Monoclonal B-cell Lymphocytosis Cases Research Articles

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Intensity of antigen expression reflects IGHV mutational status and Dohner-defined prognostic categories in chronic lymphocytic leukemia, monoclonal B-cell lymphocytosis, and small lymphocytic lymphoma

We demonstrate the prognostic utility of antigen quantitation in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and monoclonal B-cell lymphocytosis (MBL). Median antibody-bound-per-cell (ABC) of CD20, CD22, CD25, CD19, and %CD38(+) was determined in CLL (185/208), SLL (8/208) and MBL (15/208) cases by flow cytometry, then compared to Dohner-classification, immunoglobulin status (mutated, IGHV-M; unmutated, IGHV-U), CLL-IPI risk and time to first treatment (TTFT). Trisomy 12 cases showed increased %CD38-expression (p = .0379). Higher %CD38 was observed in IGHV-U versus IGHV-M (p = .0003). CD20ABC was increased in IGHV-U versus IGHV-M (p = .006). Del13q cases demonstrated lower CD22ABC (p = .0198). Cases without cytogenetic abnormality exhibited higher CD19ABC (p = .0295) and CD22ABC (p = .0078). Del17p cases demonstrated lower CD25ABC (p = .0097). High and very-high CLL-IPI risk groups were associated with high CD38-expression (p = .02) and low CD25ABC (p = .0004). Shortened TTFT was associated with high CD38-expression (p < .0001). Interestingly, high CD25ABC trended toward shortened TTFT (p = .07). Quantitative antigen expression reflects CLL-IPI risk groups and Dohner-classification.

Gene Expression and Cytokine Analyses Identify Markers of Progression from CLL-like Monoclonal B-Cell Lymphocytosis to Chronic Lymphocytic Leukemia

Gene Expression and Cytokine Analyses Identify Markers of Progression from CLL-like Monoclonal B-Cell Lymphocytosis to Chronic Lymphocytic Leukemia

Developmental DNA Methylation Subtype Predicts Progression to Treatment and Survival in High-Count Monoclonal B Lymphocytosis

Developmental DNA Methylation Subtype Predicts Progression to Treatment and Survival in High-Count Monoclonal B Lymphocytosis

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.

Association between the Risk of Low/High-Count Monoclonal B-Cell Lymphocytosis (MBL) and the Chronic Lymphocytic Leukemia (CLL) Polygenic Risk Score (PRS)

