Clonal Hematopoiesis of Indeterminate Potential (CHIP) and Chronic Lymphocytic Leukemia (CLL) Driver Genes: Risk of CLL and Monoclonal B-Cell Lymphocytosis (MBL)
Authors: Boddicker Nicholas J , O'Brien Daniel R , Braggio Esteban , Achenbach Sara J. , Chaffee Kari G. , Norman Aaron D. , Vachon Celine M. , Kabat Brian , Call Timothy G. , Parikh Sameer A. , Leis Jose F. , Ding Wei , Muchtar Eli , Cerhan James R. , Shanafelt Tait D. , Kay Neil E. , Caporaso Neil E , Slager Susan L. 
Published: October 2019

Abstract
Clonal hematopoiesis of indeterminate potential (CHIP) is defined as somatic mutations in clonal blood cells of individuals without any other hematological abnormalities and have been identified using whole exome sequencing (WES) from pre-specified genes. CHIP is reportedly associated with aging, reduced overall survival, cardiovascular disease, and hematologic disease, mainly myeloid cancers. Additionally, whole genome sequencing and WES studies in CLL cases have identified putative CLL driver genes with recurrent somatic variants or copy number alterations. Here, we investigate the associations of CHIP and mutations in CLL somatic driver genes with risk of CLL and MBL, a precursor to CLL.
Using WES, 571 individuals from 132 CLL families were sequenced from two different sites, Mayo Clinic (92 families) and National Cancer Institute (NCI, 40 families), including 251 CLLs, 74 MBLs, and 246 unaffected relatives. DNA was extracted from buccal cells (Mayo Clinic) and peripheral blood (NCI). Allele counts from gVCF files were extracted for specific genes and analyzed. Variants were annotated using the Variant Effect Predictor in Ensembl, genome build GRCh37 (http://useast.ensembl.org/index.html). Highly-sensitive flow cytometry of the blood was used to screen for MBL. CHIP was defined as in Genovese et al., 2014 as the presence of any of the following: A) disruptive mutations in ASXL1, TET2, or PPM1D; B) missense mutation p.617F in JAK2; C) disruptive or missense mutations in DNMT3Alocalized to exons 7 to 23D) any mutations reported at least 7 times in hematological malignancies in the Catalogue of Somatic Mutations in Cancer (COSMIC); and (E) No variant allelic fraction (VAF) threshold. CLL driver genes included disruptive or missense mutations in ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, or FBXW7with VAF greater than 0.1. Mutations from COSMIC and CLL driver genes were excluded if the minor allele frequency in 1000 Genomes was greater than 0.5%. Data was analyzed using logistic regression adjusting for age, sex, and site. Odds ratios (OR) and 95% confidence intervals (CI) were estimated.
Median age for CLL, MBL, and unaffected was 63, 61.5, and 54, respectively. The unaffected relatives were significantly younger than both the CLL and MBL groups (p<0.0001), with no difference between CLL and MBL (p=0.46). A total of 70 (12%) individuals from 54 different families had CHIP. CHIP was characterized in the majority of samples as mutations in DNMT3A, ASXL1, and TET2with 43, 9 and 8 individuals, respectively. As expected, CHIP was associated with age (p<0.001) but not sex (p=0.87). CHIP was present in 42 (17%) CLLs, 9 (12%) MBLs, and 19 (8%) unaffected relatives. There was a significant association between the presence of CHIP and CLL risk compared to unaffected relatives (OR= 2.19, 95% CI: 1.20 - 4.13, p=0.013) but not with MBL risk (OR=1.34, 95% CI: 0.54 - 3.11, p=0.51). Using the CLL driver genes, we found mutations in 119 (47%) CLL, 20 (27%) MBL, and 70 (29%) unaffected relatives. Mutations in CLL driver genes were associated with CLL risk compared to unaffected relatives (OR = 2.51, 95% CI: 1.64 - 3.87, p<0.0001), but there was no evidence for increased MBL risk (OR = 1.00). These results held when we subset the analysis to the Mayo only data where the DNA was extracted from buccal cells.
This study further supports the association between mutations in CHIP related genes and risk of hematologic malignancies, in particular CLL. This is the first report of the prevalence of CHIP in MBLs. Although MBL and CHIP represent clonal population of blood cells, our data suggest the clones are different. Mutations in CLL driver genes are also associated with CLL risk but show little to no evidence with increased MBL risk. Interestingly, our unaffected relatives of CLL cases also had a high frequency of CLL driver mutations. Future studies are needed to understand these acquired mutations (both in CHIP- and CLL driver- related genes) in the unaffected to MBL to CLL continuum.

Disclosures
Parikh: Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Research Funding. Ding:Merck: Research Funding. Cerhan:Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding; Nanostring: Research Funding. Shanafelt:Jansen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: 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; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.