Risk Of Chronic Lymphocytic Leukemia Research Articles

Genetic variability profiling of the p53 signaling pathway in chronic lymphocytic leukemia. Individual and combined analysis of TP53, MDM2 and NQO1 gene variants.

TP53 gene disruption, including 17p13 deletion [del(17p)] and/or TP53 mutations, is a negative prognostic biomarker in chronic lymphocytic leukemia (CLL) associated with disease progression, treatment failure and shorter survival. Germline variants in p53 signaling pathway genes could also lead to p53 dysfunction, but their involvement in CLL has not been thoroughly evaluated. The aim of this study was to determine the association of TP53, MDM2 and NQO1 gene variability with clinical and genetic data of CLL patients. Individual genotype and haplotype data of CLL patients were compared with clinical prognostic factors, cytogenetic and molecular cytogenetic findings as well as IGHV and TP53 mutational status. The study included 116 CLL patients and 161 healthy blood donors. TP53 (rs1042522, rs59758982, rs1625895), NQO1 (rs1800566) and MDM2 (rs2279744, rs150550023) variants were genotyped using different PCR approaches. Analysis of genotype frequencies revealed no association with the risk of CLL. TP53 rs1042522, rs1625895 and MDM2 rs2279744 variants were significantly associated with abnormal karyotype and the presence of del(17p). Similarly, these two TP53 variants were associated with TP53 disruption. Moreover, TP53 C-A-nondel and G-A-del haplotypes (rs1042522-rs1625895-rs59758982) were associated with an increased likelihood of carrying del(17p) and TP53 disruptions. MDM2 T-nondel haplotype (rs2279744-rs150550023) was found to be a low risk factor for del(17p) (OR = 0.32; CI: 0.12-0.82; p = 0.02) and TP53 disruptions (OR = 0.41; CI: 0.18-0.95; p = 0.04). Our findings suggest that TP53 and MDM2 variants may modulate the risk to have chromosome alterations and TP53 disruptions, particularly del(17p). To our knowledge this is the first study of several germline variants in p53 pathway genes in Argentine patients with CLL.

Clime: CLL Immune Micro-Environment and Risk of Infections Pre- and Post-Diagnosis: A Population-Based Study

Background: Immune dysfunction precedes a diagnosis of chronic lymphocytic leukemia (CLL) in some patients. More information is needed about which patients experience immune dysfunction and how it impacts disease progression, as well as the development of post-diagnosis infections and second malignancies. The objectives of this study were to 1) examine the rate of pre-diagnosis infection for individuals diagnosed with CLL compared to population controls, 2) evaluate the impact of pre-diagnosis infection on time to first treatment, 3) compare the rate of immune dysfunction before the start of treatment by stage, and 4) compare the rate of immune dysfunction before the start of treatment for each year post-treatment. Methods: We used a retrospective matched cohort study design that included all individuals in Manitoba diagnosed with CLL from 2006 to 2019. Individuals were identified from the Manitoba CLL database and the Manitoba Cancer Registry (MCR). Individuals with CLL were matched by age, sex, and area of residence (urban versus rural) to population controls identified from the Manitoba Health Population Registry. Provincial pharmacy, hospital admissions, medical claims, and MCR data were used to determine infection, second malignancies, and IgR therapy. Immune dysfunction was defined as two or more infections, a diagnosis with a secondary malignancy, or receipt of IgR therapy. The pre-diagnosis time period was defined as three to 15 months prior to diagnosis. The pre-treatment time period was defined as 12 months prior to treatment. The following analyses were used: conditional logistic regression for objective 1, competing risk for objective 2, logistic regression for objective 3, and logistic GEE models for objective 4. Results: A total of 1,776 CLL patients were matched to 17,760 non-CLL controls. Sixty-two percent were male, the mean age at diagnosis was 71 years, and 62% lived in an urban area. Fifty-one percent of CLL patients had a pre-diagnosis infection compared to 46% of controls. Compared to individuals with no prior infection, those who had a prior infection were more likely to be diagnosed with CLL (Odds Ratio (OR) 1.17, 95% confidence Interval (CI) 1.03, 1.33 for one infection, OR 1.29, 95% CI 1.15, 1.45 for two or more infections). After adjusting for the number of outpatient visits and comorbidity, individuals with two or more infections were 14% more likely to be diagnosed with CLL (OR 1.14, 95% CI 1.00-1.30). Conclusions: We found that infections that occurred in the three to 15 months before diagnosis increased the odds of being diagnosed with CLL. Work is ongoing examining time to treatment, immune dysfunction by stage, as well comparing immune dysfunction before and after treatment.

Initial Results of a Phase 1 Dose Escalation Study of LP-168, a Novel Covalent and Non-Covalent Next-Generation Inhibitor of Bruton's Tyrosine Kinase