BackgroundMBL is a precursor state to CLL, with a prevalence of ~5-8% in the general population and 15-18% in CLL families. To date, 41 single nucleotide polymorphisms (SNPs) have been found to be associated with CLL. We previously showed that a PRS of the weighted average of the number of risk alleles of these SNPs is associated with CLL risk using cases and controls from the International Lymphoma Epidemiology (InterLymph) Consortium. We validated this score in an independent sample of CLL cases and controls from the Genetic Epidemiology of CLL (GEC) Consortium, a cohort of families with ≥2 members with CLL. In the CLL families we also reported an association between the PRS and MBL risk, where 93% of MBLs were low-count MBL. Here we evaluate the PRS in an independent sample ascertained from the Mayo Clinic Biobank.MethodsThe Mayo Clinic Biobank is a large-scale bio-repository of adult patients assembled to provide a wide array of health-related research studies. Biobank participants (N=2530) were screened for MBL using a highly-sensitive, 8-color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow-cytometry assay on stored cryopreserved peripheral blood mononucleotide cells. Low-count and high-count MBL were defined as those individuals who had <85% and ≥85 clonal B-cells out of total B-cells, respectively. Individuals without MBL (“controls”) were frequency matched to MBLs based on age and sex, after excluding non-Caucasian and non-interpretable flow results. Participants were genotyped using the Illumina OmniExpress array. Standard genotyping quality-control metrics were applied. We computed the PRS as previously published and categorized it by quintiles based on values previously used with InterLymph controls. The middle quintile served as the reference category. We used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age at consent, sex, and socioeconomic status. In addition, we compared the median PRS by low-count MBL and high count MBL using the Mann-Whitney U test.ResultsWe identified 276 MBLs overall and 2109 controls; 97% (N=267) of MBLs had low-count MBL and only 3% (N=9) had high-count MBL. We then selected 844 controls frequency matched to the MBL cases on age and sex. Family history of leukemia/lymphoma was 11.3% among MBLs overall and 8.8% among controls. The median PRS was 7.88 and 7.46 (Figure) among MBLs overall and controls, respectively. The continuous PRS had a 1.75-fold increased risk for MBL overall (CI:1.5-2.1, P=2.65x10-12). Compared to the middle quintile, the highest quintile had 2.2-fold increased risk for MBL overall (CI:1.5-3.3, P=1.46x10-4) and the lowest quintile had 0.6-fold decreased risk (CI:0.4-0.96, P=0.03). The median PRS for low-count and high count MBL were 7.86 and 8.04, respectively. The continuous PRS had a 1.75-fold increased risk for low-count MBL (CI:1.5-2.1, P=4.81x10-12) and a 1.89-fold increased risk for high-count MBL (CI:0.8-4.2, P=0.13), however, the effect is not statistically significant between high-count and low-count MBL (P=0.89).ConclusionIn this independent sample, we validated our previous findings that the CLL PRS is associated with MBL risk. Although the sample size of high-count MBL was limited (N=9), there is evidence of a weak trend towards a higher PRS compared to low-count MBL and controls Larger sample size of high-count MBL is warranted in order to stratify MBL risk by low-count and high-count MBL. These results may help identify individuals at higher risk of MBL including low-count and high-count MBL, beyond the risk associated with age and family history of CLL.Figure: Polygenic risk score distribution by control and MBL status DisclosuresParikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding. Cerhan:Nanostring: Research Funding; Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board. Kay:Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding.

CpG-ODN Stimulation in Population Screening Monoclonal B-Cell Lymphocytosis

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) induce proliferation in normal and malignant lymphocytes B (LyB). This effect is widely exploited in CLL conventional chromosome banding analysis (CBA), which has become reliable only after the cultivation of CLL LyB using CpG-ODN stimulation in combination with IL-2. Monoclonal B-cell lymphocytosis (MBL) differs from CLL mainly in clone size. Only cytogenetic data on recurrent chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH) are available for population screening MBL (sMBL). In sMBL the clone of malignant LyB is typically below 10/µL. We compared CpGODN stimulation in healthy donors and in individuals with sMBL. LyB and MBL LyB count were determined by flow cytometry in 15 samples from healthy subjects and 12 MBL cases. Mitotic indices were determined and CBA was done after cultivation of samples by CpG-ODN + IL2. In MBL samples, FISH analysis was performed on isolated LyB. MBL LyB clones in sMBL cases presented less than 1% of WBC and up to 33% of LyB. The MBL group was therefore compared to the group of healthy donors. Although normal and MBL group did not differ in WBC, overall LyB, and normal LyB count, a significantly higher mitotic index was observed in MBL samples (p = 0.0139). We were able to accomplish CBA in all samples which revealed a normal karyotype in all but one case. In this particular sMBL case FISH performed on isolated LyB showed 5% trisomy 12 which was later confirmed by CBA on CpG stimulated blood sample in 15% of metaphases. Our study, which was done on MBL cases obtained by population screening, confirmed that CpGODN preferentially induced proliferation in MBL LyB over normal LyB. Therefore, CBA can also be successfully accomplished in sMBL and can be used to additionally confirm clonality as well as to improve sensitivity of FISH analysis. Due to coexistence of comparable size of normal and malignant LyB, MBL can serve as a model for exvivo studying of LyB stimulation by CpG-ODN.

Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B-cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biologic events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep-sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs(52%) at the initial time-point analyzed, including 13(27%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time-point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution have prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

Monoclonal B-cell lymphocytosis in the population of Slovenian region of Lower Carniola.

