for the scores including LDT were higher than those without LDT, although the accepted 0.70 threshold exceeded in both cases. These findings were also confirmed when the same analysis was carried out according to TTFT's explained variation. When data were further analyzed based on the combination between LDT and IGHV mutational status in the training and validation cohorts, IGHVunmut and LDT>12months group showed a predominant prognostic role over IGHVmut LTD ≤ 12 months (P=0.006) in the O-CLL validation cohort. However, this predominance was of borden-line significance (P=0.06) in the Barcelona group, while the significant prognostic impact was definitely lost in the Novara group. Overall, in this study, we demonstrated that LDT could be re-utilized together with the more sophisticated prognostic factors to manage the follow-up plans for Binet stage A CLL patients.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Prevention of Covid-19 Infection and Related Complications by Ozonized Oils.

BACKGROUND: The COVID-19 pandemic continues to ravage the human population; therefore, multiple prevention and intervention protocols are being rapidly developed. The aim of our study was to develop a new chemo-prophylactic/-therapeutic strategy that effectively prevents COVID-19 and related complications. METHODS: In in vitro studies, COVID-19 infection-sensitive cells were incubated with human oropharyngeal fluids containing high SARS-CoV-2 loads. Levels of infection were determined via intra-cellular virus loads using quantitative PCR (qPCR). Efficacies for infection prevention were determined using several antiviral treatments: lipid-encapsulated ozonized oil (HOO), water-soluble HOO (HOOws), UV, and hydrogen peroxide. In in vivo studies, safety and efficacy of HOO in fighting COVID-19 infection was evaluated in human subjects. RESULTS: HOO in combination with HOOws was the only treatment able to fully neutralize SARS-CoV-2 as well as its capacity to penetrate and reproduce inside sensitive cells. Accordingly, the feasibility of using HOO/HOOws was tested in vivo. Analysis of expired gas in healthy subjects indicates that HOO administration increases oxygen availability in the lung. For our human studies, HOO/HOOws was administered to 52 cancer patients and 21 healthy subjects at high risk for COVID-19 infection, and all of them showed clinical safety. None of them developed COVID-19 infection, although an incidence of at least 11 cases was expected. Efficacy of HOO/HOOws was tested in four COVID-19 patients obtaining recovery and qPCR negativization in less than 10 days. CONCLUSIONS: Based on our experience, the HOO/HOOws treatment can be administered at standard doses (three pills per day) for chemo-prophylactic purposes to healthy subjects for COVID-19 prevention and at high doses (up to eight pills per day) for therapeutic purposes to infected patients. This combined prevention strategy can provide a novel protocol to fight the COVID-19 pandemic.

Validation of the Alternative International Prognostic Score-E (AIPS-E): Analysis of Binet stage A chronic lymphocytic leukemia patients enrolled into the O-CLL1-GISL protocol.

OBJECTIVES: To validate the predictive value on time to first treatment (TTFT) of AIPS-E and IPS-E evaluated in an independent cohort of newly diagnosed and non-referred Binet stage A CLL patients enrolled in the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). METHODS: A cohort of 292 newly diagnosed Binet A CLL cases has been enrolled in the study. Patients from several Italian Institutions were prospectively enrolled within 12 months of diagnosis into the O-CLL1-GISL protocol. RESULTS: The majority of patients were male (62%); median age was 60.4 years, 102 cases (34.9%) showed unmutated IGHV genes, 8 cases (2.8) the presence of del(11q)/del(17p), 142 cases (48.6%) the presence of palpable lymph nodes and 146 cases (50%) and ALC > 15 × 109 /l. After a median follow-up of 7.2 years, 130 patients underwent treatment. According to the AIPS-E, 96 patients were classified as low-risk, 128 as intermediate-risk, and 68 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.71 (P < .0001) for predicting TTFT. According to IPS-E, 77 patients were classified as low-risk, 135 as intermediate-risk, and 80 as high-risk. These groups showed significant differences in terms of TTFT. The C-statistic was 0.705 (P < .0001) for predicting TTFT. CONCLUSIONS: Our data confirm an accurate prognostic utility of both AIPS-E and IPS-E at the individual patient level. These data may be useful for a precise stratification of early-stage patients.

Time to first treatment and P53 dysfunction in chronic lymphocytic leukaemia: results of the O-CLL1 study in early stage patients.

