Title: Evaluation of PINK1 Protein Expression as a Predictive Marker for the Efficacy of
Adjuvant Chemotherapy in Colorectal Cancer: A Retrospective Study
Version: 3 Date: 08 Jun 2025
Reviewer's report:
This manuscript presents a retrospective study evaluating the prognostic significance of PINK1
expression in colorectal cancer (CRC) patients undergoing adjuvant chemotherapy. The authors
comprehensively analyze immunohistochemical expression of PINK1 and LC3 in surgical
specimens, associate them with clinical outcomes, and perform transcriptomic analysis in a
subset of recurrent cases to explore potential mechanistic underpinnings. The study addresses
an important clinical issue, given the limited predictive markers for chemotherapy response
in CRC. The manuscript is well-structured and includes detailed experimental methodology,
survival analyses, and gene enrichment results. However, several concerns should be addressed
to strengthen the validity and clarity of the findings.
Major Concerns
The primary limitation is the lack of a significant association between PINK1 or LC3 expression
and recurrence-free or overall survival in the full cohort (n = 82), which weakens the general
applicability of the marker. While the finding of PINK1 as a prognostic factor in the recurrent
subgroup (n = 25) is interesting, it is derived from a small, potentially underpowered subset, and
subject to selection bias. Furthermore, recurrence was already established in this group, making
the prognostic utility of PINK1 for recurrence prediction questionable; it may instead reflect
post-recurrence tumor biology rather than serve as a predictive biomarker.
Additionally, the classification of high and low PINK1 expression based on combined staining
intensity and percentage is not fully justified. While the scoring system is explained, the cutoff
for “high” (score 3–4) versus “low” (score 0–2) lacks validation or reference to prior literature,
and it is unclear whether interobserver variability was assessed beyond consensus resolution.
Moreover, LC3 data appear less robust and are not meaningfully discussed, although they are
included in the analysis.
The RNA-seq analysis is of added value but limited by the extremely small number of
samples (n = 11), especially given the division into high and low PINK1 groups (n = 4 vs 7).
Consequently, conclusions drawn from enrichment and differential expression analysis must be
interpreted with great caution. The discrepancy between RNA and protein levels of PINK1 is
acknowledged, but deeper discussion on potential causes—such as post-translational regulation
or tumor heterogeneity—is needed. No phospho-specific PINK1 analysis was performed,
limiting mechanistic insight.
Minor Concerns
Some figures and tables lack clarity. Figure legends could be more detailed, particularly for the
immunohistochemical panels. In Figure 1, scale bars are present but not described in the legend.
The volcano plot (Figure 4a) should label key genes to enhance interpretability. Tables 1–3
present useful clinical data, but many p-values are not discussed in the main text, and important
clinical parameters such as MSI status are missing. Furthermore, typographical errors such as
“experss” in tables should be corrected.
In the methodology, the description of RNA-seq processing is somewhat vague. Details on
data normalization, software versions, and statistical thresholds for pathway enrichment should
be more explicitly stated. In addition, the use of both DAVID and GSEA is appropriate,
but the redundancy between GO and KEGG terms could be streamlined to avoid overlap in
interpretation.
The authors mention several limitations in the discussion, but a clearer statement of how these
impact the conclusions is warranted. Specifically, the heterogeneity in chemotherapy regimens
and the lack of a control (surgery-only) group limit the ability to ascribe PINK1 expression as
a marker of chemotherapy efficacy. Additionally, the retrospective nature and small size of the
recurrence subgroup compromise generalizability.
The manuscript is written in acceptable English, but several grammatical and stylistic issues
require attention. Sentences such as “High PINK1 expression was observed in 42.2% (35 cases)
of the cases” should be streamlined. Instances of word redundancy, tense inconsistencies, and
awkward phrasing (e.g., “treatment resistance by enhancing cellular stress tolerance”) should
be revised for clarity. Citations are generally appropriate and cover recent literature; however,
some statements would benefit from stronger referencing, especially those regarding mitophagy
mechanisms and PINK1’s role in cancer.