Monoclonal B-cell lymphocytosis (MBL) is a premalignant asymptomatic condition characterized by the presence of a small population of monoclonal B lymphocytes (less than 5 × 10(9) /L) in the venous blood of otherwise healthy individuals. Incidence of MBL is higher among first-degree relatives of patients with chronic lymphocytic leukemia (CLL) and also increases with age and among men. We compared the incidence of MBL found in the same environment in both an open general population and relatives of CLL patients. Samples were analyzed with 5-parameter flow cytometry using following monoclonal antibodies: kappa and lambda clonality, CD5, CD19, CD20. We analyzed 216 samples of patients in the Slovenian area of Lower Carniola aged 23-88 years. Fifty-four patients who came from families where CLL is present were compared by age and gender to 162 healthy individuals from the general population of the same region. We found 10 cases of MBL (4.6%). In 9.4% of younger (≤ 50 years) first-degree relatives of CLL patients with immunophenotypic characteristics of atypical MBL, we observed a prevailingly normal κ to λ ratio. The difference in incidence of MBL cases between relatives and the general population decreases with age. MBL in Slovenia can be coincidentally found with a similar frequency to previously studied populations of other parts of Europe. We found, however, a higher incidence of atypical MBL among first-degree relatives.

Monoclonal B-cell lymphocytosis: from literature to laboratory practice

Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic condition characterized by the presence of less than 5,000 monoclonal B-cells per microliter and the absence of clinical signs or symptoms of a B-cell lymphoproliferative disorder. Most MBL cases involve B cells presenting an identical phenotype to CLL (CLL-like MBL) with a Catovsky-Matutes score of 3 to 5 and share the same chromosomal abnormalities than CLL. Depending on the absolute B cell count, one may distinguish low-count CLL-like MBL (<500 B cells/μL) which have no evidence of progression, no reduction in overall survival, no increase in infection risk and do not require any specific follow-up. Patients with clinical CLL-like MBL (>500 B cells/μL) have a 1% to 2% per year risk of progression to CLL requiring therapy, a higher risk of infectious complications and mortality implicating an annual follow-up by hematologist. MBL may also express other less common phenotypes and are named atypical MBL in case of CD5 antigen expression (Catovsky-Matutes score: 1-2) and non-CLL-like MBL for CD5 negative cases (Catovsky-Matutes score: 0-2). Their poorer prognosis implicates imaging studies, bone marrow biopsy and cytogenetic analysis in addition to physical examination in order to rule out non-hodgkinien lymphoma, and require a more frequent follow-up. This review focuses on key concepts in the classification, diagnosis, monitoring and biology of MBL in laboratory practice.

Next-generation IgVH sequencing CLL-like monoclonal B-cell lymphocytosis reveals frequent oligoclonality and ongoing hypermutation.

Chronic lymphocytic leukemia (CLL) develops from CLL-like monoclonal B-cell lymphocytosis (MBL) which represents a low-level asymptomatic expansion of cells that phenotypically resemble CLL. Although antigen selection plays a key role during CLL development, it is not known whether this occurs in early MBL or only during progression to CLL. Recent studies suggested that MBL sometimes displays oligoclonality, but these used techniques with limited sensitivity and specificity and were not conclusive. In this study, we combine cell sorting and next-generation sequencing of rearranged immunoglobulin heavy chain variable (IgVH) genes to thoroughly assess the VH repertoire and oligoclonality of purified MBL cells. Clonal functional rearrangements or clonotypes were identified in 29 of 30 sequenced cases, with 7 or 24% having two clonotypes with unrelated CDR3 sequences. In four of the seven cases with unrelated clonotypes, VH segments from the same family were used. In addition, 6 of 29 cases showed clear evidence of ongoing VH gene hypermutation with three of these being among the seven with unrelated clonotypes. This study conclusively shows that MBL cases often contain multiple B-cell clones, the first to report ongoing VH gene mutation in MBL, and that antigen selection appears to occur in early MBL.

