Clinically Based Cetuximab Re-Challenge in Patients with RAS Wild-Type Metastatic Colorectal Cancer and Retrospective Analysis of Liquid Biopsies—Preliminary Data

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Doctor Mihaylova and colleagues conducted a retrospective analysis of liquid biopsies prospectively collected from a cohort of 11 patients with RAS/BRAF wild-type, pMMR, and HER2-negative colorectal cancer. After carefully reviewing the paper, I believe the data should not be accepted for publication. Only a minority of the patients in the study received a rechallenge therapy, limiting its contribution to advancing knowledge in this area.  Although the analysis of serial liquid biopsies is well-designed and technically sound, the data is not robust enough to draw meaningful conclusions. On the other hand, the data could provide some insights into the value of baseline and follow-up liquid biopsies during the treatment of tissue triple wild-type colorectal cancer patients. However, the small sample size presents a significant limitation, making it unlikely that these results will attract a wide readership in its current form.

I have noted some of the considerations that led to my decision.

General considerations:

The number of pages should be revised (after table 2, the page numbering restarts from 1). Additionally, please remember that italics should be used for gene nomenclature. For example, though it is clear that you are referring to KRAS, NRAS, and BRAF wild-type patients, this is not explicitly stated in the full text. Even though it may be intuitive that N/K stands for NRAS/KRAS (line 37, page 1), it should be clearly reported.

Furthermore, the significance of abbreviations throughout the text must be specified. For instance, it is not indicated that EGFR stands for Epidermal Growth Factor Receptor (line 40, page 1), and the meaning of the abbreviation “MAT” should be clarified (line 56, page 2).

Introduction:

Line 44, page 2: EGFR ectodomain mutations were not annotated among the possible causes of anti-EGFR resistance; however, the authors discuss these resistance mechanisms later in the text (line 47, page 2).

Line 47, page 2: The phrase “mutated clone of RAS” misleading, as the authors are referring to the clonal evolution of RAS-mutated cells that emerged during anti-EGFR therapy; these clones are composed of cells, not genes.

Lines 47-48, page 2: the sentence is somewhat confusing. If the authors intend to describe a general process, it should read, “After the onset of acquired resistance, the response to therapy gradually decreases.” In this case, the meaning of the subsequent sentence “with a cumulative survival of 4.4 months” is unclear. Does this mean that the median survival after first-line therapy for a RAS/BRAF wild-type patient progressing to first-line anti-EGFR therapy is 4.4 months? Based on which study? Conversely, if the authors are describing the results of a single study, they should clarify this with a sentence like, “As shown by Parseghian CM et al. in their study…”

Materials and methods:

Lines 175-76: the methodology for analyzing KRAS, NRAS, and BRAF mutational status should be clarified (Sanger method?).

Line 182: the phrase "established clinical criteria" is too vague. The clinical criteria used to select patients for rechallenge differ among the various studies that have examined anti-EGFR rechallenge. It is essential to specify the following:

1. Whether the time from the last anti-EGFR administration during first-line treatment to the emergence of progressive disease was less than 3 months for all patients, to determine if the term "rechallenge" is appropriate for everyone.
2. Whether all patients received a regimen that was free of anti-EGFR agents.
3. If the authors selected only patients who achieved at least a partial response (PR) during the first-line anti-EGFR-containing regimen.
4. Whether a minimum anti-EGFR-free interval was considered.

 Results:

The number of patients with liver-limited disease remains unclear. The authors state that 55% of patients exhibited liver-only metastases (lines 77-78); however, based on the data presented in Table 1, the percentage appears to be higher (8 out of 11 patients, or 72.7%).

The authors also report that 3 out of 11 patients underwent a cetuximab re-challenge (lines 82-83). Yet, according to the information in Table 2, only patients 1 and 2 received anti-EGFR therapy after a period of being anti-EGFR-free. Patient 6 received various anti-EGFR-containing regimens following a “start-and-stop” strategy, rather than a true re-challenge. Additionally, in both cases where re-challenge was mentioned, the first-line anti-EGFR treatment was halted after six cycles. Given that we can assume the re-assessment CT scan indicating a partial response occurred after at least 8 to 12 weeks from baseline, it seems likely that disease progression was noted during the treatment break. The authors should clarify the time interval between treatment cessation and disease progression to accurately classify the treatment strategy as re-challenge rather than re-treatment.

