Molecular portraits of colorectal cancer morphological regions

Reviewer #1 (Public Review):

In this manuscript Budinska and colleagues aim to align morphologically distinct areas of colorectal cancers with the gene expression profiles and published signatures. They observe that distinct morphotypes such as serrated or mucinous align with certain subtypes but that these contradict the bulk subtype assignment. Although these data are of interest they may not be as novel as suggested by the authors and lack clarity in terms of patient selection and subtype definition.

1. Patient selection and tumor area selection are crucial for this study but not very carefully defined. Why are some core and others not? Figure referral is an issue here (sup figure 6 where all core and non-core samples are supposed to be according to the legend of Fig 4 is likely sup fig 7 but this is then a complete copy paste of Figure 4). In the methods it is stated that the core samples are based on limited contamination of additional morphotypes (<20%) but Fig 4 suggests that all tumours listed have multiple morphotypes.

2. CMS subtype should be performed with single sample predictor rather than CMScaller.

3. A couple of surprising observations need specification. MUC2 is a strong CMS3 reporter gene yet Mucinous tumours appear to end up in CMS4 rather than 3. Can the authors show that indeed stroma cells are very evident in these samples?

4. The SE PP and CT are assigned to CMS2, but in Figure 4 this appears a lot more variable than the authors would make the reader believe. The full data are not completely clear (see point 1)

5. The tumor response rates are rather weird as this is likely dependent on the complete tumour and not so much the subareas. It is not very well described what we see in this analysis.

6. Serrated adenomas have previously been aligned with CMS4. Is this different from serrated areas in cancers?

7. The fact that iCMS2 and iCMS3 align rather well with the current analysis of the distinct regions suggests that the analysis that was reported last year is the proper way to view tumor intrinsic signatures. The authors now propose a rather similar outcome to this issue which does take away a lot of the novelty of the findings of this study.

Reviewer #2 (Public Review):

In this paper, Budinská et al. consider whether morphological heterogeneity in colorectal cancer (CRC) might impact gene-expression based classifiers typically applied to bulk CRC tissues. To investigate this, the authors generated and analysed whole transcriptome microarrray profiling data from macro-dissected morphotype-specific tumour regions, bulk tumor and surrounding normal and stromal tissues.

The authors make a number of claims based on their analyses. Namely that
(1) morphotype-specific gene expression profiles and active molecular pathways can be identified and that (2) most gene expression-based classifiers make different predictions when applied to different morphotypes within the same tumour and when applied to morphotype-specific tumor regions versus bulk tumor tissue.

Overall, the manuscript provides an interesting histological/morphological framework through which we can consider heterogeneity in colorectal carcinoma and an approach by which we might improve the performance of gene expression-based classifiers in predicting clinical behaviour and/or responses to therapy. Exploration of CRC morphotypes and their differences was quite interesting. However, more work is needed to support the claims made by the authors. While I appreciate that the authors themselves identify limitations of their study within the manuscript, I believe awareness of these limitations is not reflected in some of the claims made in the abstract and at points in the main text when discussing the use of expression-based classifiers.

Author Response:

We are grateful to the reviewers for their insightful comments, suggestions, and criticism. In the updated version of the manuscript, all these will be properly reflected. Here we briefly address the main points raised:

Reviewer #1:

1.1. Patient selection and tumor area selection are crucial for this study but not very carefully defined. Why are some core and others not? Figure referral is an issue here (sup figure 6 where all core and non-core samples are supposed to be according to the legend of Fig 4 is likely sup fig 7 but this is then a complete copy paste of Figure 4). In the methods it is stated that the core samples are based on limited contamination of additional morphotypes (<20%) but Fig 4 suggests that all tumours listed have multiple morphotypes.

The tissue samples were obtained from a hospital cohort of patients with stage II-IV colorectal cancer (at diagnostic time), with no particular selection criteria imposed, as this was an exploratory study.