Exome-Wide Sequencing Reveals Oncogenic Mutations in Both Progressive and Non-Progressive MBL

While virtually all CLL cases are preceded by an asymptomatic phase of monoclonal B cell lymphocytosis (MBL), only 1-2% of MBL cases develop to CLL requiring chemotherapy annually. Specific determinants that lead to progression are unknown thus far, but are highly relevant, since this might contribute to a better understanding of CLL biology. Recently, the application of whole genome and exome sequencing using next generation sequencing (NGS) in CLL resulted in the discovery of novel genes including mutations in SF3B1 and NOTCH1, that could potentially play important roles in CLL leukemogenesis. Additionally NGS enabled in-depth examination of genetic alterations, and therefore has shed new light on clonal evolution of driver mutations which is considered a hallmark for progression. However, the role of genetic mutations and clonal evolution in the development of MBL and progression of MBL to CLL is still largely unknown and NGS studies in MBL samples and paired longitudinal samples is currently scarce. In this study we investigated the role of genetic mutations in the development of MBL and progression of MBL to CLL.Thirty-six clinical MBL (CD19+ cell count: 0.5-5.0x109/L) cases were ascertained through flow cytometric screening of a population based cohort of non-hematology patients identified via an absolute lymphocytosis on a routine complete blood count. From thirteen cases a follow-up sample was available obtained at an average time of 4.8 years after initial study enrollment. All cases had a detectable CLL-phenotype clone but did not meet iwCLL criteria for a diagnosis of CLL. Samples with clinical follow-up were characterized as progressive or non-progressive based on whether the CLL phenotype clone expanded at the time of follow-up. CLL-phenotype cells and non-malignant monocytes were purified using flowcytometry and DNA was extracted. We performed whole-exome sequencing using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ~700 human miRNAs from miRBase (v13).A total of 76 exomes from 33 unique individuals were sequenced for this study. Six MBL cases were progressive and 27 MBL cases were non-progressive. Paired normal DNA was sequenced for all of these cases. On average, we achieved 30-40 fold coverage of the targeted regions. Over 90% of the targeted exons were covered at an average depth exceeding 3-fold. Sequencing reads were mapped to the reference human genome, and high-quality mismatches were classified as synonymous and nonsynonymous variants. We identified somatic mutations affecting 328 genes in at least one pair among these cases. Transitions comprised the majority of the somatic variants (P<10-3, chi-squared test). On average, we observed 10 somatic alterations per sample (range 3-30). These variants were not present in publicly available data from normal controls including dbSNP135, the 1000 Genomes Project, and available exome sequencing data from healthy individuals without lymphoma. We further required each of the identified genes to have three somatically acquired and/or non synonymous rare events predicted to be functionally significant. A striking heterogeneity among these cases were observed with no gene mutation occurring at a frequency exceeding 15%. The most frequently mutated genes were LAMA3 and MLL3, each mutated in 13% of the cases. A number of known cancer-related genes were also mutated in MBLs at a frequency of roughly 5-8% including MLL, NOTCH2 and TP53. Acquired mutations in MADCAM1 were identified only at progression and not at diagnosis in selected progressive MBL samples.This study represents an in-depth investigation of MBL exomes. Our data show a striking degree of heterogeneity in the genetic mutations that occur in MBLs. Mutations in known oncogenes and tumor suppressor genes occur commonly in MBL. Their presence does not indicate poor prognosis or a clear risk for progression to CLL. Our data indicate that individual genetic events are likely to be much less important than the sequential oncogenic mutations and combinations of specific genetic events for the progression of MBL to CLL. Thus, our work sheds new light on the genetic underpinnings and clonal diversity of MBL and provides a starting point for a better understanding of the genetic drivers and sequence of progression to CLL. DisclosuresNo relevant conflicts of interest to declare.

Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

A seven-gene expression panel distinguishing clonal expansions of pre-leukemic and chronic lymphocytic leukemia B cells from normal B lymphocytes.

