Weinberg, J. Brice

Abstract High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Abstract Chronic lymphocytic leukemia (CLL) is a neoplastic disease of mature B-cells with a highly heterogeneous clinical course. While European ancestry (EA) populations present an increased incidence of CLL, African ancestry (AA) populations have a younger median age of onset, higher frequency of adverse prognostic factors, and inferior clinical outcomes. Despite the considerable effort to characterize the genetic landscape of CLL, AA are overwhelmingly underrepresented. Our hypothesis is that the clinical differences observed between AA and EA populations are, in part, explained by underlying genetic features. To address this imbalance, we identified 90 AA patients diagnosed with CLL, 64% of which were untreated at sample collection. RNA and DNA were extracted from CD5+/CD19+ clonal B-cells. We performed mRNA-seq and targeted sequencing in 59 recurrently mutated somatic CLL driver genes. Differentially expressed genes were identified using edgeR. Data was compared to our previously analyzed EA CLL cohort (N=445). The median age at diagnosis was 59 years for AA and 66 for EA and 74% of AA and 50% of EA had unmutated IGHV (u-IGHV) status. When evaluating the entire AA and EA cohorts, there was a significant increased frequency of mutations in TP53, SF3B1, and NFKBIE, identified in 29%, 24%, and 20% of AA CLLs, compared to 5%, 9%, and 9%, respectively, in EA CLLs (p<0.01). When exclusively evaluating the untreated and u-IGHV cases, AA CLLs showed greater proportion of TP53 (30% vs 12%; p=0.0145), NFKBIE (30% vs 15%; p=0.0492), BIRC3 (21% vs 10%; p=0.0697), and KRAS (15% vs 7%; p=0.1683). Furthermore, there was an increase in mutations targeting relevant molecular pathways, such as NF-κB (42% vs 15%) and MAPK (18% vs 8%). Upregulation MAPK pathway was also confirmed by mRNA-seq analysis in the AA u-IGHV CLLs. Because of the high prevalence of TP53 mutations in the AA cohort, we further evaluated differential gene expression in the DNA Damage/Telomere Stress-Induced Senescence pathway. AA CLLs presented a significant downregulation of multiple genes associated with genome stability and cellular DNA damage response - DDR (including TP53 and ATM), double strand break repair (H2AFX and RAD50), telomere maintenance (POT1 and ACD), and cell cycle regulation (RB1, CCNA1, and CCNE2) (FDR<0.05). DDR is responsible for DNA repair or induction of apoptosis, with its deficiency resulting in the accumulation of chromosomal aberrations, negatively impacting clinical outcome in CLL. Disparities in cancer are influenced by numerous factors that affect disease risk, screening and diagnosis, access to treatment, and survival. We identified an increased number of genomic alterations in the AA CLL cohort, primarily inducing activation of NF-κB and MAPK pathways and DDR impairment, with the increased frequency of mutations, notably in TP53 and BIRC3, expected to negatively impact prognosis. Citation Format: Cecilia Bonolo de Campos, Daniel R. O'Brien, Chantal E. McCabe, Huihuang Yan, Geffen Kleinstern, Zhiquan Wang, Laura A. Bruins, Cristine Allmer, Nicholas J. Boddicker, Charla R. Secreto, Aaron D. Norman, Shulan Tian, Kari G. Rabe, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Richard Furman, J Brice Weinberg, James R. Cerhan, Celine M. Vachon, Neil E. Kay, Susan L. Slager, Esteban Braggio. Characterization of underlying genomic features among African ancestry populations diagnosed with chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2209.