Introduction: Inhibitors of Bruton's Tyrosine Kinase (BTKi) have been transformative therapies in chronic lymphocytic leukemia (CLL) and other lymphoid malignancies. Despite extended remissions, most patients (pts) will relapse. Covalent BTKi (cBTKi) including ibrutinib, acalabrutinib, and zanubrutinib, share a common resistance mechanism, acquisition of a C481 mutation in BTK. Non-covalent BTKi (ncBTKi) can overcome this, however, second-site mutations, notably T474 gatekeeper and kinase-dead mutations, have been observed. LP-168 is a selective next generation inhibitor of BTK that can bind wild-type and C481-mutated BTK covalently and non-covalently, respectively, with preclinical activity in resistant CLL models (submitted to ASH 2023). This dual activity is hypothesized to allow efficacy against cells with wild-type BTK through covalent activity while preventing expansion of the most common resistance mechanisms through non-covalent activity. Here we describe initial results of a phase 1 study of LP-168, with safety evaluation focusing on DLT evaluable pts, and efficacy evaluation focusing on the cohort of pts with CLL. Methods: NCT04775745 is a multicenter phase 1 dose escalation study of LP-168 monotherapy in pts with relapsed/refractory B-cell malignancies. CLL pts must have received ≥2 prior therapies. Dose escalation was performed using 3+3 design with expansion at multiple doses. Treatment emergent adverse events (TEAEs) were assessed per NCI CTCAE v5.0. Responses were evaluated using iwCLL 2018 criteria. Intra-patient dose escalation was permitted when dose levels cleared safety evaluation. Results: From 16 Sep 2021 to 10 Jul 2023, 43 DLT-evaluable pts were enrolled, including 35 with CLL, at doses from 100-300 mg daily as well as 150 mg twice daily. Data for this abstract were locked 10 Jul 2023. CLL pts received a median of 3 prior therapies, with 94% receiving a prior cBTKi and 11% a ncBTKi. 20 pts had alterations in TP53, 21 pts had C481S BTK mutations, 7 pts had gatekeeper mutations at T474 BTK, and 5 pts had mutation in PLCγ2 (Table). 33 pts have response data with median follow-up of 12.6 months (Figure). Overall response rate (ORR) including all dose levels was 54.5% (18/33)-all were partial response (PR) or partial response with lymphocytosis (PR-L). When evaluating only pts at doses of 200 mg or higher, ORR was 66.7% (10/15). In the subgroup of pts who had received only one prior BTKi including all dose levels, ORR was 77.8% (14/18). In the subgroup of pts with both BTK C481 and T474 mutations at doses of 150mg BID or 300mg QD, ORR was 75% (3/4). At the time of this report, only 5 pts with CLL have discontinued the study for progressive disease. At data lock, 43 DLT evaluable pts (with all histologies) were evaluable for safety. No DLT have been observed, and most AE have been low grade. TEAE seen in >20% of pts included diarrhea, fatigue, arthralgia, headache, constipation, cough, and dizziness; of those, grade 3 or higher AE included fatigue and constipation in 1 pt each. Serious AE were seen in 11 pts, of which 1 led to discontinuation (intracranial hemorrhage in a mantle cell lymphoma pt in complete remission). No DLT was identified at doses up to 300 mg daily and 150 mg twice daily. No atrial fibrillation/flutter has been reported. Hypertension grade 3 or higher was seen in 1 pt, and 1 pt experienced high-grade bleeding (as described). AE frequency did not increase with increasing doses. Pharmacokinetics showed plasma exposure increases dose-dependently. Steady state AUC 24h was between 47.1-94.6 ug/mL*h with limited accumulation. Steady state half-life was between 15.3-21.9 hours at all doses and C max was between 3500 and 5600 ng/mL. BTK occupancy is nearly at 100% at dose levels that were tested, with little variability among pts. Collective review demonstrates optimal Cmax to be achieved at either 200 or 300 mg dosed daily. Conclusions: LP-168 has been well tolerated in this study, with no DLTs identified at doses up to 300 mg daily. Preliminary efficacy was observed in high risk CLL pts, including those treated with one or more BTKi. Considering safety, efficacy, and PK/PD, dose expansion will occur at 200 mg and 300 mg daily following the FDA Project Optimus guidelines to determine the recommended phase 2 dose. A separate cohort will enroll pts with gatekeeper mutations to further explore efficacy. Updated biomarker, safety, and efficacy data will be presented at the meeting.

Melanoma and CLL co-occurrence and survival: role of KC history

BackgroundSurvival following melanoma and chronic lymphocytic leukemia (CLL) have both been individually associated with previous history of non-melanoma skin cancers (specifically keratinocyte carcinomas [KC]). Furthermore, melanoma and CLL have been reported to occur within the same patients. The survival experience of patients with both cancers is understudied, and the role of history of KC is unknown. Additional research is needed to tease apart the independent associations between KC and CLL survival, KC and melanoma survival, and the co-occurrence of all three cancers.MethodsA retrospective cohort study was conducted among patients who were diagnosed with melanoma and/or CLL at a comprehensive cancer center between 2008 and 2020. Multivariable Cox regression models were used to examine the association between history of KC and survival following melanoma and/or CLL with careful consideration of calendar year of diagnosis, treatment regimens and other risk factors. A nested case–control study comparing patients with both CLL and melanoma to those with only CLL or only melanoma was conducted to compare blood parameters across the three groups.ResultsA time-dependent association was observed between history of KC and favorable melanoma survival within 4 years following diagnosis and poorer survival post 7 years after melanoma diagnosis. History of KC was not significantly associated with survival following the diagnosis of CLL, after adjustment for clinical factors including historical/concurrent melanoma. Patients with co-occurring melanoma and CLL tended to be diagnosed with melanoma first and had elevated blood parameters including white blood cell and lymphocyte counts as compared with patients who were diagnosed with only melanoma.ConclusionsHistory of KC was an independent predictor of survival following melanoma but not of CLL. Additional studies are needed to determine if blood parameters obtained at the time of melanoma diagnosis could be used as a cost-effective way to identify those at high risk of asymptomatic CLL for the promotion of earlier CLL diagnosis.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/μL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

Abstract 5222: Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL)

Abstract Low count (LC) MBL is characterized by a circulating population of clonal B-cells and is a precursor state to CLL. Most individuals with LC MBL do not progress to CLL and never come to clinical attention. An active area of research is to identify factors that distinguish those individuals with MBL who progress from those who do not. Currently, 41 single nucleotide polymorphisms (SNPs) are known to be associated with CLL risk, and a portion of these SNPs have also been found to be associated with MBL risk. We hypothesize that these CLL-susceptibility SNPs associated with both MBL and CLL risk are SNPs associated with developing a B-cell clone; whereas the remaining SNPs found not to be associated with MBL risk are associated with progression of the B-cell clone (i.e., progression from MBL to CLL). To test this hypothesis, we evaluated the association of these 41 SNPs with risk of progression to CLL using a case-control study of 1,911 CLL cases and 1,579 individuals with MBL from Mayo Clinic. Individuals with MBL were identified through screening using 8-color flow cytometry assay. Individuals with LC MBL (defined by having a clonal B-cell population with the percent clonal B-cells out of total B-cell count <85%) who had an immunophenotype consistent with CLL were included as controls. CLL cases were obtained through the Mayo Clinic hematology practice. DNA was genotyped from whole blood. We analyzed the 41 CLL susceptibility SNPs with risk of progression to CLL using logistic regression and estimated odds ratio (OR) and 95% confidence interval (CI), adjusting for sex and age. We evaluated the CLL polygenic risk score (CLL-PRS), comprised of a weighted average of the 41 SNPs, with risk of CLL. Out of the 41 established CLL susceptibility SNPs, 15 were associated with CLL progression from MBL to CLL (all P<0.05). Nine of these 15 significant SNPs were not previously found to be associated with risk of MBL. The most significant SNPs were rs9880772 (OR=1.23, CI: 1.11-1.36, P=6.1 × 10−5), rs888096 (OR=1.25, CI: 1.12-1.38, P=3.0 × 10−5), and rs305065 (OR=1.28, CI: 1.14-1.43, P=1.5 × 10−5). One SNP (rs77551289) had a suggestive association (OR=1.21, P=0.069). The remaining 25 SNPs had no evidence of association with progression (ORs ranging between 0.9-1.1 and all P>0.1). The CLL-PRS was also associated with CLL risk (continuous OR = 1.25, CI: 1.15 - 1.35, P = 6.7 × 10−8). In our study accounting for the presence of B-cell clonal population, we identified 15 out of 41 CLL-susceptibility SNPs to be significantly associated with progression from LC MBL to CLL. The remaining 25 non-significant CLL-SNPs were not associated with progression and may instead be associated with development or initiation of the B-cell clone. These findings suggest that the established CLL susceptibility SNPs may play differing role (i.e., development or progression of the clone) in the etiology of CLL. Citation Format: Raphael Mwangi, Geffen Kleinstern, Sara J. Achenbach, Dennis Robinson, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie G. Waller, Nicholas J. Boddicker, James R. Cerhan, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, Tait Shanafelt, Susan L. Slager. Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5222.

