Familial Chronic Lymphocytic Leukemia Research Articles

Investigating the Influence of Germline ATM Variants on Susceptibility to Chronic Lymphocytic Leukemia and Other Malignancies

Introduction: Chronic lymphocytic leukemia (CLL) patients (pts) face an increased susceptibility to secondary cancers. Furthermore, the hereditary predisposition to CLL is influenced by factors that have yet to be fully elucidated by GWAS-identified common variants. Among the hereditary cancer risk mutations observed, germline Ataxia-telangiectasia mutated ( ATM) aberrations are among the most prevalent. Rare germline ATM variants are detected in 24% of CLL pts, surpassing the frequency observed in other hematologic neoplasms and the general population. In CLL, loss of ATM via 11q23 deletion is linked to an unfavorable prognosis and a shortened progression-free survival. The extent to which germline ATM variants predispose CLL pts to other malignancies remains unclear; to examine this potential risk we conducted a retrospective study to assess the influence of germline ATM variants on the predisposition to secondary neoplasms in CLL pts and their relatives. Methods: Five hundred and eighty-five pts seen at Dana-Farber Cancer Institute and who had NGS performed to evaluate germline ATM status, either through direct germline sequencing of saliva or by inference according to the hierarchical algorithm we have previously published (Lampson, 2022), were mailed a questionnaire that investigated: demographics; personal and family history of any cancer; non-medical radiation and Agent Orange exposure; and Ataxia-Telangiectasia syndrome (AT). Of these, 333 replied (57%). Information collected from our CLL database included FISH, IGHV status and treatment status. European ancestry was categorized based on the United Nations geoscheme (UNSD, 2019). Pt characteristics are in Table 1. Pts were stratified into 4 groups, based on ATM mutational status and del(11q): Group-1, ATM germline variants alone (N=63); Group-2, ATM germline with somatic ATM mutation and/or del(11q)(N=22); Group-3, ATM somatic aberration alone (including del(11q))(N=41); and Group-4, no ATM aberration (N=207). Results: Of the 333 pts in our cohort, baseline demographic and clinical characteristics among the 4 groups were balanced, except for unmutated IGHV status. Nineteen (86%) and 36 (86%) pts of groups 2 and 3, carrying somatic ATM aberrations, had unmutated IGHV, compared to 21 (33%) and 76 (37%) of groups 1 and 4, without somatic ATM aberrations(p<0.01). Regarding treatment, 60% of our cohort had received at least one line of therapy (median: 1 (1-7)). Among the 164 pts (49%) with history of an additional non-CLL neoplasm, 221 cancers were reported. Excluding pts with only non-melanoma skin cancers, the incidence is reduced to 31% (104 pts). The median number of second cancers per pt is 1 (1-4). Of the 134 pts with available age of onset data for the secondary malignancy, 80 (60%) were diagnosed with the secondary cancer after CLL. Comparing pts with germline ATM variants (N=85) to the ones without germline ATM variants (N=248), no significant difference in the incidence of secondary cancer was found (p=0.73). The median age of secondary cancer onset was also similar between the groups (median: 63 and 64 years in germline ATM present vs absent, p=0.28). Of the whole cohort, 283 pts (85%) reported at least one relative with a cancer history (median 2 relatives affected (1-17)). The incidence of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in pts with germline ATM variants (32%) compared to those without germline ATM variants (21%), including familial CLL (25% vs18%) (p=0.04). Familial CLL was also strongly associated with Ashkenazi Jewish ethnicity (39% incidence in Ashkenazi pts vs 16% in non-Ashkenazi, p=0.004). No difference in the incidence of other hematologic or solid malignancies was found between the relatives in the two groups. No pts reported a familial or personal history of AT. Compared to pts without any ATM aberration, time to first treatment (TTFT) was shorter in pts harboring somatic ATM events, while no difference was observed between pts with germline ATM variants (median TTFT: 82, 59, 52, 90 months for group 1, 2, 3 and 4 respectively, p=0.01) (Figure 1). Conclusions: Our results suggest a higher incidence of B-cell lymphoproliferative disorders, including familial CLL, in the relatives of CLL pts carrying germline ATM variants. The presence of these germline variants did not impact TTFT, compared to pts harboring somatic ATM mutations.

Professor Daniel Catovsky 19 September 1937-2 December 2022.

Daniel Catovsky was trained in Argentina and graduated in Medicine in 1961. He emigrated to London in 1967 with his wife, Julia Pollack, and their daughter Mina. Subsequently they had two boys, Sebastian and Michael. Daniel joined the Hammersmith Hospital and Royal Postgraduate Medical School as a Research fellow in 1967 and in 1976, he became Honorary Consultant Physician/Senior Staff Member in the Medical Research Council Leukaemia Unit under Professor David Galton. Colleagues at that time included John Goldman and Sandy Spiers. From 1976 onwards he was a member of the French–American–British (FAB) cooperative group, which had a major impact on the diagnosis and classification of haematological neoplasms. In 1987 he was awarded a Personal Chair in Haematological Malignancies at the Royal Postgraduate Medical School, Hammersmith Hospital. The next year he moved to the Royal Marsden Hospital/Institute of Cancer Research where he established an Academic Haemato-Oncology Unit, continuing as Professor of Haematology until his retirement in 2003. Thereafter, he continued to be fully engaged in research at the Institute of Cancer Research as Professor Emeritus and Fellow, impacting all around him. Through all these years Daniel had an extraordinary active life encompassing research, teaching and diagnostic and clinical activities. He served as a member, chairman or leader in many boards such as the Central Institutional Review Board of Cancer Research UK (CRUK) (2003–2005); Leukaemia Research Fund Data Monitoring Committee (Chairman 2004–2006); Scientific Advisory Board of the CLL (chronic lymphocytic leukaemia) Global Research Foundation; Data Monitoring Committee UKCLL2006 trial, Arctic and Admire trials, CLL11 trial, and Parexel independent response review panel; Scientific Advisory Board of the Hairy Cell Leukaemia Consortium; National Cancer Research Institute CLL Trials Steering Committee; Scientific Advisory Committee of the CLL Genome Consortium (Spain); Data Safety Monitoring Board for the German CLL12 trial and CLL13 trial (chairman); and a variety of editorial boards of national and international journals. In addition, he also coordinated the UK CLL 1, 2, 3, 4 and 5 trials. Daniel was President of the British Society of Haematology (1996–1997) and of the European Research Initiative on CLL (ERIC) (2006–2009). He was awarded the BSH Medal for his contributions to British haematology (2000) and the IWCLL Binet–Rai medal for outstanding contributions to CLL research (2005). His work is clearly reflected in more than 850 peer-reviewed publications and he is the eighth most cited researcher in the field of oncology in clinical medical journals. Daniel had unique qualities, being a remarkable scientist and an excellent clinician. Most of his work was devoted to CLL and other chronic lymphoid leukaemias. He was a pioneer of studies of familial CLL and identified and described distinct lymphoid disorders, a relevant issue with implications in the correct diagnosis and management of these patients. His unit had wide appeal to many and was always open to visiting physicians and scientists who came from everywhere in the world, eager to learn from him. His valuable teaching had, and still has, a major impact in many units worldwide. Besides his scientific and medical skills, Daniel was a remarkable and unforgettable man with unique attributes and amazing appeal, full of humanity, well reflected in his empathy towards patients, fellows and colleagues. We have lost Daniel, but his imprint will remain. Life would not have been the same without him, nor will it be the same from now on. A sad loss to his family, to science and to the entire community.