Background MBL is a precursor to chronic lymphocytic leukemia (CLL) and is subclassified into low-count (LC) MBL (absolute B-cell count<0.5x109/L) and high-count (HC) MBL (absolute B-cell count between 0.5 and 5x109/L). We previously reported that a polygenic risk score (PRS) based on a weighted average of 41 CLL-susceptibility variants was associated with risk of both MBL and CLL among a cohort of individuals from CLL families. Here we evaluate this PRS in an independent cohort of MBL and CLL individuals of European ancestry (EA), all of whom were ascertained agnostic to CLL family-history status. We also evaluate the PRS by MBL subtype (LC/HC), and in African American (AA) CLL cases and controls. Methods We genotyped 535 EA MBLs (139 HC-MBL, 396 LC-MBLs), 735 CLLs (640 EA, 95 AA), and 2,866 controls (2,631 EA, 235 AA) from the Mayo Clinic CLL Resource, Duke University, and Weill Cornell Medical College. We computed the CLL-PRS for each individual and used logistic regression to estimate odds ratios (OR) and 95% confidence intervals, adjusting for age and sex. To assess discriminatory accuracy, we computed the c-statistic. Among EA individuals, we calculated a trend test among LC-MBL, HC-MBL, and CLL risk using the P-value for heterogeneity from a polytomous logistic regression analysis. Moreover, we plotted a boxplot for the PRS among controls, LC-MBL, HC-MBL, and EA CLL, as well as for AA CLL cases and controls, and tested the statistical difference using the Kruskal Wallis test and Mann-Whitney test, respectively. Results We found a significant association of PRS with overall MBL risk (OR=1.87, P=1.1x10-28) with good discrimination (c-statistic=0.72). Significant associations were also found for LC-MBL (OR=1.75, P=7.5x10-19, c-statistic=0.72), HC-MBL (OR=2.22, P=1.4x10-17, c-statistic=0.74), and CLL of EA (OR=2.60, P=1.2x10-62, c-statistic=0.78), with a significant difference among these cohorts (Figure 1.A) and a significant positive trend across these cohorts (Pheterogeneity=8.4x10-6). Although we observed a 33% increased risk of CLL in AA (c-statistic=0.57), the PRS was borderline significant (P=0.07, Figure 1.B). Conclusion The CLL-PRS is a strong prediction-tool for risk of CLL and MBL among individuals of EA. Future studies are needed to improve the PRS for AAs including performing GWAS of AA in order to identify CLL-susceptibility SNPs that are more representative within known CLL loci and to discover novel CLL loci that are unique for AAs. Disclosures Parikh: GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Brander:Genentech: Consultancy, Honoraria, Other, Research Funding; Juno/Celgene/BMS: Other, Research Funding; MEI Pharma: Other, Research Funding; Ascentage: Other, Research Funding; ArQule: Consultancy, Other, Research Funding; NCCN: Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; AstraZeneca: Consultancy, Honoraria, Other, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other, Research Funding; Pfizer: Consultancy, Other; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Novartis: Consultancy, Other; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Tolero: Research Funding; Teva: Consultancy, Honoraria; DTRM: Other, Research Funding; BeiGene: Other, Research Funding; Novartis: Consultancy, Other; NCCN: Other; Verastem: Consultancy, Honoraria, Other, Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees. Furman:Acerta: Consultancy; AstraZeneca: Consultancy, Research Funding; Beigene: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Loxo Oncology: Consultancy; Oncotarget: Consultancy. Shanafelt:Mayo Clinic: Patents & Royalties: and other intellectual property; Genentech, Pharmacyclics LLC, an AbbVie Company, AbbVie, GlaxoSmithKline, and Merck: Research Funding.

Abstract Chronic lymphocytic lymphoma (CLL) has one of the highest familial risks among cancers. Monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, has a higher prevalence (13%-18%) in families with 2 or more members with CLL compared with the general population (5%-12%). Although, the rate of progression to CLL for high-count MBLs (clonal B-cell count ≥500/µL) is ∼1% to 5%/y, no low-count MBLs have been reported to progress to date. We report the incidence and natural history of MBL in relatives from CLL families. In 310 CLL families, we screened 1045 relatives for MBL using highly sensitive flow cytometry and prospectively followed 449 of them. MBL incidence was directly age- and sex-adjusted to the 2010 US population. CLL cumulative incidence was estimated using Kaplan-Meier survival curves. At baseline, the prevalence of MBL was 22% (235/1045 relatives). After a median follow-up of 8.1 years among 449 relatives, 12 individuals progressed to CLL with a 5-year cumulative incidence of 1.8%. When considering just the 139 relatives with low-count MBL, the 5-year cumulative incidence increased to 5.7%. Finally, 264 had no MBL at baseline, of whom 60 individuals subsequently developed MBL (2 high-count and 58 low-count MBLs) with an age- and sex-adjusted incidence of 3.5% after a median of 6 years of follow-up. In a screening cohort of relatives from CLL families, we reported progression from normal-count to low-count MBL to high-count MBL to CLL, demonstrating that low-count MBL precedes progression to CLL. We estimated a 1.1% annual rate of progression from low-count MBL, which is in excess of that in the general population.