Abstract 1185: Mosaic chromosomal alterations are independent predictors of chronic lymphocytic leukemia risk

Abstract Chronic lymphocytic leukemia (CLL) is strongly associated with mosaic chromosomal alterations (mCAs), a type of clonal hematopoiesis characterized by large structural chromosomal changes (e.g., gains, losses and copy neutral loss of heterozygosity). We aim to identify specific chromosomal regions that, when impacted by mCAs, are most predictive of CLL risk and evaluate their predictive utility in models that include established CLL risk factors. We utilized genotype array data from 436,784 participants in the UK Biobank (UKBB) and 35,382 participants in the Prostate, Lung, Colorectal, and Ovarian (PLCO) screening trial to identify mCAs. UKBB was split (90/10) into training and test sets, and PLCO served as an external validation set. We developed sequential regression models to predict risk of CLL, with a baseline model consisting of age, age-squared, sex, smoking status, and ancestry. Additional models evaluated mCAs, a CLL polygenic risk score (PRS), and blood cell counts. Area under the receiver operating characteristic curve (AUC) was used to evaluate the predictive utility for both 5-year and 10-year CLL risk. Any detectable mCA was associated with incident CLL (Hazards Ratio (HR)=26, 95% Confidence interval (CI)=21-31), and mCAs on each individual chromosome were associated with increased CLL risk. mCAs on segments of chromosomes 13, 12, 22, 14, and 11 had the largest effect size (e.g., chr 13q14 HR=199, 95%CI=155-257). The CLL prediction model, including baseline factors, CLL PRS, and any autosomal mCA status, showed strong predictive utility in independent UKBB (AUC=0.88) and PLCO (AUC=0.88) samples. Restricting to a subset of mCAs strongly associated with CLL did not improve performance over including any detectable autosomal mCA. After the inclusion of blood cell traits, predictive utility further increased in UKBB (AUC=0.91; no count data available in PLCO). We observed the strongest CLL predictive utility in 5-year risk compared to 10-year risk across all models. mCAs genome-wide are associated with CLL risk, with variable effects across chromosomes and specific chromosomal regions. Autosomal mCAs are strong independent predictors of CLL risk and substantially add to predictive utility over standard clinical measures. The predictive utility of autosomal mCAs is strongest within the first 1-5 years of sample collection. Evaluation of mCAs could provide added utility for risk stratification in populations at high risk of CLL. Citation Format: Aubrey K. Hubbard, Alexander DePaulis, Sruthi Srinivasan, Ian D. Buller, Shu-Hong Lin, Weiyin Zhou, Stephen J. Chanock, Neal D. Freedman, Derek W. Brown, Mitchell J. Machiela. Mosaic chromosomal alterations are independent predictors of chronic lymphocytic leukemia risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1185.

Risks and outcomes of hospitalizations in patients with chronic lymphocytic leukemia admitted with immune thrombocytopenia: an analysis of the National Inpatient Sample Database.

Immune thrombocytopenia (ITP) is a known autoimmune complication of chronic lymphocytic leukemia (CLL). Currently, there is limited data regarding the risk CLL confers on hospitalization outcomes in patients admitted with ITP.The National Inpatient Sample (NIS) database was queried using the International Classification of Diseases (ICD) codes to identify hospitalizations for ITP and then subclassified the data into hospitalizations with and without CLL. A multivariate logistic regression was designed to account for patient characteristics and comorbidities. The primary outcome was all-cause mortality. Secondary outcomes included major bleeding, gastrointestinal bleeding, intracranial bleeding, and the need for platelet transfusions, intravenous immunoglobulin, and splenectomy. Among 662,171 cases of ITP between 2005 and 2019, 15,672 had concurrent CLL. CLL patients were significantly older and had more comorbidities compared to patients without CLL. Multivariate analysis revealed CLL patients with ITP had a risk of all-cause mortality (odds ratio: 1.28, 95% CI: 1.19-1.37; p < 0.01). CLL patients also had a higher risk of complications, second-line ITP treatments, blood transfusions, and bleeding, with the exception of intracranial hemorrhage. Our study suggests CLL is an independent risk factor for increased morbidity and mortality among hospitalized patients with ITP. Prospective studies are needed to determine if refractoriness to conventional treatments for ITP can account these results.

EXPRESSION OF IMMUNOGLOBULIN SEQUENCES HOMOLOGOUS TO ANTI-SARS-CoV-2 ANTIBODIES AND HIV IN CHRONIC LYMPHOCYTIC LEUKEMIA CASES.