Predictive and Prognostic Molecular Biomarkers in Familial Chronic Lymphocytic Leukemia: A Pilot Monocentric Study

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world and the median age at diagnosis is 70 years. Family history of hemopathies and, more consistently, of CLL is a risk factor to develop the disease. The prevalence of blood diseases, and more specifically of CLL, in relatives of patients with CLL has been estimated at 13% and 7%, respectively. Familial CLL is defined as the presence of at least one first degree relative with CLL. Previous studies hypothesize the phenomenon of anticipation, which is the earlier onset and more aggressive course of a disease in the second generation of relatives, in familial CLL. Aims: The primary end point is to determine the prevalence of familial CLL in our court of patients with CLL and to confirm the phenomenon of anticipation. The secondary endpoints involve the comparison of the prevalence of clinical features and molecular biomarkers between familial and sporadic CLL; the comparison of the impact of clinical features and molecular biomarkers on patients’ clinical course in terms of time to treatment (TTT), time to next treatment (TTNT), progression-free survival (PFS) and overall survival (OS) between familial and sporadic CLL; the evaluation of response to treatment (ORR) in familial CLL compared to sporadic cases. Patients and methods: This is a retrospective monocentric study which consists of clinical and biological data collection from all patients with familial CLL. We enrolled 500 patients, which included 46 familial CLL and 454 sporadic CLL cases, diagnosed between January 1987 and April 2022. Patients’ characteristics were described by frequency tables for qualitative variables and position indicators for quantitative variables. The associations with clinical-biological parameters were analyzed using Chi-square or Fisher's exact test for the variables qualitative and Wilcoxon or Kruskal-Wallis test for quantitative variables. Survival was estimated with the Kaplan Meier method and compared by the log rank test. Results: The prevalence of familial CLL and familiarity for any other hemopathy in our population was 9.4% and 20% respectively. The median age at diagnosis was 65 years old in sporadic and 59 years old in familial CLL (p=0.018). Focusing on patients with familial CLL, our data confirmed the phenomenon of anticipation. In the first generation of patients with familial CLL (including father, mother, and older siblings), the median age at diagnosis was 69 years old (range 50-90), while in the second generation (including children and younger siblings) the median age at diagnosis was 57 years old (range 31-83). The mean difference between the two populations was of 12.6 years (17.5 years lower limit and 7.7 years upper limit), with a 95% confidence interval (p=0.00003). Concerning the IgHV mutational status, sporadic CLL showed mutated IgHV in 65% of the cases and unmutated IgHV in 35%, while familial CLL showed mutated IgHV in 35% of the cases and unmutated IgHV in 65% (p<0.001). No statistically significant difference was instead found for FISH nor for molecular biomarkers, including TP53, NOTHC1, BIRC3 and SF3B1 (Table 1). In our cohort, the time to treatment, time to next treatment and progression free survival for familial CLL appear to be shorter than for the control group, but those results are not statistically significant at the log rank test. Moreover, the overall response rate was significantly lower in patients with familial CLL: this data has never been reported by any study and it further confirms the rather aggressive course of the disease (Table 1). Familial CLL did not show any significant difference in overall survival compared to sporadic CLL. Conclusions: We confirmed the phenomenon of anticipation in our population. Moreover, we found higher rate of unmutated IgHV in familial CLL compared to sporadic controls, which would underlie a more aggressive course of the disease in this subset of patients. To define the molecular characteristics of familial CLL and to improve the significance of the survival analysis, we need to study a larger population of patients. Our monocentric experience is meant to be a pilot study which will be extended to other centers in Italy and opens the way to a better understanding of CLL clustering in families. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Rare Germline Variant in NFATC4 Associated with Familial Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) has one of the highest familial risks of any cancer, with up to eightfold increased risk in first degree relatives of CLL patients. Genome-wide association studies (GWAS) have identified multiple common single nucleotide polymorphisms (SNPs) associated with risk, but these have modest effect. Similarly, identified germline variants associated with disease account for just a small minority of CLL patients, making familial CLL an attractive target for research, that could provide direct insight into pathobiology.We used whole exome sequencing (WES; Illumina) to search for rare germline variants (<1% Minor Allele Frequency) in 36 patients from 17 families with Mendelian-like CLL inheritance, where at least two affected family members were sequenced. Median age at diagnosis was 56.4 (range 41-72), 61% were male, 10/23 (43%) were IGHV -unmutated, and 5/24 (21%) had del17p and/or TP53 mutation. We found 3819 rare variants shared within all affected members of a given family. Of those, 71 non-synonymous variants were shared across at least 2 families, and 39 of these were predicted to be pathogenic by at least two commonly-used phenotype prediction tools. None of these variants are within known somatic CLL driver genes, nor were any found in genes near known CLL GWAS SNPs. In order to prioritize these variants, we assessed whether they were enriched among CLL cases (n = 646) as compared to ethnically matched normal population controls (n=8,920), a dataset we have recently reported (Tiao, Improgo et al, Leukemia). Using Fisher's exact test with Benjamini-Hochberg (BH) correction for multiple tests, we found that 27 of the 71 shared variants across multiple families were also enriched in unrelated CLL patients compared to controls. Among those, a rare variant of nuclear factor of activated T-cells 4 (NFATC4), rs200328372, was the most highly enriched in CLL (OR 20.8, Fisher-q-BH 0.026). This variant affects a splice acceptor site predicted to have a deleterious effect on the protein (FATHMM for non-coding variants). In a pathway analysis (MSigDB, DAVID) of genes carrying shared variants across families, NFATC4 together with SOS1 and PTPN7 were associated with the T-cell receptor, B-cell receptor, and MAP-kinase pathways, suggesting a possible role in CLL pathogenesis.The NFATC4 protein, also known as NFAT3, is part of a DNA-binding transcriptional complex that translocates to the nucleus after activation through dephosphorylation by calcineurin. This complex is involved in the inducible expression of cytokine genes in T cells, including interleukin (IL)-2 and IL-4. While NFATC1 has been implicated in CLL pathogenesis and was associated with more aggressive disease in a mouse model of CLL, the role of NFATC4 has not been elucidated. To further investigate this as well as its association with familial CLL, we compared gene expression profiling (GEP; Affymetrix U133 plus 2.0 array) data between familial and sporadic CLL patients and normal B-cells. While NFATC4 expression did not differ between normal B-cells (n=16) and CLL (n=143), it was significantly down-regulated in familial (n=47) as compared to sporadic (n=96) CLL cases (Mann-Whitney, p=0.005). In the 4 cases that harbor the shared risk variant, GEP results showed additional significant down-regulation of NFATC4 expression (Kruskal-Wallis, p=0.003 for all comparisons) (Figure 1).RNA sequencing (Illumina) demonstrated NFATC4 down-regulation in all CLL samples (not specifically in familial CLL as with GEP; n=100) versus normal B-cells (n=6; Mann-Whitney, p=-0.00024), suggesting a putative role for NFATC4 as a tumor suppressor. Since NFATC4 has over 30 alternatively spliced transcripts potentially affected by the rs200328372 variant, we used Cufflinks software to look at isoform-level RNA sequencing data, available on two relatives who carried this variant, compared to CLL cases and controls. Both relatives had reduced expression of all isoforms (Figure 2), suggesting that this mutation may reduce NFATC4 expression altogether. Studies of protein expression are ongoing.In conclusion, through rare germline variant analysis in familial CLL, we found a potentially deleterious variant of NFATC4 that is enriched in CLL. Subsequently, we used RNA sequencing data to show, for the first time, that NFATC4 expression is decreased in CLL versus normal B-cells, revealing a potential tumor suppressor mechanism in CLL. [Display omitted] DisclosuresBrown:Sun BioPharma: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Redx: Consultancy; AstraZeneca: Consultancy; Pfizer: Consultancy; Janssen Oncology: Honoraria; AbbVie: Consultancy, Honoraria; Roche/Genentech: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Infinity Pharmaceuticals: Consultancy; Astellas Pharma: Consultancy.

Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL

In this study, we wished to determine whether familial chronic lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH) gene usage and occurrence of somatic mutation, to gain insight into the pathogenetic relatedness of these epidemiologically distinct forms of CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with data previously reported for sporadic CLL. In addition, we assessed the relationship of the occurrence of somatic mutation to several clinical and phenotypic features. The distribution of V genes among these cases was similar to that observed in sporadic CLL: VH3 > VH1 > VH4. Thirteen of the 23 cases (57%) showed germ line VH gene sequences, whereas somatic mutations were detected in 10 cases (43%). The average mutation frequency of these latter 10 cases of was 6.7% (ranging from 1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance of a second clone over a 20-year period was observed in 1 case, suggesting that mutations can continue to accumulate after neoplastic transformation. The presence of somatic mutations correlated with age at presentation, low white blood cell (WBC) count, and low fluorescence intensity of surface CD5, and the potential significance of these relationships is discussed. Our data indicate that familial and sporadic B-CLL display a similar pattern of immunoglobulin gene usage and frequency of somatic mutation, and are consistent with a common ontogeny and immunogenetic origin for these 2 epidemiologically distinct forms of CLL. (Blood. 2000;95:1413-1419)

Germline POT1 Variants Can Predispose to a Variety of Hematologic Neoplasms

Germline mutations in the shelterin component protection of telomeres 1 (POT1) were recently found to be associated with familial chronic lymphocytic leukemia (CLL), melanoma, glioma, and several other familial cancer syndromes. The role of POT1 mutations in myeloid neoplasms and other hematologic malignancies, however, remains unknown. To explore the role of POT1 variants in hematologic neoplasms, we analyzed POT1 variants in 3323 consecutive patients who underwent next-generation sequencing (NGS) of a panel of hematologic malignancy-associated genes at our institution and characterized the clinical and pathological characteristics of patients with germline and somatic POT1 mutations. Of 3323 consecutive patients who underwent NGS, 2770 patients had a hematologic malignancy (lymphoid n = 1299, myeloid n = 934, and both lymphoid and myeloid n = 537), while 553 patients were evaluated for non-malignant cytopenias. All 57 patients (2.06%) carrying either a POT1 disease-associated variant or variant of uncertain significance had a hematologic malignancy compared to no identified POT1 variants in 553 patients with benign cytopenias (OR = 23.5, p &amp;lt; 0.001), suggesting that the presence of POT1 variants was predictive of a hematologic malignancy. Of 57 patients, 33 had lymphoid malignancies, 23 had myeloid neoplasms, and 2 had a lymphoid and myeloid neoplasm (Fig 1). Patient variants were classified as germline or somatic using constitutional DNA sequencing, POT1 emergence/disappearance over time, or POT1 maintenance in remission. In the absence of these data, likely germline or likely somatic designations were made by assessing variant allele frequencies against clinical/pathologic characteristics. 18 patients (33%) were found to have germline or likely germline POT1 variants (29% and 42% in the lymphoid and myeloid malignancy groups, respectively). Another 6 patients (11%) had variants whose germline status could not be determined. Of the 17 unique germline POT1 variants, 10 were missense and located within mapped functional protein domains, while 7 were classified as predicted loss-of-function (pLOF) due to a disruption of start, premature stop, frameshift, or spice site alteration. Patients with hematological malignancies had a ~5-8x increased odds of having a germline pLOF POT1 variant compared to cancer-free individuals in the Genome Aggregation Database (gnomAD, n = 113,108 exomes, OR = 7.5, p &amp;lt; 0.001) or in the Penn Medicine BioBank (PMBB, n = 7877, OR = 5.0, p = 0.010), with a prevalence of 0.25% compared to 0.03% and 0.05%, respectively. Germline pLOF POT1 variants were significantly more enriched in patients with myeloid (gnomAD: OR = 6.1, p = 0.02) and lymphoid (gnomAD: OR = 9.8, p &amp;lt; 0.001; PMBB: OR = 6.5, p = 0.004) malignancies. In 33 patients with lymphoid malignancies and POT1 variants, the most common diagnoses were CLL/SLL (n = 21, germline n = 6, somatic n = 12), CD5- CD10- indolent B cell neoplasms (n = 4, germline n = 1, somatic n = 3), and multiple myeloma (n = 3, all somatic) (Table 1). Lymphoid malignancies with a germline POT1 variant had a relative paucity of additional mutations; in contrast, somatic POT1 variants frequently co-occurred with other mutations, most commonly with TP53 (Fig 2, n = 5, 23%). Among 23 patients with myeloid malignancies, patients with germline POT1 variants developed malignancies at a significantly younger age compared to those whose POT1 variants were somatic (median age 59.5 vs 70.5 years, p = 0.04). The most common diagnosis in patients with myeloid neoplasms carrying germline POT1 variants was MPN (germline n = 5, somatic n = 1). AML, MDS/MPN, and MDS occurred in 4, 3, and 1 patients respectively. All patients with myeloid neoplasms had additional disease-associated mutations, with the most common co-occurring variants in TET2 (n = 7), JAK2 (n = 6, co-occurring with 50% of germline POT1 myeloid variants), and NRAS (n = 6). In conclusion, this is the first comprehensive analysis of POT1 variants in an unselected hospital-based population undergoing molecular testing for variants associated with hematologic malignancies. Our results show that the presence of POT1 variants is predictive of having a hematologic neoplasm and that over 30% of POT1 variants in hematologic malignancy patients are germline. Our study expands the spectrum of POT1-associated familial neoplasms and highlights the needs for better recognition of familial hematologic cancer syndromes. Disclosures No relevant conflicts of interest to declare.

