AbstractRecent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanningApcin a subset of sporadic and radiation-induced intestinal tumors ofApcMin/+mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1ApcMin/+mice that were either left untreated or irradiated with 2 Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable β-catenin, which is a hallmark ofApc+allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving theApc+allele in β-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involvingApc+was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving theApc+allele as a causative radiation-related event in intestinal tumors ofApcMin/+mice, providing an accurate approach for attributing individual tumors to radiation exposure.