Causal associations between plasma proteins and prostate cancer: a Proteome-Wide Mendelian Randomization

Reviewer #1 (Public review):

Summary:

In Causal associations between plasma proteins and prostate cancer: a Proteome-Wide Mendelian Randomization, the authors present a manuscript which seeks to identify novel markers for prostate cancer through analysis of large biobank-based datasets and to extend this analysis to potential therapeutic targets for drugs. This is an area that is already extensively researched, but remains important, due to the high burden and mortality of prostate cancer globally.

Strengths:

The main strengths of the manuscript are the identification and use of large biobank data assets, which provide large numbers of cases and controls, essential for achieving statistical power. The databases used (deCODE, FinnGen, and the UK Biobank) allow for robust numbers of cases and controls. The analytical method chosen, Mendelian Randomization, is appropriate to the problem. Another strength is the integration of multi-omic datasets, here using protein data as well as GWAS sources to integrate genomic and proteomic data.

Weaknesses:

The main weaknesses of the manuscript relate to the following areas:

(1) The failure of the study to analyse the data in the context of other closely related conditions such as benign prostatic hyperplasia (BPH) or lower urinary tract symptoms (LUTS), which have some pathways and biomarkers in common, such as inflammatory pathways (including complement) and specific markers such as KLK3. As a consequence, it is not possible for readers to know whether the findings are specific to prostate cancer or whether they are generic to prostate dysfunction. Given the prevalence of prostate dysfunction (half of men reaching their sixth decade), the potential for false positives and overtreatment from non-specific biomarkers is a major problem, resulting in the evidence presented in this manuscript being weak. Other researchers have addressed this issue using the same data sources as presented here, for example, in this paper, looking at BPH in the UK Biobank population.
https://www.nature.com/articles/s41467-018-06920-9

(2) There is no discussion of Gleason scores with regard to either biomarkers or therapies, and a general lack of discussion around indolent disease as compared with more aggressive variants. These are crucial issues with regard to the triage and identification of genomically aggressive localized prostate cancers. See, for example, the work set out in: https://doi.org/10.1038/nature20788 .

(3) An additional issue is that the field of PCa research is fast-moving. The manuscript cites ~80 references, but too few of these are from recent studies, and many important and relevant papers are not included. The manuscript would be much stronger if it compared and contrasted its findings with more recent studies of PCa biomarkers and targets, especially those concerned with multi-omics and those including BPH.

(4) The Methods section provides no information on how the Controls were selected. There is no Table providing cohort data to allow the reader to know whether there were differences in age, BMI, ethnic grouping, social status or deprivation, or smoking status, between the Cases and Controls. These types of data are generally recorded in Biobank data, so this sort of analysis should be possible, or if not, the authors' inability to construct an appropriately matched set of Controls should be discussed as a Limitation.

Assessing impact:

Because of the weaknesses of the approach identified above, without further additions to the manuscript, the likely impact of the work on the field is minimal. There is no significant utility of the methods and data to the community, because the data are pre-existing and are not newly introduced to the community in this work, and Mendelian randomization is a well-described approach in common use, and therefore, the assets and methods described in the manuscript are not novel. With regard to the authors achieving their aims, without assessing specificity and without setting their findings in the context of the latest literature, the authors (and readers) cannot know or assess whether the biomarkers identified or the druggable targets will be useful in the clinic.

In conclusion, adding additional context and analysis to the manuscript would both help readers interpret and understand the work and would also greatly enhance its significance. For example, the UK Biobank includes data on men with BPH / LUTS, as analysed in this paper, for example, https://doi.org/10.1038/s41467-018-06920-9. By extending this analysis to identify which biomarkers and druggable targets are specific to PCa, and which are generic to prostate dysfunction, the authors would substantially reduce the risks of diagnostic false positives. This would help to manage the risks of inappropriate treatment or overtreatment.

Reviewer #2 (Public review):

This is potentially interesting work, but the analyses are attempted in a rather scattergun way, with little evident critical thought. The structure of the work (Results before Methods) can work in some manuscripts, but it is not ideal here. The authors discuss results before we know anything about the underlying data that the results come from. It gives the impression that the authors regard data as a resource to be exploited, without really caring where the data comes from. The methods can provide meaningful insights if correctly used, but while I don't have reasons to doubt that the analyses were conducted correctly, findings are presented with little discussion or interpretation. No follow-up analyses are performed.