Association of elevated serumfree light chains with chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis

Chronic lymphocytic leukemia (CLL) and its precursor, monoclonal B-cell lymphocytosis (MBL), are heritable. Serumfree light-chain (sFLC) measures are a prognostic factor for CLL, but their role in susceptibility to CLL is not clear. We investigated differences between sFLC measurements in pre-treatment serum from five groups to inform the association of sFLC with familial and sporadic CLL: (1) familial CLL (n = 154), (2) sporadic CLL (n = 302), (3) familial MBL (n = 87), (4) unaffected first-degree relatives from CLL/MBL families (n = 263), and (5) reference population (n = 15,396). The percent of individuals having elevated monoclonal and polyclonal sFLCs was compared using age-stratified and age- and sex-adjusted logistic regression models. In age groups >50 years, monoclonal sFLC elevations were increased in sporadic and familial CLL cases compared to the reference population (p’s < 0.05). However, there were no statistically significant differences in sFLC monoclonal or polyclonal elevations between familial and sporadic CLL cases (p’s > 0.05). Unaffected relatives and MBL cases from CLL/MBL families, ages >60 years, showed elevated monoclonal sFLC, compared to the reference population (p’s < 0.05). This is the first study to demonstrate monoclonal sFLC elevations in CLL cases compared to controls. Monoclonal sFLC levels may provide additional risk information in relatives of CLL probands.

Abstract 1226: Rare germline variants segregating in chronic lymphocytic leukemia (CLL) families

Abstract CLL is a highly heritable cancer with first degree relatives of CLL cases having a 7.5-fold increased CLL risk. Genome-wide association studies (GWAS) and linkage studies have been performed to study inherited predisposition; however a larger proportion of heritability to CLL remains unexplained. Rare coding variants might account for the missing heritability information. Inherited loss of function variants in shelterin complex genes (POT1, ACD, TERF1, TINF2, TERF2, TERF2IP- involved in telomere regulation), CDK1 (critical for cell division) and ATM (tumor suppressor gene) have been found to co-segregate in CLL families and be enriched in CLL cases using exome-wide sequencing data. Our study evaluates rare germline variants from these suspect genes segregating in CLL families who are followed at the Mayo Clinic. Using whole exome sequencing (WES), we sequenced 93 CLL families with at least 2 reported CLL cases consisting of 443 individuals: 160 with CLL, 73 with monoclonal B-cell lymphocytosis (MBL), and 210 relatives. DNA was extracted from buccal cells, coding exons were selectively captured using Agilent 50Mb and SureSelect Human All Exon V4 capture kits; sequencing was performed using Illumina HiSeq 2000. Mayo Clinic's DNASeq pipeline uses Novoalign (initial read alignment), Picard (marking duplicate reads), and the Genome Analysis Toolkit (GATK) for local realignment, recalibration, and variant calling. The variant discovery step leverages GATK's HaplotypeCaller in per sample mode and all of the samples across the cohort are jointly genotyped together. All called variants are evaluated with GATK's Variant Quality Score Recalibration tool and annotated for biological relevance (BIOR). Quality control included removing variants that had &amp;lt;75% call rate across the two capture kits, &amp;lt;8x coverage, or phred score&amp;lt;10, resulting in 317,666 remaining variants. Of these, over 80% of the coding sequence had a median read depth of 23 reads. In our pedigrees, we searched for rare variants within the genes described above. We identified suspect variants with the following criteria: 1) enriched in CLL and MBL samples compared to unaffected samples; 2) multiple affected members with the variant within a family; 3) variants present in all sequenced affecteds within the family; 4) rarely seen in an in-house database of non-cancer controls or 1K Genomes; and 5) predicted to have a functional damaging effect (using SIFT). We identified three novel rare missense variants, defined as functionally deleterious, which each co-segregated within a CLL family. Specifically, these variants from shelterin complex genes; POT1 (rs116916706), TERF2IP (rs138458227), and TERF2 (rs749171225), met the criteria. This study further highlights telomere dysregulation as a key process in CLL development. Investigating rare variants within CLL pedigrees with WES can help identify germline variants impacting predisposition to familial CLL. Citation Format: Alyssa I. Clay-Gilmour, Daniel R. O'Brien, Sara J. Achenbach, Celine M. Vachon, Kari G. Chaffee, Timothy G. Call, Jose F. Leis, Aaron D. Norman, Brian F. Kabat, Sameer A. Parikh, Neil E. Kay, Esteban Braggio, James R. Cerhan, Susan L. Slager. Rare germline variants segregating in chronic lymphocytic leukemia (CLL) families [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 1226.