Abstract Background and Significance : Chronic lymphocytic leukemia (CLL) is the most heritable hematologic malignancy; however, no common CLL predisposition genes are known. Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by small accumulations of B lymphocytes in the peripheral blood. MBL has a CLL-like immunophenotype, may progress to overt CLL, and is over represented in CLL families. Therefore, MBL observed in the context of familial CLL may be a marker of inherited risk for development of CLL. Detailed characterization of family-associated MBL may also provide mechanistic insights into the pathogenesis of familial CLL. Our strategy was to detail the biologic characteristics of CLL in family-associated MBL. Methods : Persons with MBL were identified by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. Flow cytometry was used to determine the surface immunophenotype including CD38 and intracellular ZAP-70. We defined MBL as populations of CD19+, CD5+, CD20lo, CD23+ B cells that comprised at least 2% of the CD19+ peripheral B cell compartment and did not exceed 5.0 × 109 MBL cells/L. RNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences. MBL cells were sorted in bulk for FISH for loci associated with clinical CLL. Results : Twenty-two out of 190 (12%) unaffected family members were found to have MBL. We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 32%; range 2%–97%). Nonetheless, the absolute size of the MBL clone was small, 15 of 17 individuals had < 200 × 106 MBL cells/L. CD38 expression (defined as CD38 surface expression in ≥30% of MBL cells) was observed in 8 of 18 subjects tested. ZAP-70 (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 4 of 12 participants. Among 12 subjects tested, 5 MBL expressed both surface IgD and IgM, 3 expressed IgD only, 2 expressed IgM only, and 2 did not express IgD or IgM. Analysis of immunoglobulin heavy and light chains has been completed in 8 individuals. Both immunoglobulin heavy chain variable (IgVH) region mutated (n = 12) and unmutated (n = 4) sequences were observed. Four of 8 individuals had 2 or more unrelated MBL clones (range 2–5), including one individual with both unmutated and mutated clones. Among the 16 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IgVH genes were 3–07 (3 MBL clones), 3–15 (3), and 4–34 (3). No VH1 family gene rearrangements were observed. In one individual, a VH3–07 MBL clone showed intraclonal diversification suggestive of antigen driven immunoglobulin sequence changes. Twenty productive light chain rearrangements were identified among the 16 MBL clones, with 11 Vλ and 9 Vκ genes used. We observed 6 productive rearrangements of Vλ1–51. MBL cells were bulk sorted for FISH from 9 subjects. Mono or biallelic deletion of 13q14.3 was observed in 5 subjects, the other 4 were normal. Conclusions : Our findings confirm that MBL is commonly observed among the unaffected family members from CLL kindreds. We found that some MBL clones express ZAP-70, CD38 or have unmutated IgVH genes and thus are similar to clinical CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. Small MBL clones are commonly oligoclonal. Importantly, although the immunoglobulin heavy and light chain genes rearranged in these MBL clones are all commonly used in CLL, the absence of VH1 and abundance of Vλ1–51 rearrangements do suggests differences in BCR usage between CLL and MBL. Further investigation of family associated MBL may clarify the genetics and immunobiology of familial CLL.