Identification of epitopes recognized by leukemic B cells could provide insights into themolecular mechanisms of B cell transformation in chronic lymphocytic leukemia (CLL). Theaim of this paper was to compare nucleotide sequences of immunoglobulin heavy chain variable region (IGHV) genes in CLL with known sequences directed against antigens of different origins available in public databases. Analysis was performed in thegroups of 412unselected CLL patients with productive IGHV gene using polymerase chain reaction followed by direct sequencing. Homology between CLL Ig sequences and antibodies directed against autoantigens was found in 12patients (2.9%), homology between CLL Ig sequences and antiviral antibodies- in 35patients (8.5%). Most of these sequences belonged to stereotypical clusters. Among thesequences that have homology to antiviral antibodies, themost prevalent were cases homologous with antibodies against HIV (14cases, 3.4%) and SARS-CoV-2antigens (10cases, 2.4%). None of thepatients in our cohort was HIV-infected and thestudy was conducted before theemergence of SARS-CoV-2virus. Suggestions could be made about thepossible impact of past infection of SARS-CoV-2virus on thepathogenesis of CLL. In particular, an increase in theproportion of CLL cases with theexpression of some stereotyped BCR and/or an increase of CLL risk in thelong-term period after SARS-CoV-2virus infection is not excluded. This assumption needs to be verified by epidemiological data.

Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.

Genome-Wide Association Study Identifies Variants Associated with Clinical Outcomes in Pediatric Patients with Newly Diagnosed Acute Myeloid Leukemia

Genetic aberrations in leukemic blasts are associated with clinical outcomes in patients with AML. However, the role of inherited genetic variants with regards to variation in AML treatment outcome remains obscure. To that end, we conducted a genome wide association study (GWAS) on 400 newly diagnosed pediatric AML patients to identify single nucleotide polymorphism (SNPs) that may be associated with minimal residual disease (MRD1) after induction 1 chemotherapy, event-free survival (EFS) and overall survival (OS). In this study, we utilized whole-genome genotyping with Illumina's Omni2.5Exome-8 BeadChip array to investigate SNPs for association with clinical outcome in 2 pediatric AML cohorts: the multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=233] trials. Post GWAS quality control, 1,365,439 unphased SNP genotypes were selected for imputation using TOPMed Imputation Server. Following imputation and additional quality control filtering, 6,741,200 SNPs evaluated for association with clinical outcomes (MRD1, EFS and OS). To improve statistical rigor, two GWAS approaches were utilized: i) A one-stage GWAS with both clinical trial cohorts combined (n=400) and ii) A two-stage GWAS where we utilized a two-step iterative resampling procedure (PMID: 26377241) by splitting the cohort at a 70:30 ratio into discovery and replication cohorts, balanced by clinical trial, treatment arm and initial risk group at diagnosis (100 randomizations performed). For each of the 100 iterations of the two-stage GWAS, we used the discovery cohort to perform a genome-wide analysis, followed by a replication screen. SNPs replicated in at least 20 of 100 rounds of discovery-replication were prioritized. A comparison of the two approaches showed all significant SNPs in the two-stage GWAS were also captured in the one-stage GWAS. For EFS, a Bonferroni-adjusted genome-wide significant association was observed for a SNP in CSMD1 and this SNP was replicated in 83 out of 100 discovery-replication rounds for two-stage GWAS. For CSMD1 SNP, presence of variant A allele has significantly reduced EFS (HR=3.29, 95% CI (2.177-4.975), P=1.61x10-8, Figure 1A). CSMD1 is a membrane-bound complement inhibitor that has been shown to act as a putative tumor suppressor gene in other malignancies (PMIDs:27764775, 35623420). Another significant marker worth mentioning was an intronic SNP in CDKN2B-AS1 that associated with poor outcome [EFS: HR=2.37, 95% CI (1.700-3.317), P=4.40x10-7] and [OS: HR=2.87, 95% CI (1.954-4.225), P=8.06x10-8, Figure 1B]. This marker is in high linkage disequilibrium with 26 other SNPs within the CDKN2B and CDKN2B-AS1 cluster. The product of this gene is an RNA molecule that interacts with polycomb repressive complex-1 (PRC1) and -2 (PRC2) involved in silencing other genes within this cluster through epigenetic mechanisms (PMID: 20716771). CDKN2B-AS1 has been reported to play a role in tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia and with the risk of chronic lymphocytic leukemia (PMIDs: 32976214/23770605). Though limited by numbers, this is one of the largest GWAS conducted in pediatric AML to identify SNPs associated with clinical outcomes. Our top results show that genetic variation in several genes of biological relevance may impact MRD1, EFS and OS. Future studies are aimed at looking at SNP/gene and association with gene expression collected from leukemic cells at time of diagnosis to establish AML-specific eQTLs and functional validation of the most promising candidates. Additionally, future analyses will include other subgroups such as cytogenetic risk group and validation of these findings in larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptomic Changes Associated with Rare Germline Missense ATM Variants in Chronic Lymphocytic Leukaemia

Background Germline pathogenic variants in ATM gene are associated with risk of chronic lymphocytic leukaemia (CLL) and poorer prognosis. However, genetic testing is revealing a large number of rare germline missense variants of uncertain significance (VUS) in ATM whose clinical value is unclear. In this study, we evaluated the impact of ATM germline missense variants on the CLL transcriptome. Methods The genetic germline and transcriptome data from 237 CLL cases were obtained from public CLL datasets (EGAD00001001464, EGAD0000100146612). Only rare (minor allele frequency < 0.5%) missense ATM germline variants annotated as VUS by Clinvar and Varsome databases and with pathogenic prediction by SIFT/PolyPhen were studied. The transcriptome analysis was carried out using the DEseq2, 3D RNA-seq and Enrichr tools. Adjusted p value (padj) < 0.15 was considered significant. Results Of the analysed dataset, 9 (3.8%) CLL cases carried rare germline missense predicted pathogenic VUS in ATM. Further analysis was performed within subgroups of patients based on their IGHV mutational status, as preliminary cluster analysis showed distinct gene expression profiles for these subgroups. In the IGHV mutated (m-IGHV) subgroup, six cases carried germline missense ATM VUS(s) (G204R, R717W, D817N, V1671G, T1871I, L2293R, L2293V), four had solo del(11q) and 77 were without any somatic and/or germline disruption in genes associated with DNA damage response (DDR), nor did they carry mutations in a panel of 10 recurrently mutated genes (TP53, SF3B1, BIRC3, NOTCH1, XPO1, EGR2, POT1, NFKBIE, RPS15, FBXW7) associated with inferior prognosis in CLL. In patients with unmutated IGHV (u-IGHV), three cases had ATM VUS (N796H, P1376S, D2870N), including one with concurrent del(11q), 7 had only del(11q), and 20 were without any somatic and/or germline disruption in DDR and CLL-associated genes. Transcriptome analysis of m-IGHV patients with germline missense ATM predicted pathogenic VUS revealed that the highest enrichment scores belonged to mitotic spindle signaling, reactive oxygen species pathway, cholesterol homeostasis, peroxisome, fatty acid metabolism, Wnt/β-catenin signaling, response to UV radiation and oxidative phosphorylation, and genes enriched in targets of MYC, E2F, RUNX1 and CREB1 transcription factors. Analysis of m-IGHV patients with del(11q) revealed similar gene enrichment in mitotic spindle signaling, hypoxia and in the targets of MYC, RUNX1 and CREB1 as in the m-IGHV cohort with ATM VUS, but additionally, genes enriched in TNF-α signaling via NF-κB, apoptosis, and IFN-γ response were detected. Furthermore, two m-IGHV cases had biallelic disruption of TP53; analysis of these patients revealed differentially expressed genes involved in apoptosis, cholesterol homeostasis, oxidative phosphorylation, and targets of MYC and E2F. Analysis of u-IGHV patients with germline missense ATM VUS showed no significant results compared to cases without DDR/CLL gene aberrations, probably due to the low number of cases. Enrichment of genes involved in the p53 pathway, cholesterol homeostasis and MYC targets was observed in u-IGHV patients with only del(11q). Evaluation of another independent CLL cohort is currently underway. Conclusions This preliminary analysis showed a similar impact of rare germline ATM VUS on the CLL transcriptome as del(11q), at least in m-IGHV CLL, which was characterized by differentially expressed genes involved in mitotic spindle and oxidative stress signaling and targets of MYC, RUNX1 and CREB1 transcription factors. These data highlight the need for further investigation and validation of detected transcriptomic changes in a larger cohort, which is ongoing. Grant support: MH CZ - DRO (FNOl, 00098892), IGA LF UP_2022_011