Combined somatic mutation and copy number analysis in the survival of familial CLL.

Recurrent large-scale somatic copy number alterations (SCNAs), and somatic point mutations can be analysed to stratify patients with chronic lymphocytic leukaemia (CLL) into distinct prognostic groups. To investigate the relationship between SCNAs and somatic mutations, we performed whole-exome sequencing and single nucleotide polymorphism microarray analyses on 98 CLL patients from 40 families with a high burden of CLL. Overall, 69 somatic mutations in 29 CLL driver genes were detected among 45 subjects (46%), with the most frequently mutated genes being TP53 (8·2%), NOTCH1 (8·2%) and ATM (5·1%). Additionally, 142 SCNAs from 54 subjects (57%) were detected, including losses of chromosome 13q14 (28·9%), 11q (5·6%), 17p (2·1%), and gain of chromosome 12 (4·2%). We found that patients having both an adverse point mutation in a CLL driver gene and an unfavourable SCNA tended to have poorer survival (Hazard ratio [HR]=3·17, 95% confidence interval [CI]=0·97-10·35; P=0·056) than patients having either a point mutation (HR=1·34, 95%CI=0·66-2·71; P=0·42) or SCNAs (HR=2·65, 95%CI=0·77-9·13; P=0·12). TP53 mutation carriers were associated with the poorest overall survival (HR=4·39, 95%CI=1·28-15·04; P=0·018). Our study suggests that combining SCNA and mutational data could contribute to predicting outcome in familial CLL.

Cytogenetic/mutation profile of chronic lymphocytic leukemia/malignant melanoma collision tumors of the skin

BackgroundCollision tumors are rare entities that consist of two histologically distinct tumor types arising in the same anatomic site. An association between chronic lymphocytic leukemia (CLL) and malignant melanoma (MM) has been already described. Up to now, they have been documented only at positive regional lymph nodes while we focused on collision tumor in a skin lesion.Case presentationWe characterized the genomic profile of a skin CLL/MM collision tumor in a patient with a 9-years story of CLL. Typical high-grade genomic biomarkers featured the CLL: the immunoglobulin heavy variable genes were unmutated; a clonal del(11q), involving ATM and BIRC3, was present in the peripheral blood (PB) and skin lesion, while a subclonal large del(13q)/D13S319-RB1 was detected only in the PB. Interestingly, the del(13q) clone, increased from 10% to 46% from diagnosis to relapse. NOTCH1, SF3B1, and TP53 were wild type. The MM lesion carried a BRAFV600E and a TERT promoter mutation.As the family story was consistent with a genetic predisposition to cancer, we performed mutational analysis of genes involved in familial melanoma and CLL, and of BRCA1 and BRCA2. No germinal mutation known to predispose to CLL, MM, or breast cancer was found. Interestingly, conventional cytogenetic detected a constitutional t(12;17)(p13;p13).ConclusionsOur data are consistent with distinct genetic landscape of the two tumors which were characterized by specific disease-related abnormalities. CLL cells carried poor prognostic imbalances, i.e. large deletions of the long arm of chromosomes 11 and 13, while in MM cells two functionally linked mutations, i.e. BRAFV600E and a TERT promoter occurred. Although, known germline variations predisposing to MM and/or CLL were ruled out, genetic counseling suggested the proband family was at high risk for MM.

The wide spectrum of POT1 gene variants correlates with multiple cancer types.

The POT1 protein binds and protects telomeres. Germline variants in the POT1 gene have recently been shown to be associated with risk of developing tumors in different tissues such as familial chronic lymphocytic leukemia, colorectal, glioma and melanoma tumors. Recently, we uncovered a variant in the POT1 gene (p.R117C) as causative of familial cardiac angiosarcomas (CAS) in Li-Fraumeni-like (LFL) syndrome families. Our in silico studies predicted that this protein had lost the ability to interact with TPP1 and single-stranded DNA. In vitro studies corroborated this prediction and showed that this lack of function leads to abnormally long telomeres. To better understand the POT1 gene and its role with tumorigenesis, we extended the study to LFL (with and without members affected with angiosarcomas (AS)) and sporadic AS and cardiac sarcomas. We found POT1 variants in the 20% of the families with members affected with AS and 10% of sporadic AS and sarcomas. In silico studies predicted that these new variants were damaging in the same manner as previously described for the POT1 p.R117C variants. The wide spectrum of variants in the POT1 gene leading to tumorigenesis in different tissues demonstrates its general importance. Study of the POT1 gene should be considered as routine diagnostic in these cancers.

Monoclonal B-cell lymphocytosis in blood donors in Turkey

ABSTRACTObjectives: Monoclonal B-cell lymphocytosis (MBL) is a precursor state of chronic lymphocytic leukemia (CLL) with peripheral lymphocytosis below 5 × 109/l. The diagnostic criteria exclude the presence of lymphadenopathy, organomegaly, infections, autoimmune diseases or any sign of a lymphoproliferative disorder. This prospective study was designed in order to evaluate the frequency of MBL in blood donors in Turkey.Methods: The diagnosis of MBL was identified by flow cytometry method based on the International Familial CLL Consortium Report. A total of 999 volunteers [median age 34 (18–78) years; male/female: 705/294] were included in the study.Results: Monoclonal B-cell lymphocytosis was demonstrated in 18 cases (1.8%). A total of 16 cases (1.6%) was evaluated as CLL-like MBL, while 2 (0.2%) had a non-CLL-like phenotype. The subjects were divided into three groups according to age, as <40 years, 40–60 years and >60 years. The prevalence of MBL was 1.1% below 40 years, 0.6% between 40 and 60 years and 0.1% in cases over 60 years, without statistical significance (p > 0.05).Discussion: The sensitivity of the flow cytometry method is essential and may be responsible for the variations in the prevalence of MBL in different populations which can also be attributed to study design, higher detection rates in the elderly and families with genetic predisposition to CLL.Conclusion: Large population-based studies and standardized laboratory methods are needed to determine the potential risk factors of progression to CLL, including molecular markers and genetic profile.