Furthermore, the variant allele frequency of the reported mutations should be provided to offer insights into their clonality; this information should have been included in the results section (lines 94-104).

Lastly, the authors mention a third mutation found in patient 5 at the second sample (lines 97-98, page 3). However, according to the data in Table 2, this mutation appears to be a baseline mutation. This discrepancy makes it challenging to understand what the authors mean by the “second sample.”

Discussion

Line 142, change “here” to “there.”

Line 146, the authors discuss the prognostic value of PIK3CA mutations. Given that the study focuses on the added value of liquid biopsy for anti-EGFR rechallenge, a discussion on the predictive value of PIK3CA mutations in patients treated with anti-EGFR therapy would be beneficial. Additionally, mutations at exon 20 of PIK3CA, such as the one detected in one patient from the study, have been previously associated with resistance to anti-EGFR therapy (De Roock W et al., Lancet Oncol 2010). Therefore, it would be important to include a discussion on this topic.  

Line 141: The statement “IDH2 protein was highly expressed in CRC tissues” is somewhat debatable and is partially contradicted later in the text, where it states that “IDH1/2 mutations in CRCs were uncommon”. If the authors are referring to a specific study, they would have specify which study. Suppose they are discussing a general tendency in CRC. In that case, the sentence should read, “IDH2 protein is highly expressed in CRC tissues.” A reference supporting this statement should be added or identified within the text.

Author Response

Doctor Mihaylova and colleagues conducted a retrospective analysis of liquid biopsies prospectively collected from a cohort of 11 patients with RAS/BRAF wild-type, pMMR, and HER2-negative colorectal cancer. After carefully reviewing the paper, I believe the data should not be accepted for publication. Only a minority of the patients in the study received a rechallenge therapy, limiting its contribution to advancing knowledge in this area.  Although the analysis of serial liquid biopsies is well-designed and technically sound, the data is not robust enough to draw meaningful conclusions. On the other hand, the data could provide some insights into the value of baseline and follow-up liquid biopsies during the treatment of tissue triple wild-type colorectal cancer patients. However, the small sample size presents a significant limitation, making it unlikely that these results will attract a wide readership in its current form.

I have noted some of the considerations that led to my decision.

General considerations:

The number of pages should be revised (after table 2, the page numbering restarts from 1). Additionally, please remember that italics should be used for gene nomenclature. For example, though it is clear that you are referring to KRAS, NRAS, and BRAF wild-type patients, this is not explicitly stated in the full text. Even though it may be intuitive that N/K stands for NRAS/KRAS (line 37, page 1), it should be clearly reported.

Furthermore, the significance of abbreviations throughout the text must be specified. For instance, it is not indicated that EGFR stands for Epidermal Growth Factor Receptor (line 40, page 1), and the meaning of the abbreviation “MAT” should be clarified (line 56, page 2).

Author’s Reply:

We thank the reviewer for this very reasonable comment. All gene nomenclature is in italics now, and the corrections are made for KRAS/NRAS. The "Epidermal Growth Factor Receptor" was put in the brackets to clarify EGFR, and the MAT abbreviation replaced in the text with "monoclonal antibody treatment".

We are experiencing a problem with the page numbering in our manuscript. After inserting Table 2 in landscape orientation, the template automatically resets its own numbering sequence. We trust the copy editors will be able to resolve this technical issue.

Introduction:

Line 44, page 2: EGFR ectodomain mutations were not annotated among the possible causes of anti-EGFR resistance; however, the authors discuss these resistance mechanisms later in the text (line 47, page 2).

Author’s Reply:

We thank the reviewer for this very reasonable comment, but on line 50, page 2 (in revisted version of manuscript, or line 44 in old version), we did not mention EGFR ectodomain mutations. Still, we noted „as well as the emergence of mutations in other genes that confer resistance“ and the following reference  - Parseghian CM, Loree JM, et al., Anti-EGFR-resistant clones decay exponentially after progression: Implications for anti-EGFR re-challenge. Ann Oncol, 2019, 30(2):243–9, the EGFR ectodomain mutations as possible causes of anti-EGFR resistance are in detail described. That is why we discuss EGFR ectodomain mutations as one of the possible resistance mechanisms later in the text – line 54 to line 60

Line 47, page 2: The phrase “mutated clone of RAS” misleading, as the authors are referring to the clonal evolution of RAS-mutated cells that emerged during anti-EGFR therapy; these clones are composed of cells, not genes.