Association between Cumulative Airborne Dioxin Exposure and Chronic Lymphocytic Leukemia/Non-Hodgkin's Lymphoma Risk in a Nested Case-Control Study within the French E3N Cohort

Background: Dioxins are a family of lipophilic, bio-accumulative chemical compounds belonging to persistent organic pollutants groups. The TCDD (2, 3, 7, 8 tetrachlorodibenzo-para-dioxin) which is the most toxic congener of dioxins is classified as carcinogen to humans by the International Agency for Research on Cancer. Airborne dioxins exposure is suspected to increase the risk of Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin's lymphoma (NHL) in some studies. Although dioxins are bio-accumulative in the human body, no epidemiological study had assessed the relationship between cumulative airborne dioxin exposure and CLL/NHL risk. Objective: The relationship between cumulative airborne dioxin exposure and CLL or NHL risk was assessed in a case-control study nested within the French E3N cohort (cohort of 98 995 women covered by the health insurance provider of the national education system). Materials and methods: All incident cases of CLL/NHL diagnosed between 1990 and 2008 were selected. They were identified by self-administered questionnaires as well as through reports from family members and from the national cause of death registries. All cases were validated with medical reports. Subtypes of NHL were classified according to the International Lymphoma Epidemiology Consortium working group classification of lymphoid neoplasms for epidemiologic research. 368 incident cases of CLL and NHL were matched to 368 controls on age (± 1 year) and on the department of residence reported at inclusion in 1990. Cumulative exposure to airborne dioxins was estimated for each participant at the individual address level using a Geographic Information System (GIS) based exposure index including residence-to-source distance, wind direction, annual dioxin emission intensity, length of operation and meteorological parameters (1). Airborne dioxin exposure was summarized into a cumulative exposure index from the latest inclusion date (i.e., the date of return of the first questionnaire) of the case-control pair, to the index date (i.e., the date of diagnosis of the case). Multivariate conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI). The main model was adjusted on education level used as a proxy for socioeconomic status, place of residence, body mass index (kg/m2), physical activity (metabolic equivalent activity task-hour per week (MET-h/w)) and smoking status. Results: The mean follow-up time was 11.7 years ranging from 0.2 to 19.4 years and the cumulative exposure index averaged was 3.6 µg-TEQ/m2. The mean age of women at diagnosis of CLL/NHL was 63.3 years. The subtype distribution was 23% of Diffuse Large B-cell Lymphoma (DLBCL), 19% of Follicular Lymphoma (FL), 27% of CLL & small lymphocytic lymphoma (SLL), 11% of Marginal Zone Lymphoma (MZL) and 20% of other NHL. We observed a statistically significant positive association between the cumulative dioxin exposure index and the CLL/NHL risk (OR and 95% CI: 1.2 (1.0,1.4) for a 4.4 log µg-TEQ/m2 increase corresponding to a standard deviation). Analysis by subtypes showed a significant association between the CLL/SLL subtypes and the cumulative continuous exposure index (OR and 95% CI: 1.6 (1.1,2.3)) (Table 1). Discussion/Conclusion: The present study supports a positive significant association between cumulative airborne dioxin exposure and CLL/NHL risk, and, more specifically, a positive significant association for CLL & SLL. Our findings are consistent with publications that found positive associations between dioxin exposure and CLL/NHL risk. This is the first epidemiological study investigating the relationship between cumulative airborne dioxins exposure and CLL/NHL risk using a GIS-based cumulative exposure index and a multi-source approach. These findings contribute to justify the efforts to decrease the airborne exposition to dioxins. Additional studies evaluating the association between cumulative dioxin exposure and other hematologic malignancies are required. Reference 1. Coudon, T., Danjou, A.M.N., Faure, E. et al. Development and performance evaluation of a GIS-based metric to assess exposure to airborne pollutant emissions from industrial sources. Environ Health18, 8 (2019). https://doi.org/10.1186/s12940-019-0446-x Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characteristics of Rare Germline ATM Variants in Chronic Lymphocytic Leukemia (CLL)

