Peer review of: Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact (2021)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
 In this revision, Zaidi et al. have looked more closely at the potentially novel subtype of CRC that
 is hypermutated, MSS, but not explained by any obvious mechanism.
 While it is interesting to see that the mutations in the hypermutated MSS cases that do not harbor
 a POLE exonuclease can be validated by Sanger sequencing, this raises further questions that were
 unfortunately not addressed in this manuscript:
 1) In addition to gene mutations in DNA repair genes (which the authors looked at), is this subtype
 different in any other way? Most importantly, did the authors perform a mutational signature
 analysis that could hint at the underlying mechanism?
 2) Is this novel subtype exclusive to this particular data set? If the same mutation calling methods
 were applied to the TCGA data set, would this subtype emerge as well?
3) Or is all of this possibly due to a data issue? Are these mutations introduced into these samples
some other way? Are these older FFPE samples? Are the authors using all commonly used variant
filters that remove sequencing artifacts (several are listed in methods, but are there others,
including 8OxoG)? See this paper for examples:
https://www.sciencedirect.com/science/article/pii/S2405471218300966
Reviewer #2 (Remarks to the Author):
I am happy that the authors have now responded satisfactorily to my previous concerns
Reviewer #3 (Remarks to the Author):
 I was asked to review this manuscript, which has undergone multiple revisions for Nature
 Communications, and specifically comment on the ongoing concerns of Reviewer #1.
 Overall, this is a nice descriptive manuscript with deep, paired tumor-normal sequencing of a large
 dataset of CRCs using a 205 gene targeted panel. The authors confirm known mutations of CRC
 and confirm the association between high TMB with improved prognosis.
Comments for Reviewer #1:
Reviewer #1 suggests that the authors should further examine a subset of tumors that are
hypermutated by the authors' definition but lack POLE/POLD1 mutation or MSI-H phenotype. While
I agree that examining this subtype would be interesting, this is beyond the scope of the authors'
original intent. I suspect that this small group is comprised of a combination of (1) CRCs with
isolated MSH6 deficiency, (2) the "long-tail" of MSS tumors with increased TMB, and (3) CRCs with
other mechanisms of hypermutation such as MUTYH-associated polyposis. Figuring out the
mechanism of each individual cancer would not add to the main conclusions of the current
manuscript.
 Additional Major Comment:
 I agree with comments of Reviewer #2 that hypermutation status confounds many aspects of the
 analysis. Namely, many of the mutations in HM specimens are expected to be passenger
 mutations and may not contribute to pathogenesis.
For example, there is a high likelihood that the associations between BMPR2, ZFHX3, KMT2B, and
 RYR1 and survival are false discoveries. Mutations in any these genes may be a surrogate marker
 of hypermutation, which is not accounted for in the statistical analysis. Note that RYR1 is a large
 protein with >5000 amino acids and therefore can be more susceptible to somatic mutations in the
 setting of HM for that reason alone.
 This statement is supported by the observation that mutation in each gene is associated with
 improved (rather than decreased) survival, and the association is no longer significant when
 HM/NHM is considered. This finding should be excluded from the paper, since it has the potential
 to confuse the scientific literature and lead others to perform experiments based on a false or
 misunderstood premise.
 Similarly in Table 3, most genes are more frequently mutated in the right colon, which is highly
 enriched for CRCs with HM. The data is said to have been adjusted for MSI, but it should also be
 adjusted for HM.
 In general, the authors should consider using more sophisticated statistical models for the gene-
 specific analyses that account for the mutational burden of each individual cancer.
 Additional Minor Comments:
 Line 136: The authors imply that mutations in MMR genes cause MSI phenotype in 46% of MSI
 CRCs. This sentence should be removed or clarified with deeper analysis. Abundant literature
 evidence show that about 80% of CRCs with MSI are caused by MLH1 hypermethylation. Most of
 mutations in MMR genes in the authors' dataset are expected to be due to incidental passenger
 mutations from to high mutational burden of MSI-H cancers. I see this frequently in my clinical
 NGS practice. This can be confirmed, if the authors choose, with MLH1 promoter methylation and
 MMR gene expression by immunohistochemistry (common tests the patients may have had as part
 of their routine clinical care).
 Line 141: The frequency of specific POLE and POLD1 mutations (for example, P286R) should be
 stated in the main section of the manuscript. The manuscript implies that all POLE and POLD1
 exonuclease mutations identified are pathogenic, which may be unintended or inaccurate.
 Figure 1A: The "green" and "aqua" datapoints are nearly indistinguishable. To the left of the dotted
 line, >80% of the events look orange to me, but closer examination of 1B shows that less than
 half of these cases actually have MSI. The way that black dotted lines connect 1A and 1B suggest
 that 1B is an expanded view of 1A; however, 1A and 1B have cases listed in different order.
 Perhaps the authors can work with an illustrator at Nature to ensure this figure accurately reflects
 the data for the reader.