Author’s Reply:

We appreciate your important comment regarding the accuracy of scientific terminology. We have corrected the phrase "mutated clone of RAS" to "RAS-mutated cells" (line 63) in the text to properly reflect the biological process of clonal evolution. This correction more accurately describes the phenomenon of expansion and subsequent reduction of the population of cells harboring RAS mutations after the cessation of anti-EGFR therapy.

Lines 47-48, page 2: the sentence is somewhat confusing. If the authors intend to describe a general process, it should read, “After the onset of acquired resistance, the response to therapy gradually decreases.” In this case, the meaning of the subsequent sentence “with a cumulative survival of 4.4 months” is unclear. Does this mean that the median survival after first-line therapy for a RAS/BRAF wild-type patient progressing to first-line anti-EGFR therapy is 4.4 months? Based on which study? Conversely, if the authors are describing the results of a single study, they should clarify this with a sentence like, “As shown by Parseghian CM et al. in their study…”

Author’s Reply:

We thank the reviewer for this reasonable comment. We have significantly revised and expanded this section (lines 52 to line 60 on page 2) to provide better clarity. We have replaced the confusing sentence with a more detailed explanation of Parseghian et al.'s mathematical modeling approach that describes the clonal dynamics of RAS and EGFR mutations after anti-EGFR therapy withdrawal.

Materials and methods:

Lines 175-76: the methodology for analyzing KRAS, NRAS, and BRAF mutational status should be clarified (Sanger method?).

Author’s Reply:

For the KRAS/NRAS and BRAF mutation analyses, we used the Diatech Pharmacogenetics Easy PGX line Real-Time PCR kits, respectively: ”EasyPGX® ready KRAS/NRAS/BRAF”. These CE IVD kits allowed identification of mutations in codons 12, 13, (exon 2), 59, 61 (exon 3) and 117, 149 (exon 4) in KRAS and NRAS genes, and BRAF V600 mutations (V600E, V600E complex, V600K, V600D, V600R) in exon 15 of the BRAF gene. All the tests were performed according to the manufacturer’s instructions.

This information was added in the text of the manuscript in Materials and methods section (line 236 to 240).

Line 182: the phrase "established clinical criteria" is too vague. The clinical criteria used to select patients for rechallenge differ among the various studies that have examined anti-EGFR rechallenge. It is essential to specify the following:

Author’s Reply:

We thank the reviewer for the comment. We have changed the sentence on line 182 (in revised version of the manuscript line 257 to line 261) as follows:

The anti-EGFR re-challenge approach was implemented based on following published established clinical criteria: „Anti-EGFR re-challenge involves retreating mCRC patients with anti-EGFR antibodies if they previously achieved a response or SD during prior anti-EGFR therapy. This strategy requires an EGFR-free interval between two anti-EGFR-containing lines of treatment, ideally lasting 6 months“ [17].

Whether the time from the last anti-EGFR administration during first-line treatment to the emergence of progressive disease was less than 3 months for all patients, to determine if the term "rechallenge" is appropriate for everyone.

Author’s Reply:

The re-challenge is not suitable for everyone. Thus, patients who progressed very soon while on 2^nd line therapy, less than 3 months were not suitable for re-challenge. Patients who progressed quickly on first-line anti-EGFR therapy (less than 4 months, such as patients 3 and 5) or had inadequate duration of second-line therapy were not considered suitable for re-challenge, in accordance with established protocols.

Whether all patients received a regimen that was free of anti-EGFR agents

Author’s Reply:

Yes, all patients received 2^nd line treatment that was free of anti-EGFR agents.

If the authors selected only patients who achieved at least a partial response (PR) during the first-line anti-EGFR-containing regimen

Author’s Reply:

No, according to above mentioned definition which we have used in our study patients who did not progressed which means that patients with stable disease (SD), partial response (PR) and complete response (CR) are included. Our selection criteria focused on the absence of progression rather than specifically requiring PR, which is consistent with published re-challenge protocols in the literature.

Whether a minimum anti-EGFR-free interval was considered

Author’s Reply:

Usually, treatment evaluations are done after 4 months of therapy. If the patient has not progressed at the first evaluation, it is considered suitable for re-challenge. But ideally, the EGFR-free interval between two anti-EGFR-containing lines of treatment lasts 6 months.