Abstract Abstract 1785 Background: Chronic lymphocytic leukemia (CLL) is a malignant lymphoproliferative disorder characterized by the progressive clonal expansion of CD5+ B cells. Approximately 8–10% of CLL patients have a first degree relative with CLL, indicating that genetic factors can contribute to disease susceptibility. Genome wide association (GWA) studies have identified 16 “common variants,” single-nucleotide polymorphisms (SNPs) with a population prevalence of >5%, that confer risk for CLL. Among patients with CLL, the clinical course can be highly heterogeneous. Studies have defined disease-characteristics that vary among patients that can be used as markers to help predict clinical outcome. African American (AA) CLL patients have a poorer overall survival than Caucasians. However, there is little information on whether there are distinctive, ethnic-associated disease-characteristics that contribute to differences in clinical outcome. Moreover, studies of the genetics of CLL have involved primarily Caucasian patients. For these reasons, we examined for ethnic-related differences in the distribution and prevalence of CLL-associated SNPs and adverse disease-associated characteristics among AA with CLL relative to those of Caucasian patients with this disease. Methods: The clinical and biologic characteristics of 43 AA CLL patients ascertained at Duke University and the Durham VA Medical Center were tabulated and compared to Caucasian CLL patients at the same centers. Fifteen SNPs were genotyped in this “discovery” cohort of 43 AA CLL patients using TaqMan SNP genotyping assays. As a “validation” cohort, 70 AA CLL patients ascertained by the CLL Research Consortium (CRC) were assayed using the same platform. Three control groups were considered in analysis: (1) previously published Caucasian CLL patients, (2) control AA genotypes reported in the HapMap database and (3) control AA with no history of malignancy from other GWA studies. The 3' untranslated region of CPEB1 was sequenced in AA CLL patients using conventional Sanger sequencing. Results: Among the Duke/Durham VA CLL patients, no significant differences were observed in age, WBC count, platelet count, or hemoglobin at diagnosis; lymphocyte doubling time; Rai stage; ZAP70 expression; or CD38 expression between AA and Caucasian patients. AA had a larger proportion of trisomy 12 and fewer 13q.14 deletions than Caucasians: 40.0% of AA had trisomy 12 and 15.0% had del 13q, compared to 14.6% and 40.7% of Caucasians, respectively. IGHV mutation status also varied between races, with 62.2% of AA having unmutated IGHV, compared to 38.4% of Caucasians. Disparities in clinical outcomes were similar to that reported in the SEER database: overall survival was 8.8 years in AA patients and 17.8 years in Caucasians, and time to first treatment was 2.8 years among AAs and 4.5 years for Caucasians. Risk allele frequencies among 15 genotyped SNPs showed that 14 SNPs had risk allele frequencies that statistically differed between AA patients and Caucasian CLL patients. In 9 of 14 SNPs, the risk allele was less common in AA than in Caucasians but similar to that AA controls. In the other 5 SNPs, the risk allele was more common in AA CLL than Caucasian CLL, and similar to that of AA controls in 4 of the SNPs. A single SNP, rs783540 (CPEB1) was found to have an increased frequency in AA patients with CLL when compared to both Caucasian CLL and control AA. As an exploratory analysis to identify possible pathogenic variants, the 3' UTR of CPEB1 was sequenced as there are no common non-synonymous SNPs in CPEB1. Preliminary review of the sequencing data revealed that a G insertion SNP (rs34816185) that alters a predicted miRNA binding site is in linkage disequilibrium with rs783540 (D'= 0.978). Conclusions: Most SNPs associated with CLL risk are less frequent in AA than in Caucasian CLL patients. However, rs783540 (intron 2, CPEB1) risk allele was more frequent in AA CLL than that previously reported in Caucasian CLL or AA controls. Initial data suggests that rs783540 is linked to a SNP in the 3' UTR of CPEB1, which may have functional significance. Studies to better delineate the functional and clinical implications of these germline variants are underway. Our data provide the first evidence of differential genetic risk as a determinant of CLL biology among different ethnic groups. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3881 Background: Chronic lymphocytic leukemia (CLL) is largely incurable neoplasm of the blood and has one of the highest familial risks of disease among cancers. Monoclonal B-cell lymphocytosis (MBL) with a CLL immunophenotypic profile (i.e., CLL-like MBL) is a precursor state to CLL. In the general adult population, the prevalence rate of MBL is 5–9% and increases with age. This is in contrast to the 13–18% rate of MBL in first-degree relatives of CLL patients from high-risk families (defined here as families with at least two confirmed living cases of CLL). Few longitudinal studies of MBL incidence exists. One study evaluated MBL status over time in a cohort of individuals with MBL from a rural valley in Northern Italy. Our work herein will be the first to evaluate the status of MBL over time in relatives of CLL patients from high-risk families. Methods: Using a cohort of 41 high-risk CLL families, we evaluated the MBL status and clonal blood B-cell counts in 94 relatives of CLL patients at two time points at least 1 year a part. High sensitivity, multi-color