In summary, there are likely some gems here, but the whole manuscript is essentially the output from an analytic pipeline.

Taking the researchers aims in turn:

(1) Meta-GWAS - while combining two datasets together can provide additional insights, the contribution of this analysis above existing GWAS is not clear. The PRACTICAL consortium has already reported the GWAS of 70% of these data. What additional value does this analysis provide? (Likely some, but it's not clear from the text.) Also, the presentation of results is unclear - authors state that only 5 gene regions contained variants at p<5x10-8, but Figure 1 shows dozens of hits above 5x10-8. Also, the red line in Figure 1 (supposedly at 5x10-8) is misplaced.

(2) Cross-phenotype analysis. It is not really clear what this analysis is, or why it is done. What is the iCPAGdb? A database? A statistical method? Why would we want to know cross-phenotype associations? What even are these? It seems that the authors have taken data from an online resource and have written a paragraph based on this existing data with little added value.

(3) PW-MR. I can see the value of this work, but many details are unclear. Was this a two-sample MR using PRACTICAL + FinnGen data for the outcome? How many variants were used in key analyses? Again, the description of results is sparse and gives little added value.

(4) Colocalization - seems clear to me.

(5) Additional post-GWAS analyses (pathway + druggability) - again, the analyses seem to be performed appropriately, although little additional insight other than the reporting of output from the methods.

Minor points:

(6) The stated motivation for this work is "early detection". But causality isn't necessary for early detection. If the authors are interested in early detection, other analysis approaches are more appropriate.

(7) The authors state "193 proteins were associated with PCa risk", but they are looking at MR results - these analyses test for disease associations of genetically-predicted levels of proteins, not proteins themselves.

Strengths:

The data and methods used are state-of-the-art.

Weaknesses:

The reader will have to provide their own translational insight.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In Causal associations between plasma proteins and prostate cancer: a Proteome-Wide Mendelian Randomization, the authors present a manuscript which seeks to identify novel markers for prostate cancer through analysis of large biobank-based datasets and to extend this analysis to potential therapeutic targets for drugs. This is an area that is already extensively researched, but remains important, due to the high burden and mortality of prostate cancer globally.

Strengths:

The main strengths of the manuscript are the identification and use of large biobank data assets, which provide large numbers of cases and controls, essential for achieving statistical power. The databases used (deCODE, FinnGen, and the UK Biobank) allow for robust numbers of cases and controls. The analytical method chosen, Mendelian Randomization, is appropriate to the problem. Another strength is the integration of multi-omic datasets, here using protein data as well as GWAS sources to integrate genomic and proteomic data.

Thank you for your positive feedback regarding the overall quality of our work and we greatly appreciate you taking time and making effort in reviewing our manuscript.

Weaknesses:

The main weaknesses of the manuscript relate to the following areas:

(1) The failure of the study to analyse the data in the context of other closely related conditions such as benign prostatic hyperplasia (BPH) or lower urinary tract symptoms (LUTS), which have some pathways and biomarkers in common, such as inflammatory pathways (including complement) and specific markers such as KLK3. As a consequence, it is not possible for readers to know whether the findings are specific to prostate cancer or whether they are generic to prostate dysfunction. Given the prevalence of prostate dysfunction (half of men reaching their sixth decade), the potential for false positives and overtreatment from non-specific biomarkers is a major problem, resulting in the evidence presented in this manuscript being weak. Other researchers have addressed this issue using the same data sources as presented here, for example, in this paper, looking at BPH in the UK Biobank population. https://www.nature.com/articles/s41467-018-06920-9

Thank you for your valuable comment. We fully agree that biomarker development must prioritize specificity to avoid overtreatment. While our study is a foundational step toward identifying potential therapeutic targets or complementary biomarkers for prostate cancer (PCa)—not as a direct endorsement of these proteins for standalone clinical diagnosis. Mendelian randomization (MR) analysis strengthens causal inference by design, and we further ensured robustness through sensitivity analyses (e.g. MR-Egger regression for pleiotropy, Bonferroni correction for multiple testing). These methods distinguish true causal effects from nonspecific associations. Importantly, while PSA’s lack of specificity is widely recognized, its role in reducing PCa mortality underscores the value of biomarker-driven screening. Our findings align with the need to integrate multiple markers (e.g. combining a novel protein with PSA) to improve diagnostic precision. Translating these causal insights into clinical tools remains challenging but represents a necessary next step, and we emphasize that this work provides a rigorous starting point for future validation studies.