Familial chronic lymphocytic leukemia in Israel: A disproportionate distribution among Ashkenazi Jews.

Relatives of patients with chronic lymphocytic leukemia (CLL) are at increased risk of developing CLL. Familial CLL is defined as more than one case of CLL among blood relatives, a phenomenon reported in approximately 5%-10% of all CLL patients. Given the known predisposition of CLL among Ashkenazi Jews, we studied the features of familial CLL in an Israeli population. This is a retrospective study, in which we reviewed the demographics, clinical characteristics, and outcomes of a total of 332 patients with CLL/small lymphocytic lymphoma. Familial CLL was recorded in 41 cases (12.3%) of the patients. The age at diagnosis was younger in patients with familial CLL (by almost 3.5years). Familial CLL was strongly associated with Ashkenazi Jewish origin. Patients with familial CLL more commonly presented with higher hemoglobin and lower serum β-2-microglobulin levels. No significant differences were detected between sporadic and familial CLL in disease stage, time to treatment, second cancers, or overall survival. Familial cases of CLL in an Israeli population show a disproportionate ethnic distribution toward Jews of Ashkenazi origin. The clinical characteristics and the overall outcome are not substantially different from sporadic cases.

Germ line mutations in shelterin complex genes are associated with familial chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) can be familial; however, thus far no rare germ line disruptive alleles for CLL have been identified. We performed whole-exome sequencing of 66 CLL families, identifying 4 families where loss-of-function mutations in protection of telomeres 1 (POT1) co-segregated with CLL. The p.Tyr36Cys mutation is predicted to disrupt the interaction between POT1 and the telomeric overhang. The c.1164-1G>A splice-site, p.Gln358SerfsTer13 frameshift, and p.Gln376Arg missense mutations are likely to impact the interaction between POT1 and adrenocortical dysplasia homolog (ACD), which is a part of the telomere-capping shelterin complex. We also identified mutations in ACD (c.752-2A>C) and another shelterin component, telomeric repeat binding factor 2, interacting protein (p.Ala104Pro and p.Arg133Gln), in 3 CLL families. In a complementary analysis of 1083 cases and 5854 controls, the POT1 p.Gln376Arg variant, which has a global minor allele frequency of 0.0005, conferred a 3.61-fold increased risk of CLL (P = .009). This study further highlights telomere dysregulation as a key process in CLL development.

Disruption of the SUMO Pathway in a High-Risk B-Cell Non-Hodgkin Lymphoma Pedigree

Taken together, B-cell lymphoproliferative disorders (lymphoma, multiple myeloma and leukemia) are the fourth most common form of cancer. Chronic Lymphocytic Leukemia (CLL), a specific sub-type of B-cell non-Hodgkin lymphoma (NHL), is the most common adult leukemia in Western countries. Despite its prevalence, the underlying genetic mechanisms responsible for CLL remain largely unknown. The strong familial clustering seen in CLL suggests that genetic variants contributing to its pathogenesis may be inherited. Our lab is dedicated to identifying inherited genetic risk variants associated with hematological malignancies with a specific focus on CLL. We hypothesize that there are multiple germline genetic variants (both common and rare) involved in risk of CLL. In order to identify rare variants, our lab uses the Utah Population Database (UPDB) to identify extended high-risk pedigrees with statistical excess of familial CLL. We then use next generation sequencing to identify genetic variants segregating in these high-risk pedigrees. This powerful study design for identifying rare variants and has previously been proven successful for identifying variants associated with other cancers.We have performed whole-exome sequencing in one particularly high-risk pedigree containing 3 CLL cases and a mantle cell lymphoma case within 2 generations. We have identified variants in multiple genes associated with the SUMO pathway. We find rare variants in RANBP2, SP100, PML, IL1RL2 (paralog of IL1R) and CREBRF (transcriptional regulation through RNA pol II) that are shared by all NHL cases in this pedigree. Other genes in this pathway have been identified by previous CLL genome wide association studies, specifically FAS, TNF and SUMO1. Furthermore, a strong role has been previously suggested for the SUMO pathway in cancer cell survival and tumor progression, lending support to the potential role of this pathway in NHL risk. We are currently validating our sequence findings and determining the prevalence of these variants in other NHL cases included in the UPDB. Our high-risk pedigree findings are supportive that disruption of the SUMO pathway contributes to pathogenesis of NHL. Identification of the specific variants involved will increase our understanding of the molecular mechanisms contributing to NHL and has the potential to provide genetic markers that can be used for diagnosis and new avenues for treatment of these diseases. DisclosuresNo relevant conflicts of interest to declare.

Familial leukemias.

Familial leukemia has been described for more than 50 years but only recently have modern genetic techniques allowed for the investigation of the genome. Genome-wide association studies have identified a number of genetic sites that appear to relate to susceptibility to leukemia in certain families and occasionally to susceptibility to a specific leukemia in general. Many questions remain, including susceptibility to what? An oncogenic virus? An environmental chemical? Mutation of another gene induced by a heritable mutation-promoting gene?.Clinically important facts have been learned. Chronic lymphocytic leukemia (CLL) is by far the most common familial leukemia. Patients with CLL have approximately a 10% chance of a first-degree relative developing CLL, and even a greater chance of one developing monoclonal B-cell lymphocytosis which may be an asymptomatic forme fruste of the neoplasm. Furthermore, there may be an increased incidence of breast cancer in familial CLL pedigrees which raises the question of a common etiology for neoplasms in general, or at least a previously unrecognized relationship among them.

