-->PONE-D-25-45234-->-->Performance of PREMM5, Clinical Criteria, and Immunohistochemistry for MMR Proteins in Genetic Risk Assessment of Mexican patients with Colorectal Cancer-->-->PLOS One

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“Some sequencing was performed through the GRACIAS project, with support from the Breast Cancer Research Foundation grant 24-210, and the Conquer Cancer Research Professorship in Breast Cancer Disparities (J.N. Weitzel).”

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Additional Editor Comments:

Both reviewers raise concerns regarding potential selection bias due to pre-screening/referral enrichment, which should be clearly described and its implications addressed prior to further consideration. Please also address the reviewers’ comments regarding incomplete IHC testing data and clarify/verify the statistical analyses and reporting.

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: Dear Editor,

I have reviewed the manuscript evaluating the performance of PREMM5, clinical criteria (Amsterdam II and revised Bethesda), and mismatch repair (MMR) immunohistochemistry for identifying germline pathogenic variants in Mexican patients with colorectal cancer. This study addresses an important gap by assessing genetic risk assessment tools in an underrepresented Hispanic/Mestizo population. The multicenter design, use of multigene panel testing, and focus on resource-limited clinical settings are notable strengths. The reported diagnostic performance of PREMM5 (AUC ≈0.82) and the high proportion of Lynch syndrome cases provide clinically relevant regional data.

However, several issues related to methodological transparency, potential bias, and reporting must be addressed before the manuscript is suitable for publication.

Major concerns

1. Selection bias and generalizability

The cohort consists exclusively of patients referred for genetic cancer risk assessment based on clinical criteria, abnormal IHC, or elevated PREMM5 scores, and is characterized by early age at diagnosis and a high prevalence of dMMR tumors. This referral-enriched design likely explains the high pathogenic variant yield (32.2%) and may inflate diagnostic performance estimates. The authors should clearly state that the findings are not generalizable to unselected colorectal cancer populations and temper conclusions accordingly.

2. Missing IHC data

IHC results were available for only about half of the cohort. The reasons for missing data are not described, and potential verification or selection bias is not discussed. The authors should report why IHC was unavailable and compare characteristics of patients with and without IHC results.

3. Statistical reporting clarity

The confidence interval for the PREMM5 AUC appears unusually narrow and should be verified. In addition, the primary outcome (Lynch syndrome vs. any germline pathogenic variant) should be clearly defined and used consistently throughout the manuscript.

4. Data availability

The current statement indicating that data are available upon request does not comply with PLOS ONE data sharing requirements. The dataset should be deposited in a public repository or an appropriate controlled-access justification provided.

5. Interpretation of non-Lynch gene findings

Approximately 22% of pathogenic variants were identified in non-Lynch genes. Given the variable and sometimes uncertain colorectal cancer risk associated with several of these genes, the recommendation for multigene testing should be presented more cautiously and with clearer discussion of clinical actionability.

Minor comments

• Improve grammatical consistency and reduce redundancy in the Introduction.

• Clarify technical details of sequencing and CNV detection across genes.

• Report missing percentage symbols and formatting inconsistencies in tables (e.g., Table 3).

• Consider adding a participant flow diagram to improve transparency.

• The limitations section should explicitly acknowledge referral bias, missing data, and limited generalizability.

Conclusion

This is a clinically relevant and potentially valuable study that contributes important data from an underrepresented population. However, clarification of cohort selection, improved transparency regarding missing data and statistical reporting, compliance with data availability requirements, and more cautious interpretation are necessary. I recommend major revision prior to further consideration.

