High mutational burden in colorectal carcinomas with monoallelic POLE mutations: absence of allelic loss and gene promoter methylation

Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase epsilon or delta genes (POLEandPOLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performedPOLEandPOLD1exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somaticPOLEp.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. APOLEp.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (<50 years old, nonsyndromic), and there was seen a prominent T-cell infiltration in three of them. Furthermore, a somaticPOLEp.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somaticPOLEp.V411L and aPOLD1p.E279K, respectively, were found only focally, and TMBs were low. It is commonly assumed that compromise of one allele is sufficient, but this has not been specifically addressed. Therefore, resequencing of thePOLEorPOLD1mutations was done with DNA from tumor cells isolated by laser-capture microdissection. This demonstrated that the mutations were monoallelic, and there was no evidence of a "second hit", neither by allelic loss (allelotyping with polymorphic microsatellite markers), nor by promoter methylation (Pyromark CpG assays). Taken together, including at least the more common DNA polymerase mutations in NGS panels allows for straightforward identification of hypermutator-type colorectal carcinomas which often may be "immunoreactive". This is important at least in young patients or when a metastasizing stage of disease has been reached and immune-checkpoint therapy enters deliberation.