Goodman, Barbara K.

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 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.

AbstractBackground: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein–Barr virus (EBV)–transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications.Methods: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing.Results: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications.Conclusions: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.