Background The use of next-generation sequencing in the clinic has increasingly uncovered new variants of unknown significance (VUS), which are often ignored clinically. Ataxia telangiectasia mutated (ATM) is a tumor-suppressor gene that has a critical role in DNA double-strand break repair and has been implicated in the pathogenesis of CLL. In this study, we assessed rare germline ATM variants including VUS using a novel variant calling algorithm, evaluated their impact on the clinical characteristics of CLL and functionally assessed their effect on ATM kinase activity. Methods We evaluated the entire ATM coding region via germline whole exome sequencing from 885 CLL patients representing three different previously reported cohorts (327 DFCI; 276 German CLL Study Group, and 282 ICGC), along with a cohort of ancestry matched controls. We utilized DeepVariant (Poplin et al Nature Biotech 2018) to detect germline variants, followed by a standardized quality control workflow (AlDubayan et al JAMA 2020). All individuals harbored ATM germline variants. Variants were annotated using dbSNP, Clinvar and Varsome, and variants with GnomAD population allele frequency (AF) < 1% were defined as rare. For clinical characterization of CLL patients with rare germline ATM missense variants, we focused on the DFCI cohort. For functional analysis, we evaluated ATM kinase activity by ATM and KAP1 phosphorylation in response to 1 Gy gamma-irradiation using flow cytometry in primary CLL samples with/without rare germline ATM missense variants. We also generated CRISPR/Cas9 engineered cell lines to evaluate the function of ATM p.L2307F which was frequent in our CLL cohort and which we previously demonstrated as enriched in CLL, yet generally annotated as a VUS. Results We identified a total of 130 ATM germline variants among the CLL cases, including 87 missense, and 9 splice site alterations. 80 of 87 missense variants were rare and 59 were annotated as VUS. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In 63 DFCI patients with rare missense variants, the median age at diagnosis was 55 (31-82), 21.7% had a familial history of CLL and 29% had del(11q). Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to gamma-irradiation was reduced in 12 patients assessed (8 rare and 4 non-rare) with germline ATM variants (ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P<0.01). Similar results were seen when the analysis was restricted to the 8 CLL patients with rare ATM variants. Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del 11q + ATM variant group) compared to 13 of those with del(11q) alone (del 11q group) (P<0.01) (Figure 1). Altogether, these results suggest that germline ATM variants have a functional impact on DNA damage response in primary CLL cells. We previously identified the VUS variant ATM p.L2307F as particularly enriched among CLL patients (Tiao G et al., Leukemia. 2017). We therefore generated ATM L2307F knock-in NALM-6 cell lines via CRISPR/Cas9. We generated 7 cell lines including ATM wild type (+/+), ATM monoallelic mutated (+/L2307F), ATM monoallelic knocked out (+/-), ATM monoallelic mutated with knock out of the other allele (-/L2307F), and ATM biallelic knockout (-/-). The induction of both pATM and pKAP1 levels by gamma irradiation was reduced in proportion to ATM mutational status as shown in Figure 2. Conclusions We demonstrate that rare ATM germline variants are common in CLL and affect the DNA damage response in primary CLL cells when controlling for somatic 11q deletion. We further characterize the p.L2307F variant using CRISPR and demonstrate that this allele represents a functional hypomorph in cell-based assays. Further studies are needed to assess the impact of each individual rare germline variant on the function of ATM, CLL pathogenesis and the inherited risk of CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Profiling of Cells from Patients with Chronic Lymphocytic Leukemia (CLL) Treated with Pirtobrutinib

Covalent (c) Bruton's tyrosine kinase (BTK) inhibitors (BTKi) are effective agents for treatment of chronic lymphocytic leukemia (CLL). As single agent or in combination they result in long-term disease-free survival. However, some patients are intolerant to cBTKi while in others disease relapses due to pathogenic mutation in the C481 domain where cBTKi bind. More selective and noncovalent (nc) BTKi have proven to be efficacious for this patient population. Among ncBTKi, pirtobrutinib (previously known as Loxo-305) has been successfully used in the clinic (NCT03740529) for patients with CLL after cBTKi therapy. Previously treated patients with CLL entered on this protocol from Oct 2019 till Sept 2021 and received single agent oral pirtobrutinib until disease progression. Data from 27 CLL patients are available while one is lost to follow-up; baseline BTK status was wildtype (WT) in 10 and mutant (Mt) in 16 patients. Among these 26 patients, 13 (50%) progressed while on pirtobrutinib therapy (Table1). BTK profiling results at start of therapy show that among those who progressed, 11 patients had mutant BTK (85%) and 2 patients had WT-BTK (15%) suggesting that mutant BTK may be more likely to have disease recurrence while on pirtobrutinib therapy. Most common mutations were C418S, C418R, C481F, T4741 and PLCGy2 alone or in combination. Some patients with progression developed BTK clonal evolution with new nonC481 Mt-BTK clone or increase in variant allele frequency (VAF) of mt-BTK. BTK profiling at time of DP indicated that in some of these patients the original pathological clone of C481 Mt-BTK resolved. Disease progression (13 patients) typically occurred during the first two years of therapy with median time to progression 22 cycles for patients harboring WT-BTK and 14 cycles for those with Mt-BTK. Most of these patients (12) also showed high risk CLL features of TP53 mutation (9 patients), ATM/11qdel (9 patients), Notch1 mutation (4 patients), complex karyotype (6 patients) or trisomy 12 (3 patients). There are limited therapeutic options post pirtobrutinib progression. Therefore, we conducted pharmacological profiling for these patients by ex-vivo testing CLL cells post progression. We incubated peripheral blood mononuclear cells for three days with cBTKi - ibrutinib; glutathione inhibitor (also known as mtTP53 modulator) - APR-246; Bcl-2 antagonist - venetoclax; and Mcl-1 antagonist - AZD5991; as single agent and in double or triple combinations and apoptosis was assessed by flow cytometry. Endogenous cell death was negligible even after 3 days in suspension culture indicating highly resistant cell population. Single agent venetoclax at 50 nM was most effective with a median 70% cell death (range 40-90%) after 3 days. Interestingly, cells harboring mt-BTK appeared to be more sensitive to venetoclax (p = 0.01). APR-246 alone showed heterogeneity with 10% to 85% cell death yet it was able to sensitize cells to venetoclax, ibrutinib, and AZD5991 in both WT and mutant BTK patient cohorts. Loss of C481S original clone after pirtobrutinib therapy made cells sensitive to ibrutinib. Four (4) patients, both pretreatment and during therapy or progression samples were available. Intra-patient comparison demonstrated that compared to pretreatment sample, cells were sensitized to targeted agents after 10 - 14 cycles of pirtobrutinib therapy (Figure 1). Additional samples are being tested during this ongoing clinical trial. In conclusion, progression on pirtobrutinib therapy was more common in patients with Mt-BTK. Pharmacological profiling based on BTK status at disease progression showed venetoclax and APR-246 as the most effective single agents. Combination of APR-246 or venetoclax with ibrutinib or AZD5991 showed maximum cell death. Patients were re-sensitized to ibrutinib after loss of C481S clone. Genomic and genetic profile along with pharmacological landscape provide precision medicine for these heavily pretreated patients whose disease is resistant to BTK inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MS-553, a Selective PKC-ß Inhibitor Inhibits BCR Signaling and Synergizes with Venetoclax for the Treatment of High Risk CLL

