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Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 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-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3′-UTR region of the IL-12Rβ1 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-12Rβ1 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-12Rβ1–positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 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.

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.

Supplementary Figures S1-S2 and Supplementary Tables S1-S5.

PDF file - 22K, Distribution of the most frequently utilized IGHV. The histogram shows the percentages of VH sequences used in cMBL compared to Rai0-CLL cases.

Purpose: To investigate the incidence and clinical relevance of classic and new prognostic markers, IGHV gene mutational status, and chromosomal abnormalities in clinical monoclonal B lymphocytosis (cMBL) compared with Rai stage 0 chronic lymphocytic leukemia (Rai0-CLL).Experimental Design: A group of 136 patients with cMBL and a group of 216 Rai0-CLL cases were investigated prospectively.Results:IGHV-mutated cases were significantly more frequent among cMBLs (P = 0.005), whereas the distribution of CD38 and ZAP-70 positive cases, of patients with NOTCH1 and SF3B1 mutations or exhibiting the major CLL cytogenetic abnormalities, was similar in the two groups. Moreover, no significant differences were found either in IGHV/IGHD/IGHJ gene usage or in the overall prevalence of stereotyped IGHV gene sequences. Cells from cMBL and Rai0-CLL exhibited similar gene and microRNA (miRNA) signatures; in addition, when grouped according to the IGHV mutational status, IGHV-unmutated cases showed different transcriptional signatures compared with IGHV-mutated patients, irrespective of the cMBL or Rai0-CLL classification. cMBL diagnosis per se was predictive of longer progression-free survival.Conclusions: Our study based on a prospective series of patients indicates that no major differences exist between the circulating cells from cMBL and Rai0-CLL, at least based on a comparison of the markers used in the study. This possibly suggests that the two conditions mainly differ in the initial size of the monoclonal cell population, which may influence the subsequent timing of clonal expansion and clinical manifestations. Clin Cancer Res; 19(21); 5890–900. ©2013 AACR.

PDF file - 289K, Unsupervised analyses in cMBL and Rai0-CLL cases. (A) Gene and (B) miRNA expression profiling analyses by hierarchical clustering. Information about IGHV mutational status ('+' = M, '-' = UM), CD38, ZAP-70, chromosome 12 trisomy, chromosome 17, 11, and 13 deletions ('+' = positive, '-' = negative) are included alongside the patient ID. In the legend bar: turquoise indicates Rai0-CLL and yellow cMBL samples, respectively. The color scale bar represents the relative gene expression changes normalized by the standard deviation, and the color changes in each row represent gene expression relative to the mean across the samples.

XLSX file - 33K, Features of CLL samples.

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial.Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood.Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival.Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

PDF file - 64K, Supervised analysis comparing cMBL and Rai0-CLL samples. Haetmaps of the differentially expressed (A) gene and (B) miRNA. Information about IGHV mutational status ('+' = M, '-' = UM), CD38, ZAP-70, chromosome 12 trisomy, chromosome 17, 11, and 13 deletions ('+' = positive, '-' = negative) are included alongside the patient ID. In the legend bar: turquoise indicates Rai0-CLL and yellow cMBL samples, respectively. The color scale bar represents the relative gene expression changes normalized by the standard deviation, and the color changes in each row represent gene expression relative to the mean across the samples.

PDF file - 175K, Supplementary Table S1. Clinical and prognostic parameters of cases analyzed by gene expression profiling arrays (45 cMBL and 115 Rai0-CLL) and by miRNA expression profiling arrays (39 cMBL and 111 Rai0-CLL). Supplementary Table S2. List of the 302 modulated genes identified by the supervised multiclass analysis comparing cMBL and Rai0-CLL samples stratified according to IGVH mutational status at the 90th percentile of false discovery rate (FDR) equal to 0. The SAM score(d) and the contrast values of each class are reported for each gene. Negative values indicates down-regulation, positive up-regulation. In bold italic are marked the eight genes that showed different modulation depending on Rai0-CLL or cMBL configuration. Supplementary Table S3. List of the 18 modulated miRNAs in the supervised multiclass analysis comparing cMBL and Rai0-CLL samples stratified according to IGVH mutational status at q-value 0. The SAM score(d) and the contrast values of each class are reported for each gene. Negative values indicates down-regulation, positive up-regulation.