Chronic Lymphocytic Leukemia-Associated Chromosomal Abnormalities and Risk of Secondary Malignancies

Introduction: Incidence of secondary malignant neoplasm in patients with chronic lymphocytic leukemia (CLL) is already established. Genetic predisposition has been considered to be a possible contributory factor for the preponderance of second cancers in this population. In the present study, we examined the frequency, characteristics, and clinical outcomes of secondary malignancies in patients with CLL based on their Interphase fluorescence in situ hybridization (FISH) panel.Methods: We reviewed the medical records of consecutive patients with CLL observed or treated at Ellis Fischel Cancer Center during the period from 2007-2014. We collected demographic data, CLL Rai stage, treatment history, Interphase fluorescence in situ hybridization (FISH) panel results and the presence of a secondary malignancy (skin cancers, solid tumors and hematologic malignancy excluding CLL transformation).Our aim was to investigate the association between secondary malignancy and chromosomal abnormalities and other risk factors using Chi2test for categorical variables and Wilcoxon rank-sum test for continuous variables. Cox proportional hazard models and logistic regression models were used to evaluate risk factors of developing secondary malignancy and overall survival.Results: We identified 142 CLL patients who were either observed (n=62) or had a history of treatment (n=80) for CLL. 67% were male and 33% were female. 85% of patients were Caucasians, 10% African Americans, and 5% other. Familial CLL was present in 6% of cases. 51% of patients were non-smokers. 5% of patients had cancers preceding the diagnosis of CLL (non-melanoma skin cancers=3, melanoma=1, Hodgkin’s lymphoma=1, Prostate=2). 28% of patients developed secondary malignancies after the diagnosis of CLL; among them non-melanoma skin cancers were the most common (78%), followed by cutaneous melanoma (9.5%) and papillary thyroid cancer (4.7%). Other malignancies included lung (2.3%), kidney (2.3%), prostate (2.3%) and ocular melanoma (2.3%). 12% patients had aggressive non-melanoma recurrent skin cancers that require multiple treatments along-with systemic therapy for progressive CLL.CLL-FISH panel at diagnosis was available in 98 patients. Among these patients,13q deletion was present in 45%. Within the 13q deletion group, secondary malignancy was noted in 50% during a median follow up of 7 years. All cases of papillary thyroid cancers were also present in 13q deletion. In contrast, 11q deletion was detected in 15% and trisomy 12 in 18% of patients with incidence of secondary malignancy 40% and 10%, respectively. No solid malignancy other than skin was identified in the 11q deletion and trisomy 12 groups. 17p deletion was present in 5% of cases and 60% of 17p deletion cases had secondary cancers (skin 80%). Normal FISH panel was present in 17% cases and 20% of normal FISH had secondary malignancy (solid 10%, skin 10%).The median overall survival time is 6 years for the entire cohort (IRQ: 3-9 years). Secondary malignancy was associated with worse overall survival (OS) (HR=0.57, 95% CI:0.33-0.98, p=0.04) compare to patients who did not have secondary malignancy. The risk of secondary malignancy is increased in patients with advanced age (OR=1.05, 95% CI:1.00-1.10, p=0.04) and in those who were treated with alkylating agent for CLL OR=6.62, 95% CI:2.08-21.03, p=0.001. However, there were no significant difference on risk of developing secondary malignancy based on specific chromosomal FISH result, Rai stage, smoking, family history and gender.Conclusion: Secondary malignancies are frequent in patients with detectable chromosome abnormality on FISH panel. However, the increased risk of secondary malignancy does not correlate with specific cytogenetic abnormality. Non melanoma skin cancers are exceedingly common in CLL patients and carries aggressive couse in progressive CLL patients. Larger studies are required to identify subtype of CLL based on integrated mutational and cytogenetic subgroup that are at increased risk of specific secondary malignancies. DisclosuresNo relevant conflicts of interest to declare.

Next-Generation IgVH Sequencing of Monoclonal B-Cell Lymphocytosis Reveals Clonal Heterogeneity, Ongoing Mutation, and Repertoire Differences Relative to Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) usually develops from asymptomatic monoclonal expansions of CD5 positive B-cells termed monoclonal B-cell lymphocytosis (MBL), present in the peripheral blood (PB) of approximately 5% of otherwise healthy older individuals. Although MBL only occasionally progresses to CLL, cases that do progress typically have higher MBL cell counts in the 1500-4000/µL range. Although antigen selection appears to play a central role in the development CLL, it is unclear whether this occurs at an early MBL stage or primarily during the progression of MBL to CLL. One prior study has reported clonal heterogeneity in MBL finding it in 4 of 6 low count MBL cases from familial CLL kindreds using a single cell PCR technique (Leukemia 2010,24:133-140). In this study, we assessed the VH repertoire and degree of clonal heterogeneity in sporadic MBL cases using next-generation sequencing (NGS) of the rearranged immunoglobulin heavy chain (IgH) locus.Methods: The 35 cases selected for sequencing represented residual, cryopreserved material from PB specimens submitted to ARUP for clinical phenotyping studies. All contained polytypic CD5 negative B-cells in addition to MBL/CLL phenotype cells, and had 2 or more vials for analysis. The majority (80%) had counts of MBL cells below 1000/µL (mean 294/, range 795-30 cells/µL). FACS purification of MBL cells (CD20+CD5+) and CD5 negative B-cells was performed on all samples. The IgH repertoire from the unsorted and two sorted populations was determined by NGS using the LymphoSIGHT method.Results: Five cases could not be analyzed due to insufficient numbers of MBL cells. Clonal VDJ rearrangements or clonotypes were identified in the remaining 30 based on their high frequency within the B-cell repertoire of the unsorted sample, and having a higher frequency in the sorted MBL cells relative to the sorted CD5 negative B-cells. Functional clonotypes were identified in 29 of these 30 cases. Interestingly, 5 cases had 2 functional unrelated clonotypes using different D and/or J segments that also employed different V segments. Of the 5 cases with 2 unrelated clonotypes, 3 had MBL cell counts below 1000/µL (32, 275, and 865) and 2 above (1640, 2600). Moreover, 1 of the clones in the case with 865 cells/µL represented only 25% of the MBL cells or 220 cells/µL, while 1 clone in the case with 2600 MBL cells/µL represented 18% of the MBL cells or 470 cells/µL. By flow cytometry, the CD5+ CD20+ cells in 2 of the cases with 2 functional clonotypes showed polytypic kappa/lambda expression (ratios near 1), 2 cases had uniform dim monotypic kappa expression, and 1 case showed 90% dim kappa and 10% dim lambda expression. The most frequently used VH segments were V4-34 in 6/34 or 18% of functional clonotypes, followed by V3-23 (11%), and V3-21 (9%). The V1-69 segment was used by only 1/34 (3%) functional clonotypes. The VH segments in 72% of cases with functional clonotypes were mutated (homology to germline < 98%), with 6 cases showing clear evidence of ongoing mutation by having 2 or more related clones.Conclusions: We demonstrate that MBL exhibits considerable clonal heterogeneity, with 2 distinct unrelated clones identified in 17% of 30 analyzed cases. Finding 2 distinct clones cannot be explained by a lack of allelic exclusion or the presence of 1 cell with 2 productive IgH rearrangements since each clone had different frequencies within the sorted MBL cell repertoire. This is further supported by finding the ratios of the two MBL clones in 2 cases being different in the unsorted compared to the MBL sorted cells. Clonal heterogeneity appears to occur at an early stage since the majority of clones (6/10) had cell counts below 500 cells/µL. We also found that clonal heterogeneity of MBL may not be detectable by flow cytometry or may appear as polytypic CD5+CD20+ B-cells. To our knowledge, this represents the first report of clonal heterogeneity in sporadic MBL. Our identification of infrequent use of V1-69 (1/34) supports prior studies indicating the VH repertoire of MBL is different than CLL which frequently employs V1-69. Finding evidence of ongoing VH mutation suggests antigen selection may occur in early MBL. Overall, our findings are consistent with recent observations (Cancer Cell 2011, 20;246-259) suggesting that hematopoietic stem cells from CLL patients can generate mono-or oligoclonal MBL phenotype cells that can then be selected through antigen binding for expansion. DisclosuresFaham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Heritable Predisposition To Richter Syndrome In Patients With Chronic Lymphocytic Leukemia