Inhibitors of Bruton's tyrosine kinase (BTK) have been transformative in treating patients with chronic lymphocytic leukemia (CLL) due to their ability to inhibit B-Cell Receptor (BCR) signaling. However, despite durable remissions in many patients, complete responses are uncommon and acquired mutations in BTK and its downstream target phospholipase Cγ2 (PLCγ2) lead to relapses and poor survival. In parallel, genomic high-risk features such as aberrations in TP53 represent an independent risk factor that favor progression on BTK inhibitors (BTKi). TP53 aberrations comprise of deletions in chromosome 17 (del17p) which result in the loss of one allele of TP53 and point mutations in the TP53 gene. CLL patients with del17p acquire clonal mutations that affect the other p53 allele in 82% of cases and largely express only mutant TP53 protein. Therefore, there is an urgent need to identify therapies for patients who progress on BTKi. We have observed over-expression of protein kinase C-β (PKC-β) in primary CLL patient samples including in those that have mutations in TP53. PKC-β is a downstream of the BCR and functions downstream of BTK. PKC-β is required for CLL cell survival and proliferation and therefore can be a potential target for patients with mutations in p53 that do not respond to other targeted therapies. We hypothesized that targeting PKC-β will inhibit BCR signaling downstream of BTK and may offer an effective therapeutic strategy for patients who relapse on ibrutinib. Mechanistically, we found that CLL cells (n=6 patients) in contact with stromal support upregulated their levels of PKC-β. Pre-exposure of stromal cells to MS-553, a selective PKC-β inhibitor prevented the stromal cell contact-induced increase in PKC-β within the CLL cells establishing that stromal contact was an important source of PKC-β in CLL cells. We have shown in primary CLL patient samples that exposure to MS-553 decreases activation of PKC-β and its downstream effectors GSK3-β and ERK in a dose-dependent manner regardless of mutations in p53 both in the presence and absence of stromal support. In parallel, MS-553 also decreased the levels of BCL-xL, a key pro-survival BCL2 family protein via downregulation of β-catenin. Using dynamic BH3 profiling we show that CLL samples (both WT and mutant TP53) exposed to MS-553 (5µM, 24h) increase their dependence on BCL-2 and BCL-xL for survival. This finding suggested that MS-553 could synergize with the BCL-2 inhibitor venetoclax to eliminate CLL cells. Correspondingly, cells exposed to a combination of MS-553 and venetoclax released more cytochrome c (Cyt c), used as a measure of mitochondrial dysfunction, compared to each single agent alone and led to a synergistic increase in apoptosis as measured by Annexin V positivity. These data provide strong rationale for an ongoing Phase I/II trial that evaluates MS-553 as a single agent and in combination with targeted agents such as venetoclax. Early interim data indicate that MS-553 is safe, tolerable and effective both as a single agent and in combination with venetoclax.

Preliminary Results of the Filo Phase 2 Trial for Untreated Fit Patients with Intermediate Risk Chronic Lymphocytic Leukemia Comparing Ibrutinib Plus Venetoclax (IV) Versus FCR: Results of the Month 15 MRD Evaluation

With the emergence of targeted therapies, defining the best strategy for the treatment of previously untreated CLL patients remains challenging. The aim of this phase 2 study was to compare the efficacy in first line of combined ibrutinib and venetoclax (IV) to the standard FCR regimen in fit patients with CLL of intermediate risk defined by either unmutated IGHV status, 11q deletion or complex karyotype in the absence of TP53 alteration. As previously reported (Michallet et al., Blood 2021; 138 (suppl 1):641), 120 patients were randomized 1:1 between two treatment arms, ie FCR 6 cycles or IV. After a lead-in phase of ibrutinib as a single agent from month (M)1 to M3, the total duration of treatment with the IV combined treatment was based on the response achieved at M9; if bone marrow (BM) MRD was < 0.01% using flow cytometry, the treatment was continued for 6 additional months until M15 and then stopped; if BM MRD at M9 was ≥ 0.01%, the treatment with IV was continued for 18 additional months until M27. PB MRD evaluation was performed every 6 months, whereas BM MRD was performed at M9 and M27.The primary endpoint was the percentage of patients with BM MRD < 0.01% at M27 in both arms. We present herein safety data and the preliminary results of the PB MRD evaluation at M15 . These 120 patients were enrolled from September 2019 to February 2021. The median age was 59 [34-72] and 61 [34-74] years in the FCR and IV arms, respectively. The characteristics of patients were well balanced between the 2 arms in terms of gender (male 72% FCR, 74% IV), PS ECOG 0-1 (59% FCR, 68% IV) and Binet stage (A, B and C 15%, 64%, 21% for FCR ; 8.5%, 59% and 32% for IV). 11q deletion was found in 20% and 24% of cases in the FCR and IV arms, respectively and all patients but one had unmutated IGHV. At the time of data cut-off for this interim analysis, the median follow-up was 24.8 [16.7 - 33.6] months. Sixty-three serious adverse events (SAE) were reported so far, 33 in the FCR arm and 30 in the IV arm. In the FCR arm, the most frequent SAE were tumour lysis syndrome (N=3), infections (N=12 including 4 COVID19 infections), febrile neutropenia (N=5) and secondary malignancies (N=2, including 1 myelodysplastic syndrome and 1 acute myeloid leukemia). In the IV arm, the most frequently reported SAE were tumour lysis syndrome (N=5), infections (N=8 including 4 COVID19 infections), cardiovascular events (N=7), acute renal failure (N=2) and secondary malignancies (N=2, including 1 colorectal cancer and 1 skin basal cell carcinoma). Three grade 5 adverse events were reported consisting in 1 acute myeloid leukemia in the FCR arm and 2 sudden death in the IV arm. No patient died because of COVID19 in the study. At M15, in intention to treat, in the FCR arm, 61.3 % of patients had peripherical blood MRD <0.01%, whereas MRD was ≥ 0.01% without clinical progression in 19.4% of patients and 6.5% of patients had experienced clinical progression. In the IV arm, PB MRD < 0.01% was reached in 63.8% of cases, MRD was ≥ 0.01% in 29.4% of patients and clinical progression was reported in 1.7% of patients (figure 1). In the FCR arm, 1 patient with MRD ≥ 0.01% at M9 converted to MRD < 0.01% at M15 while 2 patients had a signigicant increase of their PB MRD level from <0.01 to ≥ 0.01%. Conversely, in the IV arm, 12 patients converted from MRD ≥ 0.01% at M9 to MRD < 0.01% at M15 while 2 patients increased their PB MRD level from <0.01 to ≥ 0.01%. The two later patients had prematurely discontinued treatment before M15 evaluation. In conclusion, these preliminary results show an increase in PB MRD < 0.01% rates between M9 and M15 in the IV arm, with rates similar to those of the FCR arm at M15. However, toxicity remains significant. The primary enpoint analysis at M27 will be of great interest to try to determine the best strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rare Germline ATM Variants Influence the Development of Chronic Lymphocytic Leukemia.