Title: Evaluation of PINK1 Protein Expression as a Predictive Marker for the Efficacy of
Adjuvant Chemotherapy in Colorectal Cancer: A Retrospective Study
Version: 3 Date: 08 Jun 2025
Reviewer's report:
This manuscript presents a retrospective study evaluating the prognostic significance of PINK1
expression in colorectal cancer (CRC) patients undergoing adjuvant chemotherapy. The authors
comprehensively analyze immunohistochemical expression of PINK1 and LC3 in surgical
specimens, associate them with clinical outcomes, and perform transcriptomic analysis in a
subset of recurrent cases to explore potential mechanistic underpinnings. The study addresses
an important clinical issue, given the limited predictive markers for chemotherapy response
in CRC. The manuscript is well-structured and includes detailed experimental methodology,
survival analyses, and gene enrichment results. However, several concerns should be addressed
to strengthen the validity and clarity of the findings.
Major Concerns
The primary limitation is the lack of a significant association between PINK1 or LC3 expression
and recurrence-free or overall survival in the full cohort (n = 82), which weakens the general
applicability of the marker. While the finding of PINK1 as a prognostic factor in the recurrent
subgroup (n = 25) is interesting, it is derived from a small, potentially underpowered subset, and
subject to selection bias. Furthermore, recurrence was already established in this group, making
the prognostic utility of PINK1 for recurrence prediction questionable; it may instead reflect
post-recurrence tumor biology rather than serve as a predictive biomarker.
Additionally, the classification of high and low PINK1 expression based on combined staining
intensity and percentage is not fully justified. While the scoring system is explained, the cutoff
for “high” (score 3–4) versus “low” (score 0–2) lacks validation or reference to prior literature,
and it is unclear whether interobserver variability was assessed beyond consensus resolution.
Moreover, LC3 data appear less robust and are not meaningfully discussed, although they are
included in the analysis.
The RNA-seq analysis is of added value but limited by the extremely small number of
samples (n = 11), especially given the division into high and low PINK1 groups (n = 4 vs 7).
Consequently, conclusions drawn from enrichment and differential expression analysis must be
interpreted with great caution. The discrepancy between RNA and protein levels of PINK1 is
acknowledged, but deeper discussion on potential causes—such as post-translational regulation
or tumor heterogeneity—is needed. No phospho-specific PINK1 analysis was performed,
limiting mechanistic insight.
Minor Concerns
Some figures and tables lack clarity. Figure legends could be more detailed, particularly for the
immunohistochemical panels. In Figure 1, scale bars are present but not described in the legend.
The volcano plot (Figure 4a) should label key genes to enhance interpretability. Tables 1–3
present useful clinical data, but many p-values are not discussed in the main text, and important
clinical parameters such as MSI status are missing. Furthermore, typographical errors such as
“experss” in tables should be corrected.
In the methodology, the description of RNA-seq processing is somewhat vague. Details on
data normalization, software versions, and statistical thresholds for pathway enrichment should
be more explicitly stated. In addition, the use of both DAVID and GSEA is appropriate,
but the redundancy between GO and KEGG terms could be streamlined to avoid overlap in
interpretation.
The authors mention several limitations in the discussion, but a clearer statement of how these
impact the conclusions is warranted. Specifically, the heterogeneity in chemotherapy regimens
and the lack of a control (surgery-only) group limit the ability to ascribe PINK1 expression as
a marker of chemotherapy efficacy. Additionally, the retrospective nature and small size of the
recurrence subgroup compromise generalizability.
The manuscript is written in acceptable English, but several grammatical and stylistic issues
require attention. Sentences such as “High PINK1 expression was observed in 42.2% (35 cases)
of the cases” should be streamlined. Instances of word redundancy, tense inconsistencies, and
awkward phrasing (e.g., “treatment resistance by enhancing cellular stress tolerance”) should
be revised for clarity. Citations are generally appropriate and cover recent literature; however,
some statements would benefit from stronger referencing, especially those regarding mitophagy
mechanisms and PINK1’s role in cancer.