BackgroundApproximately 2-8% patients with chronic lymphocytic leukemia (CLL) will transform to diffuse large B-cell lymphoma (DLBCL, Richter syndrome [RS]). Clinical characteristics and molecular markers at the time of CLL diagnosis are associated with the risk of RS; however, there are no data regarding germline genetic variations and the risk of RS. Genomewide association studies (GWAS) have shown several single-nucleotide polymorphisms (SNPs) that are associated with a higher risk of familial CLL. It is not known whether any of these polymorphisms also predispose to RS. MethodsSince 2002, all consecutive patients with newly diagnosed (<9 months diagnosis) CLL at Mayo Clinic were offered enrollment into a prospective genetic epidemiology study. Patients completed extensive epidemiologic questionnaires and baseline clinical, laboratory, and biologic data were abstracted using a standard protocol. Genotyping of germline tissue at diagnosis was performed using an Illumina iSelect panel and Affymetrix 6.0 SNP chip. All patients were prospectively and longitudinally followed at defined time-points with systematic collection of data on treatments, second cancers, and RS. All patients with biopsy-proven DLBCL during follow-up were considered to have undergone transformation into RS. Time to RS was calculated from CLL diagnosis date until RS or until last follow-up date for those with no RS. SNPs were modeled in two ways: ordinal and dominant. Cox regression was used to estimate hazard ratio (HR) for individual SNPs with time to transformation. ResultsThirteen of the GWAS-discovered SNPs associated with risk of developing CLL were available and genotyped on 620 CLL patients. Median age at diagnosis of CLL was 62 years (range 27-88), and 428 (69%) were male. Three hundred and ten (51%) patients were low (0) Rai stage, 271 (45%) were intermediate (I-II) Rai stage, and 22 (4%) were advanced (III-IV) Rai stage. The immunoglobulin heavy chain gene was unmutated in 189 (40%) patients; 157 (32%) patients expressed ZAP-70, 163 (29%) expressed CD38 and 104 (31%) expressed CD49d. Fluorescence in-situ hybridization (FISH) revealed that 210 (41%) patients had del13q, 90 (18%) patients had trisomy 12, 37 (7%) had del11q, 23 (5%) had del17p and 141 (28%) had no detectable FISH abnormalities. As of last follow-up, 239 (39%) patients received therapy for CLL.After a median follow-up of 5.9 years (range 0-11), 15 (2.4%) patients developed biopsy-proven RS. The median time to RS in these 15 patients was 4.5 years (range 1.0-8.7 years). The ordinal HR for the 13 SNPs tested, their corresponding genes, and p-values are shown in Table 1. Germline polymorphisms in a single SNP, rs4987852, encoding for BCL2 (chromosome 18), was significantly associated with an increased risk of RS (ordinal HR=3.9; 95% CI=1.6-9.8; p-value=0.004). This allele was present in 48/605 (8%) non-transformed CLL patients compared to 4/15 (27%) of patients with RS. Time to RS according to the Kaplan-Meier analysis for rs4987852 is shown in Figure 1. This SNP is located in a region in which t(14;18) translocation breakpoints commonly occur in follicular lymphoma and overexpression of BCL2 leads to an increased incidence of B-cell lymphomas in mice.Table 1Reference SNP Cluster IDChromosomeChromosome regionGeneOrdinal Hazard Ratiop-valuers1339798522q13SP140.SP1100.790.59rs1748346622q13ACOXL.BCL2L111.520.29rs376982522q33CASP10.CASP80.590.16rs757819922q37HDLBP1.730.16rs75797822q37FARP20.710.58rs937880566p25IRF40.920.83rs79440041111p15C11orf21.TSPAN321.080.82rs7356651111q24GRAMD1B.SCN3B1.150.74rs71694311515q21RFXDC21.450.42rs22929821616q24IRF80.720.47rs43682531818q21PMAIP11.250.58rs49878521818q21BCL23.900.004rs48023221919q13PRKD2.STRN41.60.21 [Display omitted] ConclusionOur results suggest that inherited genetic polymorphisms predispose CLL patients to develop RS. Specifically, SNP (rs4987852) present on the BCL2 gene on chromosome 18 in CLL is associated with an increased risk of transformation to RS. These observations require replication in other CLL cohorts. Disclosures:Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.