Genes In Chronic Lymphocytic Leukemia Research Articles

Sequence variation of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene in chronic lymphocytic leukemia and healthy volunteer subjects.

Epstein-Barr virus-related malignancies have been linked to variations in the sequences of EBV genes, notably EBNA1. Therefore, the purpose of this study was to examine the DBD/DD domain and USP7 binding domain sequences at the C-terminus of the EBNA1 gene in patients with chronic lymphocytic leukemia (CLL). This study included 40 CLL patients and 21 healthy volunteers. Using commercial kits, total DNA was extracted from buffy coat samples, and each sample was tested for the presence of the EBV genome. The C-terminus of EBNA1 was then amplified from positive samples, using nested PCR. Sanger sequencing was used to identify mutations in the PCR products, and the results were analyzed using MEGA11 software. The mean ages of CLL patients and healthy individuals were 61.07 ± 10.2 and 59.08 ± 10.3, respectively. In the EBNA-1 amplicons from CLL patients and healthy individuals, 38.5% and 16.7%, respectively, harbored mutations in the DBD/DD domain of the C-terminal region of the EBNA1 gene (P = 0.378). The mutation frequency at locus 97,320 was significantly higher in CLL patients than in healthy individuals (P = 0.039). Three EBV subtypes based on residue 487 were detected. The frequency of alanine, threonine, and valine in both groups was 88, 8, and 4 percent, respectively (P = 0.207). Moreover, all of the isolates from healthy donors had alanine at this position. The findings indicated that the presence of threonine or valine at residue 487 as well as a synonymous substitution at residue 553 in the C-terminal region of EBNA1 might be involved in the pathogenesis of EBV in CLL patients.

ZMYM3 Mutations Cooperate with NOTCH1 Alterations, Reduce Histone H4 Acetylation and Promote Apoptosis Evasion in Chronic Lymphocytic Leukemia

Introduction: Recent next-generation sequencing (NGS) studies have identified up to 200 recurrently mutated genes in chronic lymphocytic leukemia (CLL), the vast majority of which appear mutated in <5% of CLL cases. Among the long list of CLL candidate driver genes is ZMYM3, a gene mutated in 2-4% of CLL patients. Since the impact of ZMYM3 mutations is unknown, we aimed to identify their clinical and biological consequences in CLL. Methods: 487 CLL patients were analyzed by targeted NGS using a customized panel of 54 CLL-related genes to characterize the mutational profile of ZMYM3 mutated patients ( ZMYM3MUT) and the clinical impact of these variants. In addition, we introduced ZMYM3 truncating mutations into the CLL-derived HG3 cell line with two genetic backgrounds: wild-type (WT), and NOTCH1 mutated cells ( NOTCH1MUT), to generate cells with either single ZMYM3MUT, NOTCH1MUT or combined ZMYM3MUTNOTCH1MUT. In these CLL models, we evaluated the impact of these alterations in gene expression, apoptosis, growth and DNA damage response (DDR). Results: A total of 32 ZMYM3 variants were identified in 30 CLL patients, most of which were loss-of-function mutations (24/32; 75%). These variants were distributed throughout the protein sequence, with a particular recurrent frameshift mutation occurring in 6 patients (p.P48fs) that was subsequently reproduced in our CLL model. Attending to their mutational profile, we identified that 70% (21/30) of ZMYM3MUT patients harbored mutations in the NOTCH pathway, either in NOTCH1 (60%) or in negative regulators such as MED12, FBXW7 and SPEN, suggesting a cooperation between ZMYM3 dysfunction and NOTCH1 signaling mutations in CLL evolution. Interestingly, ZMYM3MUT patients exhibited significantly shorter time to first treatment (TFT) than ZMYM3WT cases (median: 35 vs 52 months; p=0.010). Furthermore, ZMYM3 mutations shortened TFT of early stage CLL patients, including Binet A (48 vs 108 months, p=0.002) or Rai 0/1 (48 vs 91 months, p=0.016), as well as in low-risk cytogenetics del(13q) patients (median: 45 vs 93 months; p=0.033). We next addressed the biological implications of ZMYM3 mutations and their co-occurrence with NOTCH1 mutations in HG3 cells. First, we performed RNA-sequencing and analyzed the number of differentially expressed genes (DEGs) (|fold change|>2, adjusted p-value<0.05). Comparing with WT cells, we detected 155 DEGs in ZMYM3MUT and 83 DEGs in NOTCH1MUT, whereas ZMYM3MUTNOTCH1MUT cells showed 690 DEGs,indicating that the combination of both mutations profoundly dysregulate gene expression. Immune regulation, apoptosis and DNA damage response were some of the biological processes dysregulated by ZMYM3 mutations (p<0.05). Furthermore, considering the suggested role of ZMYM3 in chromatin remodeling (Puente et al, Nature. 2015) and that ZMYM3MUT cells showed altered expression of histone acetylation related genes (p<0.05), we identified that ZMYM3 mutations reduced global acetylation levels of histone H4 (p<0.05), which may underlie the ZMYM3-related changes in gene expression. To further define the functional effects of ZMYM3 mutations, we assessed in vitro their impact in the main dysregulated pathways identified by RNA-seq. First, we identified that ZMYM3 mutations impair DNA damage response, as reflected by the difficulties to arrest cell cycle in G2/M phase after irradiation. Moreover, we detected more γH2AX foci (p<0.05) in ZMYM3MUTcells, indicating persistent DNA damage; and fewer RAD51 and BRCA1 foci than WT cells (p<0.001) after irradiation, suggesting defective DNA damage response. In parallel, we identified that ZMYM3 mutations promote apoptosis evasion, especially in the context of NOTCH1 mutations (p<0.001), which led to enhanced growth of ZMYM3MUTNOTCH1MUT cells (p<0.05). Of note, ZMYM3MUT cells showed down-expression of caspases (-8, -7 and -3) and slightly higher levels of anti-apoptotic proteins (BCL2, MCL1 and BCL-XL), which may explain this apoptosis resistance. Conclusions: Mutations in ZMYM3 are mainly loss-of-function, associate with NOTCH1 signaling mutations and shorten TFT in CLL patients, suggesting that ZMYM3 mutational status may be a useful marker in the management of early stage CLL patients. Moreover, ZMYM3 mutations reduce histone H4 acetylation and cooperate with NOTCH1 mutations to dysregulate gene-expression, leading to impair DNA damage response and apoptosis evasion.