flow cytometry was performed on fresh or previously frozen blood samples in three laboratories: Duke University, the US Food and Drug Administration, and Mayo Clinic. The number of cell events collected for flow cytometry evaluation varied from 1 to 5 x105 cells. To assure concordance across flow cytometry laboratories, 15 samples were evaluated at multiple labs. The concordance of MBL calling was a 100%. We defined three monotypic B-cell subsets as CLL-like MBL (CD5+ CD20dim), atypical MBL (CD5+ CD20+), and non-CLL like MBL (CD5−CD20+). Results: Of the 94 relatives, the median age at consent was 57 years (range 42–93) and 39% were male. At the initial MBL screening, 70 (74%) relatives had normal immunophenotype (NIPT) and 24 (26%) had MBL (1 atypical MBL, 3 non-CLL like MBL, and 20 CLL-like MBL). Seven of the 24 individuals with MBL had monoclonal B-cell counts available at baseline screening; median = 147.4 cells/uL (range 22.7–1663). The 94 relatives underwent repeat evaluation with a median follow up time of 1.8 years (range 1–6). At the second evaluation, we found an increase in the prevalence of MBL, with 11 of the 70 (12%) initially NIPT relatives developing a new MBL clone (10 CLL-like, 1 non-CLL like). These 11 incident MBL cases were from 9 families with 2 families each having 2 relatives changing MBL status. The median number of monoclonal B-cells in these 11 incident MBL cases was 0.58 cells/uL (range 0.38–60.3). The 23 out of the 24 relatives with baseline MBL (20 CLL-like MBL and 3 non-CLL like MBL) had a persistent MBL clone. Only 6 of these 23 individuals had monoclonal B-cell counts available at both time points, and among these 6, the clonal B-cell counts decreased at follow-up screening; median difference −64.1 cells/uL (range −1204.3– 10.5). The second evaluation of the initial atypical MBL relative had too few clonal cells for flow assessment. Conclusion: We found that our CLL-like MBL clones persisted over the period of observation in relatives from high-risk CLL families; this finding is similar to that previously reported using a population-based study conducted in Northern Italy. Notably, 12% of relatives from high-risk CLL families developed incident MBL during just 2 years of follow up. Although these MBL clones are all low count (< 1500 cells/uL), these data provide further evidence that MBL is a marker of inherited predisposition to CLL. It will be important to see how our rate of change of MBL status compares to that of the general population as we continue to follow our cohort of CLL families. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 1232 Poster Board I-254 B-Chronic Lymphocytic Leukemia (CLL) shows a strong familial risk and also co-aggregates with other indolent lymphomas. Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition characterized by small B-cell clones, most with a surface phenotype similar to that of chronic lymphocytic leukemia (CLL). These clones are detectable at low absolute lymphocyte cell (ALC) numbers in otherwise healthy individuals using sensitive 6 or 8 color flow cytometry analysis and can be a precursor to CLL. In the general population, MBL increases with age with a prevalence of 5% in individuals over age 65. In contrast, the rate of MBL is 13-18% in first degree relatives of CLL patients in high risk families. In the largest study to date, we report the characteristics of MBL among 430 first-degree relatives of CLL patients (with no associated lymphoproliferative diseases [LPD]) in 132 high risk families. Patients and Methods Individuals studied came from “high risk” families (defined as those with two or more confirmed cases of CLL) that are participating in the Genetic Epidemiology of CLL Consortium. Multi-parameter flow cytometry analysis with a detection sensitivity of 0.02% was performed on either fresh or cryopreserved PBMC and MBL was classified according to previously published criteria (Marti et al, Br. J Haematol 130:325, 2005). Both the ALC and the absolute B cell count (B-ALC) were calculated for each individual. Survival analysis was used to compute the probability of developing MBL with age using the life table method. Results The overall rate of MBL was 17% (73/430) among first–degree relatives but the probability for developing MBL by age 90 was 51%. Males had a slightly higher (but non-significant) risk for MBL than females. MBL patients had significantly higher ALC and B-ALC than did those with normal immunophenotype. The mean ALC was 2.5×109/L among MBL patients, and 20% had an ALC greater than 3 ×109/L. The B-ALC count averaged 0.51 ×109/L. Ninety percent of the MBL cases had a CLL-like phenotype (CD20dim, CD5+, CD23+). Conclusions MBL is found at a very high rate in families selected for having 2 or more patients with CLL suggesting that MBL reflects inherited predisposition to CLL. Although most of the MBL cases in these families had low cell counts and thus have a presumed low likelihood of progressing to CLL or other LPD, a higher proportion of them had lymphocytosis compared to those with normal immunophenotype. We hypothesize that if MBL is an early step in the process of development of CLL, then germ line genes are likely to be acting early in carcinogenesis with more “hits” required before CLL develops. By looking for genes associated with MBL or CLL in families or in the population, we could substantially increase our power to identify germ line genes predisposing to CLL. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.