Purpose: Germ-line variants in CHEK2 have been associated with increased breast, thyroid, prostate, kidney, and colorectal cancer risk; however, the prevalence of somatic inactivation of CHEK2 in common cancer types is less clear. The aim of this study was to determine if somatic mutation and/or epigenetic modification play a role in development of sporadic breast, colon, or ovarian cancers. Experimental Design: We undertook combined genetic and epigenetic analysis of CHEK2 in sporadic primary breast, ovarian, and colon tumors [all exhibiting chromosome 22q loss of heterozygosity (LOH)] and cancer cell lines. Expression of Chk2 was assessed by immunohistochemistry in 119 ovarian tumors. Results: Two novel germ-line variants were identified; however, none of the primary tumors harbored somatic mutations.Two CpG clusters previously implicated in CHEK2 silencing were investigated for evidence of hypermethylation. No methylation was detected at the distal CpG island. The proximal CpG cluster was methylated in all tumor and normal DNA, suggesting that this might not represent a true CpG island and is not relevant in the control of CHEK2 expression. Twenty-three percent of ovarian tumors were negative for Chk2 protein by immunohistochemistry, but there was no significant correlation between LOH across the CHEK2 locus and intensity of Chk2 staining (P = 0.12). Conclusions: LOH across the CHEK2 locus is common in sporadic breast, ovarian, and colorectal cancers, but point mutation or epigenetic inactivation of the retained allele is uncommon. Loss of Chk2 protein in ovarian cancer was not associated with allelic status, suggesting that inactivation does not occur as a consequence of haploinsufficiency.The CHEK2 gene has recently been identified as a breast cancer susceptibility gene (1 -3), and some germ-line variants may increase the risk of colorectal, thyroid, prostate, and kidney cancer in specific populations (4, 5). CHEK2 encodes the human homologue of the Cds1 and RAD53 checkpoint kinases and plays a central role in the DNA damage checkpoint pathway. In response to ionizing radiation-induced DNA damage, Chk2 is activated by the ataxia-telangiectasia mutated (ATM) protein and subsequently phosphorylates several substrates, including p53, BRCA1, Mdm2, Cdc25A, and Cdc25C, leading to cell cycle arrest, activation of DNA repair mechanisms, or apoptosis. Activated Chk2 has been identified in early precursor lesions of urinary bladder, breast, lung, and colon carcinomas (but not in normal tissues) before the occurrence of genomic instability and malignant conversion (6), suggesting that DNA damage checkpoints are activated in the early stages of tumorigenesis. Therefore, mutations in CHEK2, or other genes involved in the ATM-Chk2-p53 pathway, may allow tumorigenic cells to evade normal cell cycle checkpoints, leading to aberrant cell proliferation and survival, increased genomic instability, and ultimately tumor progression.Although numerous studies have investigated for CHEK2 germ-line mutations in high-risk c...