Treatment Patterns and Outcomes of Patients with Chronic Lymphocytic Leukemia with TP53 Mutations

Background: Aberration of the TP53 gene in chronic lymphocytic leukemia (CLL) is a well-established marker of poor prognosis and a predictor of chemorefractory disease. Identification of a TP53 gene mutation is clinically useful in determining which patients may benefit from small-molecule inhibitors such as Bruton tyrosine kinase (BTKi) and BCL2 inhibitors (BCL2i). However, the treatment patterns in routine practice and outcomes of patients harboring TP53 mutations in CLL is not well reported. We present a descriptive analysis of CLL patients treated at Mayo Clinic with TP53 mutations including their demographics, treatment patterns, and outcomes. Methods: Patients with CLL who had a TP53 mutation detected by Sanger sequencing between June 2014 and April 2021 at Mayo Clinic Rochester were identified from a hematopathology database. Patient demographics, clinical and pathologic characteristics, treatment, and follow-up information were abstracted from the Mayo Clinic CLL database and by chart review. Date of TP53 mutation test was baseline. Discrete variables were compared using Chi-square or Fisher's exact tests; continuous variables were compared using Kruskal-Wallis test. Overall survival (OS) was calculated from date of TP53 mutation test until date of death from any cause or last known date alive. Rates of Richter transformation (RT) by prior treatment exposure were estimated by cumulative incidence accounting for competing risk of death. Results: A total of 145 patients were identified; 70% were male, and the median age at diagnosis was 59 years (range: 36-92). At diagnosis, 15% of patients had Rai stage III-IV, 77% had unmutated IGHV, 38% had del17p, 92% had high or very high CLL-IPI, and 62% had ≥3 cytogenetic abnormalities (Table 1). A TP53 gene mutation was identified prior to any CLL treatment in 63 patients (43%, Group 1) and after at least one prior line of treatment in 82 patients (57%, Group 2). The median time from CLL diagnosis to the first detected TP53 mutation was 3 months in Group 1 and 96 months in Group 2. There was significant heterogeneity in types of TP53 mutations with the majority of variants (n=102) being unique to an individual. At the time of TP53 mutation detection, Group 2 patients more frequently had advanced Rai stage and unmutated IGHV status (Table 1). Del17p by FISH was present prior to or at time of TP53 mutation identification (+/- 3 months) in 54% of patients in Group 1 and 52% in Group 2. Group 1 had a median follow-up of 26 months (from TP53 mutation detection date); 46/63 patients had initiated a first-line (1L) treatment (39 BTKi-based, 1 BCL2i-based, 2 BTKi+BCL2i-based, 4 chemoimmunotherapy [CIT]). In Group 2, the median number of prior lines of therapy was 2 (range 1-16). Prior treatment exposure included 38 (46%) CIT only, 41 (50%) BTKi-based ± CIT, and 3 (4%) BTKi-based + BCL2i-based ± CIT. Median follow-up was 21 months; 56/82 patients had initiated a new line of therapy (subsequent 1L). For 38 patients with prior CIT only exposure, 28 had subsequent 1L including 21 BTKi-based, 2 BCL2i-based, and 5 CIT. For 44 patients with prior BTKi exposure, 28 had subsequent 1L including 5 BTKi-based, 5 BCL2i-based, 9 BTKi+BCL2i-based, and 9 CIT. The median OS was not reached in Group 1 and was 40.6 months (95% CI 30.1-NE) in Group 2 (HR=0.21, 95% CI=0.10-0.45) (Figure 1). In Group 2, the median OS for patients with prior CIT only exposure was 55.5 (95% CI 30.1-NE) months and for patients with prior BTKi exposure was 33.3 (95% CI 14.7-NE) months (HR=0.59, 95% CI=0.31-1.12) (Figure 1). Three patients in Group 1 and 13 patients in Group 2 (3 prior CIT only, 10 prior BTKi) developed RT during the follow-up period; the cumulative incidence rates of RT at 2- and 5-year were 1.7% and 4.6%, respectively, in Group 1, and 10.8% and 21.0%, respectively, in Group 2. Conclusions: Regardless of prior treatment exposure, enrichment for high-risk features was present at time of TP53 mutation detection. Majority of patients were treated with novel agents after known TP53 mutations. OS after TP53 mutation was inferior in patients who had been previously treated, particularly those who had BTKi exposure already prior to TP53 mutation detection, compared to previously untreated patients. Future studies are needed to understand the characteristics of clonal evolution before and after treatment and the clinical impact of specific TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Landscape of NOTCH1 mutations and co-occurring biomarker alterations in chronic lymphocytic leukemia