Tumor regions were marked for macro-dissection by an experienced pathologist following the standard practice for whole-tumor transcriptomics studies. The subregions (morphological regions) were marked by the same experienced pathologist for macro-dissection (in an adjacent section) and reassessed later with respect to their “morphological purity”. It is impossible to macro-dissect regions containing a single morphological pattern. Hence, those regions which contained significant amount (>=20%) of other morphologies were considered “non-core”, while the rest were called “core” regions. This distinction applies to morphological regions solely and not to whole-tumor samples.

Indeed, the reference in caption to Figure 4, should refer to Supp. Fig. 7 (which needs to be updated).

1.2. CMS subtype should be performed with single sample predictor rather than CMScaller.

We agree that a single-sample predictor for CMS is needed, however CMScaller is the de facto classifier for CMS (>130 citations) so we used it to illustrate the practical implications.

1.3. A couple of surprising observations need specification. MUC2 is a strong CMS3 reporter gene yet Mucinous tumours appear to end up in CMS4 rather than 3. Can the authors show that indeed stroma cells are very evident in these samples?

We do not have a direct estimation of the amount of stromal cells, but the high scores of the various fibroblast-related signatures in mucinous regions (Fig2 B, D) indicate that, indeed, there is an enrichment in stroma. In the follow-up study we plan to perform specific staining as well as spatial transcriptomics of these regions to further investigate our findings.

1.4. The SE PP and CT are assigned to CMS2, but in Figure 4 this appears a lot more variable than the authors would make the reader believe. The full data are not completely clear (see point 1).

In the paper, we transparently state that PP, SE, and CT were assigned to CMS2 in 62.5%, 41.7% and 41.9% of cases, respectively. These proportions referred to all samples for which CMSCaller made a prediction. In Fig.4, we also show the proportion of cases in which CMSCaller did not predict any subtype.

1.5. The tumor response rates are rather weird as this is likely dependent on the complete tumour and not so much the subareas. It is not very well described what we see in this analysis.

We did not compute any response rates but simple prognostic scores as (weighted, if weights were provided) means of genes in the specific signatures (see Methods). The question addressed was whether these scores were comparable between whole tumor and corresponding tumor regions (within same tumor). Given the observed (relative) variability, the more important follow-up question - which we cannot answer with our limited survival data – is whether a higher score in a region in comparison with whole-tumor is indeed indicative of a higher risk of relapse.

1.6. Serrated adenomas have previously been aligned with CMS4. Is this different from serrated areas in cancers?

We do not have data from adenomas to compare with the serrated carcinoma regions. But a comparison of (regions of) both traditional serrated and sessile serrated adenomas to serrated carcinoma would be interesting.

1.7. The fact that iCMS2 and iCMS3 align rather well with the current analysis of the distinct regions suggests that the analysis that was reported last year is the proper way to view tumor intrinsic signatures. The authors now propose a rather similar outcome to this issue which does take away a lot of the novelty of the findings of this study.

Our goal was not to propose another stratification paradigm for colorectal cancer, but rather to study the associations between morphology and transcriptome and its implications in practice. As such, our analyses are not limited to molecular subtypes and the respective observations are but a small part of our findings. Indeed, the intrinsic subtypes (iCMS 2/3) are stable and robust, as they are based on the genes expressed in epithelial cells, and they may well prove to be of clinical importance too. However, they do not cover all aspects (e.g. fibroblasts subtypes) and, as stated in Joanito et al. Nat Gen 54, pages 963–975 (2022), “iCMS, MSI status and CMS jointly inform the molecular classification of CRC”. Last, in our opinion, the molecular classification of CRC, while a useful point of view in tumour classification, is not covering all the necessary perspectives on tumour heterogeneity.

Reviewer #2:

2.1. Overall, the manuscript provides an interesting histological/morphological framework through which we can consider heterogeneity in colorectal carcinoma and an approach by which we might improve the performance of gene expression-based classifiers in predicting clinical behaviour and/or responses to therapy. Exploration of CRC morphotypes and their differences was quite interesting. However, more work is needed to support the claims made by the authors. While I appreciate that the authors themselves identify limitations of their study within the manuscript, I believe awareness of these limitations is not reflected in some of the claims made in the abstract and at points in the main text when discussing the use of expression-based classifiers.

We will improve the manuscript to stress the exploratory nature of our analyses and their limitations.