PDF file - 1024K, Supplementary Figure 1. Purification of normal human B cell subset populations. Supplementary Figure 2. CLL miRNA expression profile is homogeneous and distinct from multiple myeloma and Sezary syndrome. Supplementary Figure 3. Quantitative PCR validation of miRNAs differentially expressed between CLL and normal memory B cells. Supplementary Figure 4. Expression of miR-29c* in M-CLL and UM-CLL. Supplementary Figure 5. Validation of some miRNAs differentially expressed in IGHV or FISH classes. Supplementary Figure 6. Cluster analysis of averaged miRNA expression in the different FISH subgroups of CLL.

PDF file - 145K, Q-RT-PCR validation of the microarray expression data of LOC400986 and miR-130a. The microarray and Q-RT-PCR data of LOC400986 (A) and miR-130a (C) scaled in the range 0-1 are shown in the charts together with Pearson correlation coefficient. Boxplots of LOC400986 and miR-130a Q-RT-PCR expression comparing Rai0-CLL vs cMBL cases are represented in charts B and D respectively with the corresponding P-values (Wilcoxon rank sum test).

Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL.Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease.Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups.Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. ©2010 AACR.

XLSX file - 248K, microRNAs exhibiting differential expression between CLL and normal antigen-experienced B cells.

PDF file - 171K, Distribution of unfavorable events in Rai0-CLL and cMBL and correlation with prognosis. (A) Boxplot of concomitant unfavorable events (y-axis)in cMBL and Rai0-CLL; (B) Kaplan-Meier estimated curves of time to first treatment in the whole cohort stratified according to the number of concomitant events (as indicated in each of the four legends). The p-value and the hazard ratios (with the 95% confidence interval) of the proportional hazard model are indicated above each plot.

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.

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.

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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 naive 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.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repair, cell migration, autophagy, and cell metabolism, the TP53 tumor suppressor gene is a key player for cellular homeostasis. TP53 gene is mutated in more than 50% of human cancers, although its overall dysfunction may be even more frequent. TP53 mutations are detected in a lower percentage of hematological malignancies compared to solid tumors, but their frequency generally increases with disease progression, generating adverse effects such as resistance to chemotherapy. Due to the crucial role of P53 in therapy response, several molecules have been developed to re-establish the wild-type P53 function to mutant P53. PRIMA-1 and its methylated form PRIMA-1Met (also named APR246) are capable of restoring the wild-type conformation to mutant P53 and inducing apoptosis in cancer cells; however, they also possess mutant P53-independent properties. This review presents the activities of PRIMA-1 and PRIMA-1Met/APR246 and describes their potential use in hematological malignancies.

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. This work was financially supported by grants to Antonino Neri [from Associazione Italiana Ricerca sul Cancro (AIRC) (IG16722, IG10136, and the “Special Program Molecular Clinical Oncology-5 per mille” #9980, 2010/15)]; to Giovanna Cutrona and Gilberto Fronza [from the Italian Ministry of Health 5 × 1000 funds 2014, 2015, 2016, and from the Compagnia S. Paolo Turin Italy (project 2017.0526)]; to Manlio Ferrarini (the “Special Program Molecular Clinical Oncology-5 per mille” #9980 and AIRC I.G. n.14326); to Fortunato Morabito (the “Special Program Molecular Clinical Oncology-5 per mille” #9980 and AIRC and Fondazione CaRiCal co-financed Multi-Unit Regional Grant 2014 n.16695); Elisa Taiana was supported by a fellowship (#19370) from the Fondazione Italiana Ricerca sul cancro (FIRC)

Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repair, cell migration, autophagy, and cell metabolism, the TP53 tumor suppressor gene is a key player for cellular homeostasis. TP53 gene is mutated in more than 50% of human cancers, although its overall dysfunction may be even more frequent. TP53 mutations are detected in a lower percentage of hematological malignancies compared to solid tumors, but their frequency generally increases with disease progression, generating adverse effects such as resistance to chemotherapy. Due to the crucial role of P53 in therapy response, several molecules have been developed to re-establish the wild-type P53 function to mutant P53. PRIMA-1 and its methylated form PRIMA-1Met (also named APR246) are capable of restoring the wild-type conformation to mutant P53 and inducing apoptosis in cancer cells; however, they also possess mutant P53-independent properties. This review presents the activities of PRIMA-1 and PRIMA-1Met/APR246 and describes their potential use in hematological malignancies.