Reviewer #2: The authors present an analysis of multiple screening strategies as applied to patients with colorectal cancer for the purpose of identifying those at increased likelihood of being identified on germline genetic testing as having Lynch syndrome, or a germline P/LP conferring other syndromic genetic cancer risk. The strategies analyzed here have been previously validated. This analysis is somewhat novel in its focus on a cohort of predominantly Mexican descent and presents data potentially useful to the CRC clinical community. Prior to being considered further, I recommend the follow comments/questions be addressed:

1 - in the Introduction the term "Genetic Cancer Risk Assessment" or GCRA is introduced, however what this actually is does not appear to be explained and needs to be defined. Is GCRA the clinical intake whereby patient personal and family health history are document? Is it the process whereby collected patient clinical information is reviewed for whether it meets guidelines for genetic testing? Is the GCRA the process by which patients are compared against Amsterdam, Bethesda, PREMM5 or IHC to assess whether they meet these criteria for germline testing? Does it include the process of germline genetic testing to assess the future cancer risk of a patient with CRC? Please clarify.

2 - In the Introduction is stated "GCRA is considered a standard of care for selected patients with colorectal cancer". Depending on the answer to the above re: GCRA, it is the standard of care for all patients with CRC to have their cancer risk assessed by collection, analysis and comparison of their clinical information to confirm their eligibility for germline testing. NCCN guidelines indicate germline testing should be considered in all patients with CRC. If the primary intent of this work is to validate PREMM5 as a strategy to be applied in resource constrained geographies for selectively allotting germline testing only to those patients at predicted higher relative risk for having a P/LP, this should be clarified at the outset.

3 - In the Methods the inclusion criteria are outlined, e.g. >=18 yo, diagnosis of CRC, age at diagnosis <50yo, etc. These inclusion criteria represent a "pre-screen" of patients with CRC which has implications for the total number/percentage of patients with P/LP identified after subsequent application of the screening strategies applied. The fact that these inclusion criteria functioned to pre-screen the total population of patients with CRC at the two participating institutions and select only certain qualifying CRC patients for the study needs to be explained in the text. Please add this to the Methods.

4 - To the comment above, how many patients with CRC were "pre-screened" using the inclusion criteria in order to obtain the 208 patients that were enrolled? i.e. what was the total population of patients with CRC seen at these two institutions during the enrollment timeframe of the study?

5 - Methods paragraph 3 is noted patients were consented to the study then underwent GCRA. Was prescreening using inclusion criteria performed before or after obtaining consent, or were only those patients who met the inclusion criteria invited to give their consent to be part of the study? Here again, there is a need to define what it means that patients "underwent GCRA". Please clarify.

6 - Methods section paragraph 4 notes lack of genetic testing coverage for PMS2 exons 11-15 and PTEN promoter region. This should be reiterated in the Discussion as a limitation as it impacts interpretation of the overall P/LP prevalence in the tested population with some studies suggesting >10% of PMS2 P/LP occurring in exons 11-5.

7 - Also in Methods paragraph 4, second to last line indicates "...MSH2 and MSH6 were also analyzed for number variants...", where I believe "copy-number variants" is intended, please correct if so.

8 - Discussion section, first line states "individuals with CRC who met clinical criteria for GCRA". Again, what does GCRA mean? and what are the clinical criteria that were met for a patient to undergo GCRA? This needs to be clarified as it is currently unclear if the clinical criteria are the inclusion criteria, the criteria represented by each of the screening strategies (PREMM5, Amsterdam, Bethesda, IHC), NCCN criteria or something else.

9 - Discussion section, second paragraph, "...77.6% of hereditary CRC cases were attributed to Lynch syndrome..." should be changed to, "77.6% of selected hereditary CRC cases...", as all patients were prescreened prior to enrollment and all enrolled patients were screened via PREMM5, Amsterdam, Bethesda or IHC prior to germline testing.

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Reviewer #1: Yes: Mahmut Cerkez Ergoren

Reviewer #2: Yes: Edward Esplin

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Performance of PREMM5, Clinical Criteria, and Immunohistochemistry for MMR Proteins in Genetic Risk Assessment of Mexican patients with Colorectal Cancer

PONE-D-25-45234R1

Dear Dr. Chávarri-Guerra,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #2: Yes

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-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: Yes

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Reviewer #2: The author have adequately addressed my previously provided comments and recommendations, nothing further to add.

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Reviewer #2: Yes: Edward D. Esplin

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