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.

NEAT1 Long Isoform Is Highly Expressed in Chronic Lymphocytic Leukemia Irrespectively of Cytogenetic Groups or Clinical Outcome.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated IGHV genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach.

BACKGROUND: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5- B cells from the spleen and peripheral blood (PB). METHODS: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5- cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. RESULTS: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5- B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. CONCLUSIONS: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rß1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rß1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.

Surrogate molecular markers for IGHV mutational status in chronic lymphocytic leukemia for predicting time to first treatment.

ZAP-70 is a marker of clinical outcome in chronic lymphocytic leukemia (CLL), however its assessment suffers from a lack of standardization consensus. To identify novel markers able to surrogate IGHV mutational status, CD19(+)CD5(+)-B-lymphocytes from 216 patients enrolled in a prospective study (ClinicalTrial.gov Identifier:NCT00917540), underwent gene expression profiling. Samples were split into CLL-Training (n=102) and CLL-Validation (n=114) sets, and an independent supervised analysis for IGHV mutational status was performed considering all genes with gene expression equal or above that of ZAP-70. Thirty-one genes (23 up- and 8 down-regulated) and 23 genes (18 up- and 5 down-regulated) satisfied these criteria in the CLL-Training and CLL-Validation sets, respectively, and 20 common genes (15 up and 5 down) were found to be differentially regulated in both sets. Two (SNORA70F, NRIP1) of the down-regulated and 6 (SEPT10, ZNF667, TGFBR3, MBOAT1, LPL, CRY1) of the up-regulated genes were significantly associated with a reduced risk of disease progression in both sets. Forcing the afore-mentioned genes in a Cox multivariate model together with IGHV mutational status, only CRY1 (HR=2.3, 95% CI: 1.1-4.9, P=.027) and MBOAT1 (HR=2.1, 95% CI: 1.1-3.7, P=.018) retained their independent prognostic impact, supporting the hypothesis that these genes may potentially act as surrogates for predicting IGHV mutational status.

Association between gene and miRNA expression profiles and stereotyped subset #4 B-cell receptor in chronic lymphocytic leukemia.

In this study we investigated specific biological and clinical features associated with chronic lymphocytic leukemia (CLL) patients carrying stereotyped BCR subset #4 (IGHV4-34) among a prospective cohort of 462 CLL/MBL patients in early stage (Binet A). All subset #4 patients (n = 16) were characterized by the IGHV mutated gene configuration, and absence of unfavorable cytogenetic lesions, NOTCH1 or SF3B1 mutations. Gene and miRNA expression profiling evidenced that the leukemic cells of subset #4 cases showed significant downregulation of WDFY4, MF2A and upregulation of PDGFA, FGFR1 and TFEC gene transcripts, as well as the upregulation of miR-497 and miR-29c. The transfection of miR-497 mimic in primary leukemic CLL cells induced a downregulation of BCL2, a known validated target of this miRNA. Our data identify biological characteristics associated with subset #4 patients, providing further evidence for the putative role of BCR in shaping the features of the tumor cells in CLL.

Insulin growth factor 1 receptor expression is associated with NOTCH1 mutation, trisomy 12 and aggressive clinical course in chronic lymphocytic leukaemia.

IGF1R is emerging as an important gene in the pathogenesis of many solid and haematological cancers and its over-expression has been reported as frequently associated with aggressive disease and chemotherapy resistance. In this study we performed an investigation of the role of IGF1R expression in a large and representative prospective series of 217 chronic lymphocytic leukaemia (CLL) patients enrolled in the multicentre O-CLL1 protocol (clinicaltrial.gov #NCT00917540). High IGF1R gene expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high CD38 expression (p<0.0001), trisomy 12 (p<0.0001), and del(11)(q23) (p=0.014). Interestingly, higher IGF1R expression (p=0.002) characterized patients with NOTCH1 mutation (c.7541_7542delCT), identified in 15.5% of cases of our series by next generation sequencing and ARMS-PCR. Furthermore, IGF1R expression has been proven as an independent prognostic factor associated with time to first treatment in our CLL prospective cohort. These data suggest that IGF1R may play an important role in CLL biology, in particular in aggressive CLL clones characterized by IGHV-UM, trisomy 12 and NOTCH1 mutation.