Results:

The number of patients with liver-limited disease remains unclear. The authors state that 55% of patients exhibited liver-only metastases (lines 77-78); however, based on the data presented in Table 1, the percentage appears to be higher (8 out of 11 patients, or 72.7%).

Author’s Reply:

We thank the reviewer for identifying this discrepancy. You are correct - based on the data in Table 1, 8 out of 11 patients (72.7%) exhibited liver-only metastases, not 55% as incorrectly stated in the original text. We have corrected this error on lines 95-96, changing "55%" to "72.7%" to accurately reflect the data presented in Table 1.

The authors also report that 3 out of 11 patients underwent a cetuximab re-challenge (lines 82-83). Yet, according to the information in Table 2, only patients 1 and 2 received anti-EGFR therapy after a period of being anti-EGFR-free. Patient 6 received various anti-EGFR-containing regimens following a “start-and-stop” strategy, rather than a true re-challenge. Additionally, in both cases where re-challenge was mentioned, the first-line anti-EGFR treatment was halted after six cycles. Given that we can assume the re-assessment CT scan indicating a partial response occurred after at least 8 to 12 weeks from baseline, it seems likely that disease progression was noted during the treatment break. The authors should clarify the time interval between treatment cessation and disease progression to accurately classify the treatment strategy as re-challenge rather than re-treatment.

Author’s Reply:

Thank you for your important observation. After careful review, we agree with your assessment regarding patient 6 and have revised our manuscript accordingly.

Regarding Patients 1 and 2: In Table 2, Patient 1 and Patient 2 were re-evaluated after 6 courses of chemotherapy and anti-EGFR therapy with a response assessment of partial remission. At this moment, samples were taken to assess the mutational status on circulating DNA. The patients did not interrupt chemotherapy and targeted therapy after the 6th course; they continued until disease progression. For patient 1, first-line chemotherapy plus anti-EGFR therapy lasted for 12 cycles (more than 6 months), and anti-EGFR-free therapy (2nd line) lasted more than 6 months. For patient 2, first-line chemotherapy and anti-EGFR therapy consisted of 9 cycles (more than 5 months of therapy).

Regarding Patient 6: We agree with the reviewer's assessment that patient 6 did not receive a true re-challenge. This patient received 15 cycles of 1st line FOLFOX plus Cetuximab, and due to Oxaliplatin-induced neurological toxicity, the patient switched to maintenance treatment with De Gramont and Cetuximab.

We have revised our manuscript to correctly state that only 2 out of 11 patients (18%) received true Cetuximab re-challenge, and we have clarified the treatment approach for patient 6 as a continuous anti-EGFR strategy with chemotherapy modifications rather than re-challenge.

Furthermore, the variant allele frequency of the reported mutations should be provided to offer insights into their clonality; this information should have been included in the results section (lines 94-104).

 Author’s Reply:

We thank the reviewer for this valuable suggestion. We have added the variant allele frequencies (VAF) of the detected mutations to the results section (lines 120-124) as requested. These values (32.4% for PIK3CA, 1.0% for KRAS, and 27.2% for IDH2) provide important context regarding the clonality of these mutations.

Lastly, the authors mention a third mutation found in patient 5 at the second sample (lines 97-98, page 3). However, according to the data in Table 2, this mutation appears to be a baseline mutation. This discrepancy makes it challenging to understand what the authors mean by the “second sample.”

Author’s Reply:

This is a technical error. Thank you for mentioning it. In patient 5 the baseline sample was found to have IDH2: c.418C >T. Change was made on the line 123.

Discussion

Line 142, change “here” to “there.”

Author’s Reply:

The change was done.

Line 146, the authors discuss the prognostic value of PIK3CA mutations. Given that the study focuses on the added value of liquid biopsy for anti-EGFR re-challenge, a discussion on the predictive value of PIK3CA mutations in patients treated with anti-EGFR therapy would be beneficial. Additionally, mutations at exon 20 of PIK3CA, such as the one detected in one patient from the study, have been previously associated with resistance to anti-EGFR therapy (De Roock W et al., Lancet Oncol 2010). Therefore, it would be important to include a discussion on this topic. 

Author’s Reply:

We thank the reviewer for this very reasonable comment. The study by De Roock and the discussion about the predictive value of PIK3CA mutations were added to the article (lines 196-198 and lines 200-205).