A progression-risk score to predict treatment-free survival for early stage chronic lymphocytic leukemia patients

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-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 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.

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.

Skeletal erosion has been found to represent an independent prognostic indicator in patients with advanced stages of chronic lymphocytic leukaemia (CLL). Whether this phenomenon also occurs in early CLL phases and its underlying mechanisms have yet to be fully elucidated. In this study, we prospectively enrolled 36 consecutive treatment-naïve patients to analyse skeletal structure and bone marrow distribution using a computational approach to PET/CT images. This evaluation was combined with the analysis of RANK/RANKL loop activation in the leukemic clone, given recent reports on its role in CLL progression. Bone erosion was particularly evident in long bone shafts, progressively increased from Binet stage A to Binet stage C, and was correlated with both local expansion of metabolically active bone marrow documented by FDG uptake and with the number of RANKL + cells present in the circulating blood. In immune-deficient NOD/Shi-scid, γcnull (NSG) mice, administration of CLL cells caused an appreciable compact bone erosion that was prevented by Denosumab. CLL cell proliferation in vitro correlated with RANK expression and was impaired by Denosumab-mediated disruption of the RANK/RANKL loop. This study suggests an interaction between CLL cells and stromal elements able to simultaneously impair bone structure and increase proliferating potential of leukemic clone.

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

A risk score based on three biological features (CD38, ZAP-70, and IGHV mutational status) was previously developed to predict progression-free survival (PFS) in untreated Binet A CLL patients. Here we perform a score validation analysis in a prospective and independent cohort of patients. Biological markers (CD38, ZAP-70, and IGHV mutational status) and gene expression profiles (GEP) of leukemic cells from CLL patients included in a prospective multicenter observational study (O-CLL1-GISL protocol, clinicaltrial.gov ID:NCT00917549) were used to assess the value and reproducibility of this score. To date, 468 Binet A patients were classified as low- (0 positive marker), intermediate- (1 positive marker), or high-risk (2 or 3 positive markers) using the progression risk score. The 3-year PFS probability was 91.7%, 82.9%, and 57.4% for low-, intermediate-, and high-risk (P < 0.0001) cases, respectively. These values were similar to those found in the original cohort. At Cox multivariate analysis, Rai stage, absolute lymphocyte count, progression risk score, and β-2 microglobulin maintained an independent prognostic impact on PFS. This score remained a predictor of progression when analysis was limited to 371 Rai 0 cases (P < 0.0001). Finally, the cells from the different CLL risk groups showed differences in their gene expression patterns. These results confirm the ability of this progression risk score to predict PFS among Binet A patients. The utility of the score was also extended by demonstrating that it retains prognostic value when applied exclusively to Rai 0 patients. Specific transcriptional patterns were significantly associated with risk groups.

Several studies have proposed telomere length and telomerase activity as prognostic factors in chronic lymphocytic leukemia (CLL), whereas information addressing the role of telomere-associated genes is limited. We measured relative telomere length (RTL) and TERT expression levels in purified peripheral CD19(+) B-cells from seven healthy donors and 77 untreated CLLs in early stage disease (Binet A). Data were correlated with the major biological and cytogenetic markers, global DNA methylation (Alu and LINE-1), and clinical outcome. The expression profiles of telomere-associated genes were also investigated. RTL was decreased in CLLs as compared with controls (P < 0.001); within CLL, a progressive and significant RTL shortening was observed in patients from 13q- through +12, 11q-, and 17p- alterations; short telomeres were significantly associated with unmutated IGHV configuration and global DNA hypomethylation. Decreased RTL was associated with a shorter time to first treatment. A significant upregulation of POT1, TRF1, RAP1, MRE11A, RAD50, and RPA1 transcript levels was observed in CLLs compared with controls. Our study suggests that impairment of telomere/telomerase system represents an early event in CLL pathogenesis. Moreover, the correlation between telomere shortening and global DNA hypomethylation supports the involvement of DNA hypomethylation to increase chromosome instability. © 2014 Wiley Periodicals, Inc.

NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1 c.7541_7542delCT is by far the most frequently observed NOTCH1 mutation in the disease. To estimate the prevalence and clonal evolution of NOTCH1 c.7541_7542delCT mutation, and prospectively investigate its clinical significance in early stage CLL and clinical monoclonal B cell lymphocytosis (cMBL), we analyzed by next generation sequencing (NGS) 384 cases at diagnosis enrolled in the GISL O-CLL1 multicenter trial. The patient cohort included 100 cMBL and 284 Binet stage A CLL cases, 48 of whom were also longitudinally investigated at progression or during follow-up (32 and 16, respectively) in absence of treatment. Deep sequencing of the NOTCH1 mutation hotspot was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. NOTCH1 mutation was validated by an extremely sensitive PCR-based approach and Sanger sequencing. The association between NOTCH1c.7541_7542delCT and clinical, molecular and biological variables, as well as its impact on progression free survival (PFS), were tested. Deep sequencing analysis of NOTCH1 mutation hotspot in our cohort (median depth of coverage 1510x, ranging from 605 to 2842) revealed a mutant allele frequency ranging from 0.02% to 75% of total reads in 145 cases. The occurrence of the mutation was subsequently assessed by an extremely sensitive ARMS (amplification refractory mutation system)-PCR, which allowed to confirm the presence of delCT in the 49 cases with frequency of mutated sequencing reads greater than 0.7%, specifically in 11% of cMBL (11/100) and 13.4% of CLL patients (38/284). Furthermore, mutated samples were subjected to DNA Sanger sequencing: in line with the expected sensitivity of the method, the mutation was identified only in samples with higher mutation loads according to NGS (mutant allele frequency ≥ 7%, n=25). Our data revealed that often NOTCH1 mutational activation affected a neoplastic sub-clone, especially in cMBL patients. NOTCH1 mutated patients utilized unmutated IGHV genes more frequently, and had higher expression of CD38 and ZAP-70 (P=3.2e-11, P=2.6e-08, P=3.4e-05, respectively). Trisomy 12 was more frequent in this patient group (P=5.4e-04), whereas 13q14 deletion was less represented than in the NOTCH1 wild-type patients (P=2.8e-03). NOTCH1 mutation was associated with the occurrence of stereotyped HCDR3 (P=5.6e-03); in addition, compared with other major BCR subsets, CLL subset #10 was significantly enriched in NOTCH1 mutations (P=0.032). The prevalence of the analyzed dinucleotide deletion was not significantly different between cMBL and CLL patients, even if only Rai 0 cases (28/197 cases, 14.2% mutation frequency) were considered. The percentages of variant sequencing reads in NOTCH1-mutated cases were slightly higher in CLL (median 19.6%) than in cMBL (median 4.2%), a finding confirmed by a regression analysis that highlighted the association of the CLL presentation with higher percentages of NOTCH1 delCT reads (P=0.033). NOTCH1-mutated cases, both at sub-clonal and clonal levels, displayed a significant reduction in median PFS (P=0.0018), although NOTCH1 mutation prognostic value, in multivariate analysis, was not independent if 11q and/or 17p deletion, IGHV mutational status, and cMBL or CLL status were considered. Finally, sequential analyses in a representative fraction of cases of our dataset indicated that (i) NOTCH1 mutation did not occur during the course of the disease and that (ii) the mutational load in positive cases was stable over time. These findings highlight the importance of using high sensitive methods for an accurate detection of NOTCH1 mutation in cMBL/early stage CLL. This is required for a better prognostic stratification and also to obtain useful information for potential therapeutic approaches, since sub-clonal mutations in untreated CLL can possibly anticipate the dominant genetic composition of the relapsing tumor. Disclosures: No relevant conflicts of interest to declare.

Genomic and epigenomic studies of chronic lymphocytic leukemia (CLL) are reshaping our understanding of the disease and have provided new perspectives for a more individualized diagnosis and new potential therapeutic targets. In this study, the global promoter methylation profile was determined in highly purified B-cells from 37 (Binet stage A) CLL patients, using high-resolution methylation microarrays (27,578 CpG). Overall, the methylation pattern correlated with the major biological (ZAP-70 and CD38), and molecular (IGHV mutation) markers, distinguishing CLL cases according to IGHV mutational status. Cell adhesion molecules were enriched in the signature of unmutated (UM) versus mutated (M-) CLL. Moreover, in M-CLL CpG hyper-methylation in three genes, including SPG20, was significantly anti-correlated with the corresponding gene expression level. Finally, the correlation between the methylation pattern and clinical parameters was investigated. Notably, out of 42 methyl-probes that were significantly associated with progression free survival (PFS), hyper-methylation of SPG20 was also positively associated with PFS. These data support the notion that epigenetic changes have clinical impact in CLL and may contribute to the identification of novel candidate disease-associated genes potentially useful to predict the clinical outcome of early stage CLL patients.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

