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To test the traditional model of tumor progression, Darwinian-type evolution, against the more recent Big Bang model, we selected 6 microsatellite-stable colorectal standard-type adenocarcinomas and their synchronous lymph node and liver metastases. Somatic genomic variants were identified by whole-exome sequencing (WES) of large tumor fragments from the primaries and 1 liver metastasis each, and used to design targeted resequencing next-generation sequencing (NGS) panels, 1 per case. Targeted deep resequencing (mean coverage, 2725; median, 2222) was performed with DNA from punch samples (1-mm tissue microarrayer needles) obtained from different regions of the primaries and their metastases. In total, 255 genomic variants were interrogated in 108 punch samples. Clonal heterogeneity was infrequent: a pattern of clonal heterogeneity consistent with a role in metastasis formation was observed only in 1 case in a single gene (p. Asp604Tyr of the PTPRT gene). However, when comparing variant allele frequencies (VAFs) of genomic variants in adjacent positions on chromosomes ("matched genomic variant loci") across punch samples, differences that exceeded 2 SD of the NGS assay variations (ad hoc dubbed VAF dysbalance) were observed in 7.1% of the punch samples (2.6%-12.0% per case), which indicates an intricate intermixing of mutated and nonmutated tumor cells ("intrinsic heterogeneity"). Additional OncoScan array analyses on a subset of the punch samples (31 in total) showed gross genomic aberrations as a possible explanation in only some (39.2%) of the matched genomic variant loci with VAF dysbalance. Our study provides a fairly direct (statistical model-free) view of the genomic states of microsatellite-stable colorectal carcinomas and their metastases, and suggests that Darwinian-type tumor evolution is not the key pathway of the metastasizing disease; instead, we recorded an "intrinsic" genomic heterogeneity, which may echo an initial Big Bang-like event., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase ε or δ genes (POLE and POLD1, 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 performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.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 somatic POLE p.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somatic POLE p.V411L and a POLD1 p.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 the POLE or POLD1 mutations 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.