NOTCH1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and has emerged as a marker of poor prognosis. In addition to coding NOTCH1 mutations involving exon 34, non-coding NOTCH1 mutations involving the 3′ UTR have been described in a limited number of chronic lymphocytic leukemia (CLL) patients and were associated with adverse outcomes. In this study, 1574 CLL patients were assessed using targeted sequencing with a 29 gene panel and the results were correlated with prognostic characteristics. NOTCH1 mutations were detected in 252 (16%) patients, including both coding (220/252, 14%), non-coding (24/252, 1.5%) and a mixture of coding and non-coding (8/252, 0.5%) NOTCH1 mutations. NOTCH1 mutations were more commonly seen in patients with unmutated IGHV, ZAP70 positivity and CD38 positivity. Mixed NOTCH1 mutations were also more commonly seen in patients with unmutated IGHV and ZAP70. There was no association between mixed NOTCH1 mutations and CD38 expression in this cohort. The most common cytogenetic alteration detected in patients with coding and mixed NOTCH1 mutations was trisomy 12, whereas del13q was the most common cytogenetic alteration detected in patients with non-coding NOTCH1 mutation. The most common gene mutations co-occurring with coding NOTCH1 mutations were: TP53 (23.2%), SF3B1 (16.4%) and SPEN (10%). The most common gene mutations co-occurring with non-coding NOTCH1 mutations were: SF3B1 11(34.4%), ATM 4(12.5%) and TP53 4(12.5%). CLL patients with clonal coding and non-coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 (4.3 vs 10.0 years and 0.9 vs 10.0 years respectively, p < 0.05). Similarly, CLL patients with subclonal coding NOTCH1 mutations had a significantly shorter time-to-first treatment than patients with wild type NOTCH1 (5.6 vs 10.0 years, p < 0.05). CLL patients with subclonal non-coding NOTCH1 mutations also had a shorter time-to-first treatment than patients with wild type NOTCH1 mutations, however, the difference was not significant (5.1 vs 10.0 years, p = 0.15). These data confirm that both coding and non-coding NOTCH1 mutations carry adverse prognostic impact and need to be included in sequencing assays performed for the prognostic workup of CLL patients.