// Adriana Amaro 1, * , Giovanna Angelini 1, * , Valentina Mirisola 1 , Alessia Isabella Esposito 1 , Daniele Reverberi 1 , Serena Matis 1 , Massimo Maffei 1 , Walter Giaretti 1 , Maurizio Viale 2 , Rosaria Gangemi 2 , Laura Emionite 3 , Simonetta Astigiano 4 , Michele Cilli 3 , Beatrice E. Bachmeier 5 , Peter H. Killian 5 , Adriana Albini 6 , Ulrich Pfeffer 1 1 Molecular Pathology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 2 Biotherapy, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 3 Animal Facility, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 4 Immunology, IRCCS AOU San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 5 Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany 6 Scientific and Technology Park, IRCCS MultiMedica, Milan, Italy * These authors have contributed equally to the work Correspondence to: Adriana Albini, email: adriana.albini@multimedica.it Ulrich Pfeffer, email: patologia.molecolare.integrata@gmail.com Keywords: breast cancer, invasion, apocrine breast cancer, metastasis, aneuploidy Received: May 16, 2016 Accepted: August 13, 2016 Published: September 10, 2016 ABSTRACT The acquisition of an invasive phenotype is a prerequisite for metastasization, yet it is not clear whether or to which extent the invasive phenotype is linked to other features characteristic of metastatic cells. We selected an invasive subpopulation from the triple negative breast cancer cell line MDA-MB-231, performing repeated cycles of preparative assays of invasion through Matrigel covered membranes. The invasive sub-population of MDA-MB-231 cells exhibits stronger migratory capacity as compared to parental cells confirming the highly invasive potential of the selected cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype. ArrayCGH, DNA index quantification and karyotype analyses confirmed a common genetic origin of the parental and invasive subpopulations and revealed discrete structural differences of the invasive subpopulation including increased ploidy and the absence of a characteristic amplification of chromosome 5p14.1-15.33. Gene expression analyses showed a drastically altered expression profile including features of apocrine breast cancers and of invasion related matrix-metalloproteases and cytokines. The invasive cells showed accelerated proliferation, increased apoptosis, and an altered pattern of chemo-sensitivity with lower IC50 values for drugs affecting the mitotic apparatus. However, the invasive cell population is significantly less tumorigenic in orthotopic mouse xenografts suggesting that the acquisition of the invasive capacity and the achievement of metastatic growth potential are distinct events.

Objective: We performed an external and multicentric validation of the predictive value of abdominal computed tomography (aCT) on time to first treatment (TTFT) in early stage chronic lymphocytic leukemia (CLL) patients. Methods: aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O-CLL1GISL trial (clinicaltrial.gov ID:NCT00917549). Results: Fifty-five patients showed an abnormal aCT. Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT (P < 0.0001). At multivariate analysis, aCT (P = 0.011), b-2 microglobulin (P = 0.019), and CD38 expression (P = 0.047) correlated with TTFT. Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B-cell lymphocytosis (cMBL). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT (P < 0.0001). At multivariate analysis, only aCT (P = 0.011) correlated with TTFT. Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas (SLL); nonetheless, TTFT was similar for cMBLs and SLLs. Conclusion: Our results confirm the ability of the abnormal aCT to predict progression in early stage cases.

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia among adults. Despite its indolent nature, CLL remains an incurable disease. Herein we aimed to monitor CLL disease engraftment and, progression/regression in a xenograft CLL mouse model using ultra-small superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI). Spleen contrast enhancement, quantified as percentage change in signal intensity upon USPIO administration, demonstrated a difference due to a reduced USPIO uptake, in the spleens of mice injected with CLL cells (NSG-CLL, n=71) compared to controls (NSG-CTR, n=17). These differences were statistically significant both after 2 and 4weeks from CLL cells injection. In addition comparison of mice treated with rituximab with untreated controls for changes in spleen iron uptake confirmed that it is possible to monitor treatment efficacy in this mouse model of CLL using USPIO-enhanced MRI. Further applications could include the preclinical in vivo monitoring of new therapies and the clinical evaluation of CLL patients.