(2) There is no discussion of Gleason scores with regard to either biomarkers or therapies, and a general lack of discussion around indolent disease as compared with more aggressive variants. These are crucial issues with regard to the triage and identification of genomically aggressive localized prostate cancers. See, for example, the work set out in: https://doi.org/10.1038/nature20788

Thank you for pointing this out. We acknowledge that our original analysis did not directly address this critical issue due to a key data limitation: the publicly available GWAS summary statistics for PCa (from openGWAS and FinnGen) do not provide genetic associations stratified by phenotypic severity or molecular subtypes. This limitation precluded MR analysis of proteins specifically linked to aggressive disease. To partially bridge this gap, we integrate evidence from recent studies in the revised Discussion section to explore the relevance of potential biomarkers to aggressive PCa.

(3) An additional issue is that the field of PCa research is fast-moving. The manuscript cites ~80 references, but too few of these are from recent studies, and many important and relevant papers are not included. The manuscript would be much stronger if it compared and contrasted its findings with more recent studies of PCa biomarkers and targets, especially those concerned with multi-omics and those including BPH.

Thank you for your professional comments. We have rigorously updated the manuscript to include more recent publications and we systematically compare and contrast our findings with these recent studies in the revised Discussion section.

(4) The Methods section provides no information on how the Controls were selected. There is no Table providing cohort data to allow the reader to know whether there were differences in age, BMI, ethnic grouping, social status or deprivation, or smoking status, between the Cases and Controls. These types of data are generally recorded in Biobank data, so this sort of analysis should be possible, or if not, the authors' inability to construct an appropriately matched set of Controls should be discussed as a Limitation.

We thank the reviewer for raising this important methodological concern. We have expanded the Limitations section to state it.

Reviewer #2 (Public review):

This is potentially interesting work, but the analyses are attempted in a rather scattergun way, with little evident critical thought. The structure of the work (Results before Methods) can work in some manuscripts, but it is not ideal here. The authors discuss results before we know anything about the underlying data that the results come from. It gives the impression that the authors regard data as a resource to be exploited, without really caring where the data comes from. The methods can provide meaningful insights if correctly used, but while I don't have reasons to doubt that the analyses were conducted correctly, findings are presented with little discussion or interpretation. No follow-up analyses are performed.

In summary, there are likely some gems here, but the whole manuscript is essentially the output from an analytic pipeline.

We thank the reviewer for the thoughtful evaluation of our work.

Taking the researchers aims in turn:

(1) Meta-GWAS - while combining two datasets together can provide additional insights, the contribution of this analysis above existing GWAS is not clear. The PRACTICAL consortium has already reported the GWAS of 70% of these data. What additional value does this analysis provide? (Likely some, but it's not clear from the text.) Also, the presentation of results is unclear - authors state that only 5 gene regions contained variants at p<5x10-8, but Figure 1 shows dozens of hits above 5x10-8. Also, the red line in Figure 1 (supposedly at 5x10-8) is misplaced.

Thank you very much for your feedback. Although the PRACTICAL consortium constituted the majority of PCa GWAS data, our meta-analysis integrating FinnGen data enhanced statistical power enabling robust detection of low-frequency variants with minor allele frequencies. Moreover, FinnGen's Finnish ancestry (genetic isolate) helps distinguish population-specific effects. The presentation of results showed the top 5 gene regions contained variants at p < 5×10^-8. We apologize for not noticing that the red line was not displayed correctly in the original figures included in the manuscript. We have updated it in the revised manuscript.