Germline missense variants of unknown significance in cancer-related genes are increasingly being identified with the expanding use of next-generation sequencing. The ataxia telangiectasia-mutated (ATM) gene on chromosome 11 has more than 1,000 germline missense variants of unknown significance and is a tumor suppressor. We aimed to determine if rare germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared with other hematologic malignancies and if they influence the clinical characteristics of CLL. We identified 3,128 patients (including 825 patients with CLL) in our hematologic malignancy clinic who had received clinical-grade sequencing of the entire coding region of ATM. We ascertained the comparative frequencies of germline ATM variants in categories of hematologic neoplasms, and, in patients with CLL, we determined whether these variants affected CLL-associated characteristics such as somatic 11q deletion. Rare germline ATM variants are present in 24% of patients with CLL, significantly greater than that in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). Patients with CLL with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of patients with CLL, is associated with a three-fold increase in rates of somatic 11q deletion, and is a hypomorph in cell-based assays. Germline ATM variants cluster within CLL and affect the phenotype of CLL that develops, implying that some of these variants (such as ATM p.L2307F) have functional significance and should not be ignored. Further studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

CLL-527 Significance of TP53 Codon 72 in a Cohort of Egyptian Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. This heterogeneity is partly explained by the diverse genetic aberrations identified in patients. Due to the high heterogeneity of CLL, clinicians use prognostic biomarkers and risk scoring systems to guide treatment decisions. The codon 72 arginine-to-proline polymorphism (rs1042522) at the p53 locus affects cancer development, as well as response to therapy and survival of cancer patients. The aim of the present study was to investigate the correlation of TP53 codon 72 Arg/Pro polymorphism with the evolution of the disease and prognostic markers in CLL patients attending the oncology centre at Mansoura University (OCMU). Newly diagnosed 100 CLL patients were included in the study with median age (64 years), 65% were males while 35% were females. The median follow-up period was 30 months and during this period, 26 deaths were observed and 31 of patients lost to follow-up. Mean white blood cell count was 57(27-121) x109/L, mean platelet count was 139.7(83.5-193) x109/L, and mean hemoglobin level was 10.9 (9.3-12.6) g/dL. CD38 was expressed in 57% of patients. 13q14 del was detected in 27% of patients (9/33), 11q22 del in 12% of patients (4/33), and 17p del in 8.7% of patients (6/69). We examined TP53 codon 72 polymorphism by restriction fragment length polymorphism (RFLP) in CLL patients and in 87 healthy controls. Analysis of the TP53 codon 72 polymorphism in 100 CLL patients resulted in frequencies of 0.65 and 0.35 for the G and C allele, respectively (Arg/Arg-44 cases, 44%; Arg/Pro-42 cases, 42%; Pro/Pro-14 cases, 14%). In the comparative analysis of the three groups, the patients with the Pro/Pro genotype had a higher number of male patients; 92% of male patients had Pro/Pro genotype. Likewise, patients with the Pro/Pro genotype showed a significant association with CD38 expression and 17p deletion (p = 0.016 and 0.017, respectively) compared to the other groups. Our results can suggest that Pro/Pro genotype contributes to increased susceptibility to CLL risk in our population. Also, it has been observed that genotype (Pro/Pro) has been associated with expression of CD38, 17p del and male gender.

Interleukin-10-1082A>G (rs1800896) Single Nucleotide Polymorphism is Not a Risk Factor of Chronic Lymphocytic Leukemia in Sudanese Population.

The present study was conducted to examine the association between the IL-10-1082A>G (rs 1800896) polymorphism and risk of Chronic Lymphocytic Leukemia and to assess the correlation between this polymorphism and clinicopathological characters. A case-control study was conducted in Khartoum state, Sudan, during the period from April 2017 to April 2018, involved 110 CLL patients and 80 healthy volunteers as a control group. Physical examination, complete blood count, and immunophenotype were performed in all patients to confirm the diagnosis. Clinical staging such as Rai and Binet were studied. CD38 and ZAP70 were performed by flow cytometry. Blood samples were collected from all participants; DNA was extracted by using ANALYTIKJENA Blood DNA Extraction Kit and analyzed IL-10-1082A>G polymorphism by using Allele Specific-Polymerase Chain Reaction. The statistical analysis was performed using statistical package for social sciences version 23.0 software. Frequency of AA, AG, and GG genotypes was 32.7%, 55.5%, and 11.8% for the patient group and 31.25%, 51.25%, and 17.5% in the control group, respectively. The genotype of IL-10 (-1082A>G) did not associate with susceptibility of CLL in our population. The study showed that the G allele of the IL-10 gene (-1082A>G) is associated with the male sex. However, no significant association was found between -1082A>G genotype and clinicopathological characters. Our results do not support the involvement of the IL-10 -1082A>G promoter gene polymorphism in the increased CLL susceptibility. IL-10-1082G allele (IL-10-1082AG or IL-10-1082GG) was found more frequently in males. Furthermore, no association was observed between the IL-10-1082A>G polymorphism and clinical stages systems as well as established poor prognostic markers. Finally, within the group of patients with CLL, there was no difference in the age at diagnosis and hematological parameters according to genotype distributions.