Line 141: The statement “IDH2 protein was highly expressed in CRC tissues” is somewhat debatable and is partially contradicted later in the text, where it states that “IDH1/2 mutations in CRCs were uncommon”. If the authors are referring to a specific study, they would have specify which study. Suppose they are discussing a general tendency in CRC. In that case, the sentence should read, “IDH2 protein is highly expressed in CRC tissues.” A reference supporting this statement should be added or identified within the text.

Author’s Reply:

We thank the reviewer for this very reasonable comment. We have removed the contradictory statement about IDH2 protein expression in CRC tissues that appeared on line 141. The revised text now focuses consistently on the uncommon nature of IDH1/2 mutations in CRC and provides appropriate references to support this information.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1. MATERIAL AND METHODS

1. The manuscript would benefit from a more detailed description of inclusion and exclusion criteria, as well as clarification regarding the rationale behind the limited sample size.

2. More details on the methodologies used for MSI and HER2 testing would enhance methodological transparency.

3. If I understand correctly, some samples (from the beginning of plasma collection - 2022) were stored at -20^oC. It would be valuable to address potential limitations associated with storing plasma samples at -20°C (not at -80^oC) over two years, as this may compromise cfDNA integrity.

4. Can you clarify whether copy number variations or gene fusions were assessed, as they are clinically relevant in mCRC?

5. Genes should be written in italics.

 

2. RESULTS

1. Data is presented in text tables only. A visual representation of ctDNA mutation dynamics across time points would help to clearly illustrate individual patient trajectories and molecular events monitoring.

2. A timeline indicating when samples were collected relative to therapy lines and progression events would help the reader better understand ctDNA dynamics.

3. I think the work would benefit from integrating molecular findings with the clinical course of patients.

4. Among 7 samples collected during progression, only one had a KRAS mutation detected. Considering the storage of the material at -20^oC, I suggest adding a short discussion of possible technical or biological reasons for possible false-negative results.

 

3. DISCUSSION

1. The discussion is mainly limited to RAS/PIK3CA/IDH2 mutations, omitting other known mechanisms of anti-EGFR resistance, but it may be affected also by non-genomic mechanisms, like TME changes.

2. The cohort is dominated by liver metastases, which are often associated with lower circulating ctDNA levels. In my opinion, it would be valuable to add information that this may explain the low percentage of detected mutations.

Author Response

 

1. MATERIAL AND METHODS
2. The manuscript would benefit from a more detailed description of inclusion and exclusion criteria, as well as clarification regarding the rationale behind the limited sample size

Author’s Reply:

We thank the reviewer for this comment. The revised manuscript provides a detailed description of the enrolled patients in the Materials and Methods section.

1. More details on the methodologies used for MSI and HER2 testing would enhance methodological transparency.

Author’s Reply:

We thank the reviewer for this suggestion to enhance methodological transparency. We have added more detailed information regarding our MSI and HER2 testing methodologies to the Materials and Methods section of the revised manuscript.

 

1. If I understand correctly, some samples (from the beginning of plasma collection - 2022) were stored at -20oC. It would be valuable to address potential limitations associated with storing plasma samples at -20°C (not at -80oC) over two years, as this may compromise cfDNA integrity.

Author’s Reply:

We thank the reviewer for this point. We agree that storing plasma samples at -20°C instead of -80°C for up to two years could affect cfDNA quality. There appears to be a technical error in our materials and methods section. All plasma samples in our study were actually stored at -80°C, not at -20°C as incorrectly stated in the manuscript. We have corrected this error in the revised version.

1. Can you clarify whether copy number variations or gene fusions were assessed, as they are clinically relevant in mCRC?

Author’s Reply:

We thank the reviewer for this important question. We would like to clarify that our applied methodology (Myriapod® NGS Cancer panel) does not detect gene fusions or copy number variations (CNVs). The panel we used is designed primarily for detection of single nucleotide variants and small insertions/deletions in the 16 clinically relevant genes mentioned in Materials and Methods section.

1. Genes should be written in italics.

Author’s Reply:

This has been done in the revised manuscript.

2. RESULTS
3. Data is presented in text tables only. A visual representation of ctDNA mutation dynamics across time points would help to clearly illustrate individual patient trajectories and molecular events monitoring.