(2) Cross-phenotype analysis. It is not really clear what this analysis is, or why it is done. What is the iCPAGdb? A database? A statistical method? Why would we want to know cross-phenotype associations? What even are these? It seems that the authors have taken data from an online resource and have written a paragraph based on this existing data with little added value.

We thank you for raising this issue. The iCPAGdb (interactive Cross-Phenotype Analysis of GWAS database) is an integrative platform that systematically identifies cross-phenotype associations and evaluates genetic pleiotropy by leveraging LD-proxy associations from the NHGRI-EBI GWAS Catalog. The pathogenesis and progression of prostate cancer constitute a complex pathophysiological continuum characterized by dynamic multisystem interactions, extending beyond singular molecular pathway dysregulation to encompass coordinated disruptions across endocrine regulation, immune microenvironment remodeling, and metabolic reprogramming. Therefore, it is indispensable for discriminating primary pathogenic drivers from secondary compensatory responses, ultimately informing the development of precision therapeutic strategies.

(3) PW-MR. I can see the value of this work, but many details are unclear. Was this a two-sample MR using PRACTICAL + FinnGen data for the outcome? How many variants were used in key analyses? Again, the description of results is sparse and gives little added value.

We thank you for raising this issue. Two-sample MR refers to an analytical design where genetic instruments for the exposure (plasma proteins) and genetic associations with the outcome (PCa) are derived from non-overlapping populations. This ensures complete sample independence between exposure and outcome datasets to avoid confounding biases, regardless of whether the outcome data originate from single or multiple cohorts. The meta-analysis of PRACTICAL and FinnGen GWAS generates 27,210 quality-controlled variants (p < 5×10^-8, MAF ≥ 1%, LD-clumped r² < 0.1) used in key analyses.

(4) Colocalization - seems clear to me.

(5) Additional post-GWAS analyses (pathway + druggability) - again, the analyses seem to be performed appropriately, although little additional insight other than the reporting of output from the methods.

The post-MR druggability and pathway analyses serve two primary scientific purposes: (1) therapeutic prioritization - systematically evaluating which MR-identified proteins represent tractable drug targets (either through existing FDA-approved agents or compounds in clinical development) with direct relevance to cancer or PCa management, and (2) mechanistic hypothesis generation - mapping these candidate proteins to coherent biological pathways to guide future functional validation studies investigating their causal roles in prostate carcinogenesis.

Minor points:

(6) The stated motivation for this work is "early detection". But causality isn't necessary for early detection. If the authors are interested in early detection, other analysis approaches are more appropriate.

We appreciate your insightful feedback. While early detection is one motivation for this work, our primary goal extends to identifying causally implicated proteins that may serve as intervention targets for PCa prevention or therapy. Establishing causality is critical for distinguishing biomarkers that drive disease pathogenesis from those that are secondary to disease progression, as the former holds greater specificity for early detection and prioritization of therapeutic targets. While we acknowledge that validation for early detection may require additional methodologies, MR analysis provides a foundational step by prioritizing candidate proteins with causal links to disease. This approach ensures that downstream efforts focus on biomarkers and targets with the greatest potential to alter disease trajectories, rather than merely correlative markers.

(7) The authors state "193 proteins were associated with PCa risk", but they are looking at MR results - these analyses test for disease associations of genetically-predicted levels of proteins, not proteins themselves.

In MR, the exposure of interest is the lifelong effect of genetically predicted protein levels. This approach is designed to infer causality while avoiding confounding and reverse causation, as genetic variants are fixed at conception and unaffected by disease processes. When we state “193 proteins were associated with PCa risk,” we specifically refer to proteins whose genetically predicted levels (based on instrument SNPs from protein QTLs) show causal links to PCa. Importantly, MR does not measure the direct association between observed protein concentrations and disease. Instead, it estimates the lifelong causal effect of protein levels predicted by genetics. This distinction is critical for disentangling cause from consequence. For example, a protein elevated due to tumor progression would not be identified as causal in MR if its genetic predictors are unrelated to PCa risk.

We acknowledge that clinical translation requires further validation of these proteins in observational studies measuring actual protein levels. However, MR provides a robust first step by prioritizing candidates with causal roles, thereby reducing the risk of investing in biomarkers confounded by disease processes.