Please review this paper.
user
Title: Evaluation of PINK1 Protein Expression as a Predictive Marker for the Efficacy of
Adjuvant Chemotherapy in Colorectal Cancer: A Retrospective Study
Version: 3 Date: 08 Jun 2025
Reviewer's report:
This manuscript presents a retrospective study evaluating the prognostic significance of PINK1
expression in colorectal cancer (CRC) patients undergoing adjuvant chemotherapy. The authors
comprehensively analyze immunohistochemical expression of PINK1 and LC3 in surgical
specimens, associate them with clinical outcomes, and perform transcriptomic analysis in a
subset of recurrent cases to explore potential mechanistic underpinnings. The study addresses
an important clinical issue, given the limited predictive markers for chemotherapy response
in CRC. The manuscript is well-structured and includes detailed experimental methodology,
survival analyses, and gene enrichment results. However, several concerns should be addressed
to strengthen the validity and clarity of the findings.
Major Concerns
The primary limitation is the lack of a significant association between PINK1 or LC3 expression
and recurrence-free or overall survival in the full cohort (n = 82), which weakens the general
applicability of the marker. While the finding of PINK1 as a prognostic factor in the recurrent
subgroup (n = 25) is interesting, it is derived from a small, potentially underpowered subset, and
subject to selection bias. Furthermore, recurrence was already established in this group, making
the prognostic utility of PINK1 for recurrence prediction questionable; it may instead reflect
post-recurrence tumor biology rather than serve as a predictive biomarker.
Additionally, the classification of high and low PINK1 expression based on combined staining
intensity and percentage is not fully justified. While the scoring system is explained, the cutoff
for “high” (score 3–4) versus “low” (score 0–2) lacks validation or reference to prior literature,
and it is unclear whether interobserver variability was assessed beyond consensus resolution.
Moreover, LC3 data appear less robust and are not meaningfully discussed, although they are
included in the analysis.
The RNA-seq analysis is of added value but limited by the extremely small number of
samples (n = 11), especially given the division into high and low PINK1 groups (n = 4 vs 7).
Consequently, conclusions drawn from enrichment and differential expression analysis must be
interpreted with great caution. The discrepancy between RNA and protein levels of PINK1 is
acknowledged, but deeper discussion on potential causes—such as post-translational regulation
or tumor heterogeneity—is needed. No phospho-specific PINK1 analysis was performed,
limiting mechanistic insight.
Minor Concerns
Some figures and tables lack clarity. Figure legends could be more detailed, particularly for the
immunohistochemical panels. In Figure 1, scale bars are present but not described in the legend.
The volcano plot (Figure 4a) should label key genes to enhance interpretability. Tables 1–3
present useful clinical data, but many p-values are not discussed in the main text, and important
clinical parameters such as MSI status are missing. Furthermore, typographical errors such as
“experss” in tables should be corrected.
In the methodology, the description of RNA-seq processing is somewhat vague. Details on
data normalization, software versions, and statistical thresholds for pathway enrichment should
be more explicitly stated. In addition, the use of both DAVID and GSEA is appropriate,
but the redundancy between GO and KEGG terms could be streamlined to avoid overlap in
interpretation.
The authors mention several limitations in the discussion, but a clearer statement of how these
impact the conclusions is warranted. Specifically, the heterogeneity in chemotherapy regimens
and the lack of a control (surgery-only) group limit the ability to ascribe PINK1 expression as
a marker of chemotherapy efficacy. Additionally, the retrospective nature and small size of the
recurrence subgroup compromise generalizability.
The manuscript is written in acceptable English, but several grammatical and stylistic issues
require attention. Sentences such as “High PINK1 expression was observed in 42.2% (35 cases)
of the cases” should be streamlined. Instances of word redundancy, tense inconsistencies, and
awkward phrasing (e.g., “treatment resistance by enhancing cellular stress tolerance”) should
be revised for clarity. Citations are generally appropriate and cover recent literature; however,
some statements would benefit from stronger referencing, especially those regarding mitophagy
mechanisms and PINK1’s role in cancer.
assistant