Chronic lymphocytic leukemia (CLL) is a clonal disease of B lymphocytes manifesting as an absolute lymphocytosis in the blood. However, not all lymphocytoses are leukemic. In addition, first-degree relatives of CLL patients have an ~15 % chance of developing a precursor condition to CLL termed monoclonal B cell lymphocytosis (MBL), and distinguishing CLL and MBL B lymphocytes from normal B cell expansions can be a challenge. Therefore, we selected FMOD, CKAP4, PIK3C2B, LEF1, PFTK1, BCL-2, and GPM6a from a set of genes significantly differentially expressed in microarray analyses that compared CLL cells with normal B lymphocytes and used these to determine whether we could discriminate CLL and MBL cells from B cells of healthy controls. Analysis with receiver operating characteristics and Bayesian relevance determination demonstrated good concordance with all panel genes. Using a random forest classifier, the seven-gene panel reliably distinguished normal polyclonal B cell populations from expression patterns occurring in pre-CLL and CLL B cell populations with an error rate of 2 %. Using Bayesian learning, the expression levels of only two genes, FMOD and PIK3C2B, correctly distinguished 100 % of CLL and MBL cases from normal polyclonal and mono/oligoclonal B lymphocytes. Thus, this study sets forth effective computational approaches that distinguish MBL/CLL from normal B lymphocytes. The findings also support the concept that MBL is a CLL precursor.

Risk of non-hematologic cancer in individuals with high-count monoclonal B-cell lymphocytosis.

It is unknown whether individuals with monoclonal B-cell lymphocytosis (MBL) are at risk for adverse outcomes associated with chronic lymphocytic leukemia (CLL), such as the risk of non-hematologic cancer. We identified all locally-residing individuals diagnosed with high count MBL at Mayo Clinic between 1999 and 2009 and compared their rates of non-hematologic cancer to that of patients with CLL and two control cohorts: general medicine patients and patients who underwent clinical evaluation with flow cytometry but who had no hematologic malignancy. After excluding individuals with prior cancers, there were 107 high count MBL cases, 132 CLL cases, 589 clinic controls, and 482 flow cytometry controls. With 4.6 years median follow-up, 14 (13%) individuals with high count MBL, 21 (4%) clinic controls (comparison MBL p<0.0001), 18 (4%) flow controls (comparison MBL p=0.0001), and 16 (12%) CLL patients (comparison MBL p=0.82) developed non-hematologic cancer. On multivariable Cox regression analysis, individuals with high count MBL had higher risk of non-hematologic cancer than flow controls (HR=2.36; p=0.04) and borderline higher risk than clinic controls (HR=2.00; p=0.07). Patients with high count MBL appear to be at increased risk for non-hematologic cancer, further reinforcing that high count MBL has a distinct clinical phenotype despite low risk of progression to CLL.

Immunophenotypic analysis reveals heterogeneity and common biologic aspects in monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is the presence of small B-cell clones in the peripheral blood of healthy subjects. Most MBL have the characteristic phenotype of chronic lymphocyte leukemia (chronic lymphocytic leukemia (CLL)-like MBL), and depending on the number of monoclonal B-cells, may characterize a preclinical stage of the CLL. However, there are also MBL with an atypical (CD5(+) CD20(+/bright) CD23(dim/-) ) or a CD5(neg) phenotype, which remain largely unexplored. We performed an extended immunophenotypic, cytogenetic, and hematologic analysis in 75 CLL-like, 39 atypical, 50 CD5(neg) , and 7 biphenotypic MBL cases to detect differences or similarities among the MBL subsets. The phenotypic analysis showed expression variations in many surface markers and a wide spectrum of disease-specific phenotypes within each MBL subtype. Interphase fluorescent in situ hybridization analysis showed a different panel of aberrations according to the phenotype. Overall, del(13q14) and +12 were the most common abnormalities (39%), whereas del(11q13), del(17p13), and del(6q23) were detected only in 3, 1, and 0 cases, respectively. A comparison of MBL with overt chronic lymphoproliferations revealed common aspects in the preclinical state, regarding both the kind of cytogenetic aberrations detected and the lymphocyte composition. Our findings highlight not only the heterogeneity among MBL subsets but also indicate common biologic features which differentiate MBL from clinical disease.

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.

Monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition defined by the presence of a small (<5 x 10(9)/L) clonal B-cell population in the peripheral blood in the absence of lymph-node enlargement, cytopenias or autoimmune diseases. It is found in approximately 3-12% of normal persons depending on the accuracy of analytical techniques applied. According to the immunophenotypic profile of clonal B-cells, the majority of MBL cases (75%) are classified as chronic lymphocytic leukemia (CLL)-like. This form may progress into CLL at a rate of 1-2% per year. It is thought that CLL is always preceded by MBL. The remaining MBL cases are defined as atypical CLL-like (CD5+/CD20(bright)) and CD5(-) MBL. The MBL clone size is quite heterogenous. Accordingly, two forms of MBL are identified: i) high-count, or 'clinical' MBL, in which an evidence of lymphocytosis (<5 x 10(9)/L clonal B-cells) is seen, and ii) a low-count MBL, in which a normal leukocyte count is found and that is identified only in population-screening studies. Both forms of MBL may carry the cytogenetic abnormalities that are the hallmark of CLL, including 13q-, 17p- and trisomy 12. Consistent with the indolent phenotype of this condition, genetic lesions, such as TP53, ATM, NOTCH1 and SF3B1 mutations, usually associated with high-risk CLL, are rarely seen. Overall, no prognostic indicator of evolution of MBL to overt CLL has been found at present time. However, taking into account this possibility, a clinical and lab monitoring (at least annually), is recommended.

Next-Generation Sequencing Reveals Oncogenic Driver Mutations In Both Progressive and Non-Progressive Monoclonal B Lymphocytosis

Essentially all cases of CLL are preceded by an asymptomatic expansion of a monoclonal B cell population, a condition that has been termed monoclonal B-cell lymphocytosis (MBL). MBL can be found in the peripheral blood of 3 to 5% of normal older adults. Although MBL is precursor state of CLL, the majority of MBL do not progress to CLL. Thus, MBL is being increasingly recognized and diagnosed in routine clinical practice, but the prognostic and treatment implications of MBL are unclear.The application of whole genome and exome sequencing has yielded important insights into somatic genetic mutations that contribute to the biology of CLL. Such work has identified a role for recurrent mutations in a number of novel genes including NOTCH1 and SF3B1 in CLL. However, the role of genetic mutations in the development of MBL and progression of MBL to CLL is completely unknown. Our central hypothesis is that acquired somatic mutations are responsible for the progression of MBL to CLL and determine an individual patient's disease course. Therefore, we applied exome sequencing to a cohort of MBL patients to understand the mutation profile of pre-emergent CLL and the determinants of progression from MBL to CLL.Thirty-six clinical MBL (CD19+ cell count: 0.5 – 5.0 x109/L) cases were ascertained through flow cytometric screening of a population based cohort of non-hematology patients identified via an absolute lymphocytosis on a routine complete blood count. Thirteen cases had a follow-up sample obtained at an average time of 4.8 years after initial study enrollment. All cases had a detectable CLL-phenotype clone but did not meet iwCLL criteria for a diagnosis of CLL. Samples with clinical follow-up were characterized as progressive or non-progressive based on whether the CLL phenotype clone expanded at the time of follow-up. CLL-phenotype cells and non-malignant monocytes were purified using FACS and DNA was extracted. We performed whole-exome sequencing using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13).A total of 86 exomes were sequenced for this study. Seven MBL cases were progressive and 29 MBL cases were non-progressive. Paired normal DNA was sequenced for all of these cases. Variants were filtered both through pair-wise comparisons of MBL and monocyte sequencing data and by prioritizing acquired mutations identified in know tumor suppressor or oncogenes. In all, we generated over 8 GB of sequencing data using high throughput sequencing on the Illumina platform. The number of mutations per exome were similar between progressive and non-progressive cases. Numerous non-synonymous variants in known tumor suppressor or oncogenes were identified in non-progressive cases including: NOTCH2, PIK3R1, SYK, MLL2, CDK12, CIITA, and CD79A. Non-synonymous variants identified exclusively in progressive cases included CREBBP and BRCA2.This study represents the first in-depth investigation of MBL exomes. Our data show that individual events in known oncogenes and tumor suppressor genes occur commonly in non-progressive MBL. Their presence does not indicate poor prognosis. These findings imply that sequential oncogenic mutations and combinations of specific genetic events are likely required for the progression of MBL to CLL. The knowledge of genetic variants that collectively contribute to the development of CLL would allow the development of risk stratification strategies for patients with MBL. Disclosures:No relevant conflicts of interest to declare.