R-Loop Associated Mitotic Stress Confers Vulnerabilities in Splicing Factor Mutant Leukemia

The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated.CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value <0.0001) and mitotic stress (t-test p-value <0.0001), confirming the causative role of R-loop in CIN.To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN.To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability.As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism.We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells.Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. DisclosuresWu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

Impact of Berberine on the Expression of Apollon Gene in Chronic Lymphocytic Leukemia

Background: The incidence of B-chronic lymphocytic leukemia (B-CLL) resulting from the clonal accumulation of apoptosis-resistant malignant B lymphocytes is growing in the adult population of Iran. Inhibitors of apoptosis proteins (IAPs) are considered as factors that can delay the onset of CLL cell apoptosis. Berberine is an isoquinoline alkaloid isolated from Cotridis rhizoma that exhibits anti-tumor activities through various mechanisms. Objectives: In this study, we investigated the impact of berberine on the level of Apollon expression in peripheral blood mononuclear cells (PBMCs) of 12 cases newly diagnosed with CLL and 6 healthy donors. Methods: At first, the level of Apollon expression was assessed in PBMCs of CLL patients compared to the healthy donors. Peripheral blood mononuclear cells were cultured in RPMI-1640 medium with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin for 48 hours, and the effect of berberine (25 µM) on the level of Apollon expression in CLL patients was assessed and compared to that of healthy donors. Results: We found that the expression level of Apollon was not significantly different between CLL patients and healthy donors (P = 0.640). Moreover, berberine induced no significant differences in Apollon expression as compared to the untreated (control) group (P = 0.545 and P = 0.267 in CLL patients and healthy donors, respectively). Conclusions: Overall, our results suggest that berberine has no direct effect on the expression of Apollon gene in CLL patients, and pro-apoptotic impacts of berberine may be exerted through other mechanisms.

Abstract 884: Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL)

Abstract Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies &amp;gt;0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.

Pathogenetic Implications from the Analysis of Immunoglobulin Genes in Chronic Lymphocytic Leukemia of Mediterranean Ancestry: The Lesson of the IGHV3-21 Gene.

Studies of the characteristics and the mutational status of the immunoglobulin (Ig) genes expressed by Chronic Lymphocytic Leukemia (CLL) suggest a potential role for antigenic stimulation in the pathogenesis and/or evolution of the disease. We analyzed IGHV repertoire and mutational status in 553 CLL patients from the Mediterranean area. The most commonly represented IGHV genes mirror the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3 and IGHV4-59 that are underrepresented. In addition, the analysis of the IGHV3-21 gene, frequently present in Northern European CLL, showed the following peculiarities: 1) this gene was expressed only in a limited number of cases in our Mediterranean cohort (16/553; 2.9%); 2) based on HCDR3 cluster analysis, IGHV3-21-utilizing cases could be grouped in two different subsets of similar frequency. The first subset (common-HCDR3 subset) included seven cases (out of 16), which carried a very short and virtually identical HCDR3 aminoacid sequence bearing strong similarities to the Northern European IGHV3-21 CLL (Tobin et al, Blood. 2002 Mar 15;99(6):2262–4). All cases used the IGHJ6 gene; 4/7 were unmutated; 6/7 carried the Vλ2-14 (IGLV3-21) light chain gene with a similar LCDR3; all expressed CD38 and had a progressive disease. The second subset (non-homogeneous-HCDR3 subset) included 9 cases and was characterized by long and heterogeneous HCDR3 rearrangements, with diverse IGHJ and IGV light chain gene usage and variable CD38 expression; five out of 9 cases were unmutated; the clinical course of these patients was variable (only 4/9 progressed). The first subset, with the evidence of a similar CDR3 in so many different CLL cohorts, suggests the existence of a specific, yet undefined, antigenic element that may subsequently lead to a more aggressive clinical course. The dramatically different frequency of this subset as compared to Northern Europe suggests that the potential selecting agent may be less frequently encountered in the Mediterranean area. In contrast, the second subset included cases with heterogeneous HCDR3 rearrangements and a variable clinical outcome. This second subset may mirror the heterogeneity and low frequency of expression of IGHV3-21 rearrangements in the normal repertoire of peripheral blood B cells, as shaped by the germline composition, molecular mechanisms and distinct selective influences.

The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia.

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1 Delta2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT.

to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 genein chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP acci-dent (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups ofpatients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main andalternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). TheIGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data wereanalyzed with the SPSS software package, version 20.0. Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57),did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expressionof B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relativeexpression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01-90.51). The expression of SORL1-Δ2transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmu-tated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene inpatients of the main and the control groups did not differ. Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients withunmutated IGHV genes can be considered as a negative prognostic marker.

Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

Bioinformatic Prediction of miRNAs Targeting APRIL and BAFF Genes in Chronic Lymphocytic Leukemia

Bioinformatic Prediction of miRNAs Targeting APRIL and BAFF Genes in Chronic Lymphocytic Leukemia

Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

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.

Systematic tracking of altered modules identifies the key biomarkers involved in chronic lymphocytic leukemia.

Key genes in chronic lymphocytic leukemia (CLL) were investigated through systematically tracking the dysregulated modules from protein-protein interaction (PPI) networks. Microarray data of normal subjects and CLL patients recruited from ArrayExpress database were applied to extract differentially expressed genes (DEGs). Additionally, we re-weighted the PPI network of normal and CLL conditions by means of Pearsons correlation coefficient (PCC). Furthermore, clique-merging method was applied to extract the modules and then the altered modules were screened out. The intersection genes were selected from miss and add genes in the altered modules. The common genes were screened from the intersection genes and DEGs in CLL. A total of 734 DEGs were screened by statistical analysis. In this investigation, there were 1,805 and 703 modules in normal as well as disease PPI network. In addition, 875 altered modules were obtained which included 145 miss genes, 353 add genes and 85 intersection genes. Finally, in-depth analysis revealed 9 mutual genes between the intersection genes and DEGs in CLL. Our analysis revealed several key genes associated with CLL by systematically tracking the dysregulated modules, which might be candidate targets for diagnosis and management of CLL.

Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.

We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients& methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM). Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p<0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p<0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Abstract 3443: Full-length characterization of transcript isoforms to investigate cancer-associated mutations

Abstract Mutations in the splicing factor SF3B1 in various cancers have been associated with characteristic alterations in splicing. SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 on a junction level have been observed, these patterns have not been systematically examined on an isoform level. Isoform-level analyses are imperative for building a better understanding of altered splicing in a cell; exon connectivity is best resolved using long reads. As such, we have resequenced cDNA from CLL samples with and without K700E mutation in SF3B1, as well as a normal B cell, with nanopore sequencing technology. By converting changes in current caused by blockage of DNA threading through a nanopore into sequence, nanopore sequencing can sequence complete molecules of DNA that exceed 200kb in length. We have developed a novel workflow to perform isoform-level differential splicing analyses leveraging the full-length transcript data that nanopore affords. We report results from nanopore sequencing data that are concordant with known SF3B1 biology from short read sequencing as well as altered splicing patterns that are only observed definitively using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E identifies a bias toward increased alternative 3' splice sites than alternative 5'. In addition, we observe a relative decrease in the expression of genes with intron retention events in CLL samples compared to B cell. We also discover a previously unreported finding of enrichment in intron retention events in SF3B1WT relative to SF3B1K700E and no enrichment between CLL SF3B1MT and B cell, suggesting that mutated SF3B1 in CLL modifies the intron retention landscape to be more similar to a normal phenotype. Gene ontology analyses reveal enrichment in nuclear export in genes associated with intron retention events, and we postulate that many of these are detained introns of transcripts that are kept in the nucleus. With full-length cDNA sequence, we are also able to better detect premature termination codons and estimate the proportion of transcripts subject to nonsense-mediated mRNA decay. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the utility of nanopore sequencing for cancer and splicing research. Citation Format: Alison Tang. Full-length characterization of transcript isoforms to investigate cancer-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3443.

Expression of autophagy related genes in chronic lymphocytic leukemia is associated with disease course

Autophagy leads cells to different fates in various cell types and under diverse contexts. Chronic lymphocytic leukemia (CLL), an incurable hematologic neoplasm, has highly variable course and its heterogeneity prompts interest in exploring autophagic trajectories in CLL. We detected the mRNA levels of two autophagy-related genes, BECN1 and ATG5, assessed the association between expression levels and clinical characteristics, and did survival analysis. One hundred and six patients with CLL and fifty healthy controls were enrolled in the present study. CLL samples were found higher expression levels of BECN1 and ATG5 mRNA compared with healthy controls. Further confirmation at the protein level performed in a small cohort of patients, which included Beclin1, ATG5 and LC3-II showed the same trend. What’s more, high expression at the mRNA level correlated with early Binet stage, isolated 13q deletion and negative CD38, which were associated with favor prognosis, suggesting that autophagy differs in CLL due to the presence of heterogeneity and high levels of these two genes may reflect better outcomes. Survival analysis did show patients with high expression of ATG5 mRNA had longer treatment free survival from the date of sampling.