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 study, Zaidi et al. performed deep targeted sequencing using a custom 205 gene AmpliSeq 
assay, including MSI status, in 2105 CRC cases with matched normals. The clinical annotation of 
the cases includes disease-specific survival data. Their main findings are the discovery of novel 
genes mutated in CRC, including PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, TGFBR2. Within hyper-
mutated tumors, they found a correlation between mutation rate and outcome. They also found 
TP53 mutations to be associated with the poorest prognosis. 

While the data set certainly has potential, there are several major issues with the manuscript. The 
analyses generally follow the pattern of the TCGA manuscript without too much novelty 
Furthermore, because the authors did not include the variant calls, their findings are currently not 
reproducible and a careful review of the data therefore not possible. In the absence of the 
mutation data, it appears that there are some issues with the mutation data that need to be 
corrected. 

The authors should address the following aspects to improve the study: 
1. The authors state that the data will be made available via dbGaP. In addition, however, the 
variant calls as well as all clinical attributes used in the analysis should be made available in the 
supplemental material and/or through a variant archive. For example, the data set could be 
deposited into the cBioPortal for Cancer Genomics. 

2. The authors find a rate of 19% for hypermutated tumors. This is much higher than reported in 
other studies (8.3% in TCGA, 8.7% in Yaeger et al). The overall mutation rate of the cohort was 
also higher than reported in previous studies (14.24 mutations per sample in 205 genes, vs 12.85 
mutations per gene in Yaeger at al., a study that sequenced more than twice the number of 
genes), suggesting potential issues with the mutation calls. To make the data better comparable to 
other data sets, the mutation rates should be reported as mutations per megabase. 

3. The authors found that a relatively large fraction (nearly one third) of hypermutated tumors 
were neither MSI-H nor had a POLE mutation. This number is surprising but could be interesting if 
real. Running MSIsensor, or a similar method, as an additional method to validate this finding 
could be helpful. This finding may, however, also be related to the apparent issue of an increased 
mutation count. 

4. The outcome analysis, which found BMPR2, ZFHX3, KMT2B, and RYR1 to be associated with 
better outcome is biased by the fact that these genes are more commonly mutated in 
hypermutated cases. While the authors acknowledge this (“In analyses stratified by hypermutated 
status, no genes were significantly associated with CRC-specific survival after accounting for 
multiple comparisons”), the presentation of the finding about these 4 genes is misleading and 
should be under-emphasized. 

5. In the pathway analysis, the authors should restrict analysis to only known or likely driver 
variants. They currently consider all variants, which, especially in hypermutated tumors, includes 
many variants of unknown significance. 

6. Identifying statistically recurrently mutated genes in hypermutated tumors is notoriously 
difficult, so the claim to have found novel genes is rather weak. Furthermore, genes such as 
TGFBR2 or BMPR2 harbor homopolymer stretches, which are often affected by frameshift 
insertions or deletions in MSI cases. 

7. The authors describe CTNNB1 mutations in Figure 4. Did the study identify the exon 4 in-frame 
deletions as well that were recently described in colorectal cancer? These mutations had previously 

been missed in other studies due to the mutation callers used. 

8. APC Mutation frequency in non hypermutated cases (62.7%) appears to be low compared to 
TCGA (81%) and Yaeger et al. (79%). 

9. The regression model used to evaluate differences in mutation status between tumor stage, 
tumor site, and sex lacks clarity, as it is unclear how these variables were corrected for in the 
model. 

10. Figure 3 does not convey any novel information. 

Reviewer #2 (Remarks to the Author): 

Zaidi et al. perform targeted sequencing for a panel of 205 colorectal cancer-associated genes in 
>2000 colorectal tumours to identify a number of recurrently mutated genes that had not met 
significance thresholds and to identify a number of genes that confer a favourable prognosis when 
mutated. They also show that approximately 1/3 hypermutated tumours that ought to benefit from 
checkpoint inhibitors do not have detectable micro satellite instability or POLE mutations and so 
might be missed by existing clinical assays. Previous studies such as MSK-IMPACT had instead 
termed any hypermutated samples without POLE mutations as ‘MSI-H/hypermutated’, which was 
likely misleading. 

This paper should be viewed in context of the recent MSK-IMPACT study which performed 
amplicon-based sequencing on 1134 mostly metastatic colorectal tumours. They were able to 
determine mutations, copy-number alterations and DNA-level rearrangements for a subset of 
genes. 
The main differences between Zaidi et al.’s study and MSK-IMPACT is that a) cohort size is larger 
thus providing enhanced statistical power to identify recurrent mutations and b) Zaidi et al. only 
report SNVs and indels, c) Zaidi et al. superimpose survival data (although similar data has been 
reported by the TCGA project). 

To my mind the authors have not sufficiently followed-up or given due attention to the novel 
findings of this project and have instead focussed too much on results that have been previously 
described in substantial detail by TCGA/MSK-IMPACT (e.g. improved survival in HM tumours, 
patterns of pathway activation). 

There is a need to expand on: 
Biological relevance - recurrent mutations in PRKCI, SPZ1, MUTYH, MAP2K4 (e.g. component of 
MAPK pathway, mutants cells shown to be sensitive to MEK inhibitors), FETUB 

Translational relevance - survival association with BMPR2, ZFHX3, KMT2B, and RYR1; proportion of 
hypermutated tumours probably missed by existing assays (e.g. speculate on how these tumours 
must be identified in a cost-effective way) 

Specific comments 

There are a number of strengths to the study including the size of the cohort, the robust mutation 
calling strategy using consensus calls from more than one mutation calling algorithm, successful 
validation of a random subset of mutation calls using Sanger sequencing and well justified 
selection of hypermutated samples 

The major weakness with the study is the lack of copy number alteration data which is a significant 
problem - recent work has shown that it is predominantly CNV that drives precursor lesion 

transformation and subclonal evolution in cancers (Cross et al, 2018). Indeed the title of the paper 
is thus somewhat misleading as it is only the SNV somatic mutation landscape that is described in 
this paper. 

This is a particular problem in 2 sections: 
1. Page 11 the section ascribing the residual transcriptional activity of p53, as only 5% of 
mutations carried biallelic SNV's. Therefore a large assumption is made that the single allele 
mutations were associated with LOH in the other allele. This doesnt appear to have been shown. 

2. Page 15 - the section discussing the favourable prognostic implication of BMPR2 (quoting 
Voorneveld et al 2014). The latter paper was very clear that the role of BMP signalling is skewed to 
pro-tumorigenic following loss of SMAD4. Although SNV mutation of SMAD4 is measured, this 
doesnt account for chrom 18q loss which is the most common way of impacting this pathway in 
CRC. 

Other comments. 
It would be interesting to do further analysis (?CNV, ?methylation) on the 40 or so tumours with 
very low SNV mutation burden (<5 mutations). What is driving these lesions? 

The hyper and ultramutated tumours without evidence for POLE or MMR are interesting and 
clinically relevant, as discussed in the manuscript. However the reader is left somewhat hanging as 
to the possible cause of this molecular phenotype. Were other replication fork genes in the 
targeted panel? Should these tumours undergo more extensive omic analysis to look for novel 
drivers of mutator phenotype? 

There is lack of clinicopathological information regarding tumours - age, sample histology e.g. 
mucinous/signet ring, confirmation that all samples were reviewed by expert histopathological 
prior to extraction, length of follow-up available for determination of survival data 

Some of the ‘novel’ recurrent mutations have been previously described - TGFBR2 
(https://www.ncbi.nlm.nih.gov/pubmed/17985359)