Abstract 2287: Incidence of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis in Olmsted county, 2000-2010: impact of the 2008 International Workshop on CLL Guidelines.

Abstract Background: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is the most common adult leukemia in the United States. Prior studies in Olmsted County, Minnesota, showed a CLL age and sex adjusted incidence rate of 6.6 per 100,000 during 1985-89. Subsequently, the definition of CLL is now based on the 2008 International Workshop on CLL (IWCLL) Guidelines. Individuals having a CLL immunophenotype by flow cytometry are classified as CLL if the absolute B-cell count (ABC) is &amp;gt;5 x 109/L or classified as monoclonal B-cell lymphocytosis (MBL) if ABC is &amp;lt;5 x 109/L. To assess the impact of these changes on the incidence of CLL and clinically discovered MBL, we conducted an incidence study in Olmsted County for the years 2000-2010. Methods Using diagnostic indexes available through the Rochester Epidemiology Project and the Mayo CLL database, we identified all CLL and clinically identified MBL cases from 2000-2010. Incidence rates and demographics were compared between the older National Cancer Institute-Working Groups 1996 guidelines (NCI-WG 96) defined as absolute lymphocyte count (ALC)&amp;gt; 5 x 109/L and the IWCLL 2008 guidelines defined as ABC &amp;gt; 5 x 109/L. Incidence rates were age and sex adjusted using the 2010 US white population. Time to treatment (TTT) survival analyses was from time of diagnosis to date of first treatment or last follow-up and Cox regression analysis was used. Results Using the NCI-WG 96 classification, there were 8 MBL cases and 115 CLLs in whom 61% were Rai stage 0, 34% were Rai stage 1-2, and 5% were Rai stage 3-4. Using the IWCLL 2008 classification, there were 44 MBL cases and 79 CLLs in whom 43% Rai stage 0, 49% were Rai stage 1-2 and 8% Rai stage 3-4. Median ALC at diagnosis was 8.1 x 109/L for CLL and 5.9 x 109/L for MBL. Median ABC count at diagnosis was 7.1 x 109/L for CLL and 2.8 x 109/L for MBL. The age and sex adjusted incidence rate (per 100,000) for CLL for 2000-2010 was 10.0 using NCI-WG 96 and 6.8 using IWCLL 2008. Rates for MBL were 0.66 using NCI-WG 96 and 3.5 using IWCLL 2008. The median TTT for CLL was 9.2 years using NCI-WG 96 and 6.5 years by IWCLL 2008. Discussion This study demonstrates that reclassification of CLL using the IWCLL 2008 guidelines decreases CLL incidence relative to the NCI-WG 96 criteria. For the first time, this population based study also establishes an incidence rate for clinically identified MBL. Approximately 50% of previously labeled Rai 0 CLL are now reclassified as MBL. Re-classification increases the percent of Rai stage 1-2 and decreases the percent of Rai 0 cases at diagnosis. Reclassification also substantially shortens the median TTT for newly diagnosed CLL cases classified by the 2008 criteria relative to the NCI-WG 96 criteria. In summary, the IWCLL 2008 reclassification has significant implications for incidence studies, staging, and prognosis of CLL and MBL. Citation Format: Aaron D. Norman, Timothy G. Call, Curtis A. Hanson, Neil E. Kay, Clive S. Zent, Wei Ding, James R. Cerhan, Sara J. Achenbach, Kari G. Rabe, Celine M. Vachon, Emily J. Hallberg, Tait D. Shanafelt, Susan L. Slager. Incidence of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis in Olmsted county, 2000-2010: impact of the 2008 International Workshop on CLL Guidelines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2287. doi:10.1158/1538-7445.AM2013-2287