Author’s Reply:

We thank the reviewer for this reasonable comment. After careful consideration, we believe our limited dataset does not lend itself well to additional graphical representation beyond the two tables already provided. With only three detected genetic alterations out of 21 samples from 11 patients, creating a visual representation of ctDNA mutation dynamics would include mostly negative results with very few molecular events to track. Therefore, the type of the manuscript was defined as Communication.

1. A timeline indicating when samples were collected relative to therapy lines and progression events would help the reader better understand ctDNA dynamics.

Author’s Reply:

We thank the reviewer for this valuable suggestion. We appreciate the potential value of including a detailed timeline visualization of sample collection relative to therapy lines and progression events. However, given the preliminary nature of our data and the concise format requirements of a Communication manuscript, we have chosen to present our methodology and results in a more condensed format through Table 2, which provides the essential information about treatment sequences, responses, and corresponding ctDNA findings for each patient.

1. I think the work would benefit from integrating molecular findings with the clinical course of patients.

Author’s Reply:

We thank the reviewer for this comment. Given the preliminary nature of our data and the limited sample size, we provided basic observations about only three detected genetic alterations to treatment responses. In a future expanded study with more patients and longitudinal samples, we hope to perform more comprehensive analyses that might reveal correlations between molecular profiles and treatment outcomes in the context of anti-EGFR re-challenge.

 

1. Among 7 samples collected during progression, only one had a KRAS mutation detected. Considering the storage of the material at -20oC, I suggest adding a short discussion of possible technical or biological reasons for possible false-negative results.

Author’s Reply:

We thank the reviewer for highlighting this critical point. We would like to clarify that there was an error in our materials and methods section - all plasma samples were actually stored at -80°C, not at -20°C as incorrectly stated. We apologize for this inaccuracy, which has been corrected in the revised manuscript.

Regarding the low detection rate of KRAS mutations during progression, we implemented strict quality control measures and excluded samples with poor DNA quality, as mentioned in the Results section ("three additional samples were excluded due to poor quality", line 117).

The limited detection of mutations during progression could be attributed to several biological factors, including: (1) alternative resistance mechanisms not related to RAS pathway activation, (2) potential non-genetic mechanisms of resistance, and (3) the presence of resistance mutations in other genes not covered by our panel. These biological explanations align with emerging evidence that anti-EGFR resistance often involves diverse molecular alterations beyond RAS mutations.

To address this point, we have added a brief discussion of these potential biological factors to the Discussion section (line 173 to line 191).

 

DISCUSSION

1. The discussion is mainly limited to RAS/PIK3CA/IDH2 mutations, omitting other known mechanisms of anti-EGFR resistance, but it may be affected also by non-genomic mechanisms, like TME changes.

Author’s Reply:

We thank the reviewer for this very reasonable comment, but describing non-genomic mechanisms, like TME changes, is outside the scope of our preliminary study, which is hopefully being published as a "Communication".

1. The cohort is dominated by liver metastases, which are often associated with lower circulating ctDNA levels. In my opinion, it would be valuable to add information that this may explain the low percentage of detected mutations.

Author’s Reply:

We thank the reviewer for this suggestion. We have included the following text in the discussion:

“There is evidence of a relationship between the site of metastasis in CRC and ctDNA. Patients with peritoneal metastases have lower ctDNA levels than other metastatic sites due to the plasma-peritoneal barrier [16]. In comparison with patients with peritoneal metastases, CRC patients with liver metastases have higher detectable ctDNA levels [17].” (line 165 to line 168).

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1) The aim of this study was to evaluate the efficacy of anti-EGFR therapy re-challenge in patients with RAS wild-type mCRC through prospective clinical assessment and retrospective liquid biopsy analysis. It sounds too grandiose and the results obtained are not enough. Please specify the goal and bring it into line with the data obtained.
2) Table 2 should be placed immediately after the mention.
3) Could the authors please provide the most visual results in the form of a diagram in addition to the tables?
4) The efficacy and applicability of Cetuximab should be mentioned in the introduction.
5) For all reagents, Cat, country of origin, etc. should be provided

Author Response

Comments and Suggestions for Authors

1) The aim of this study was to evaluate the efficacy of anti-EGFR therapy re-challenge in patients with RAS wild-type mCRC through prospective clinical assessment and retrospective liquid biopsy analysis. It sounds too grandiose and the results obtained are not enough. Please specify the goal and bring it into line with the data obtained. - 

Author’s Reply:

We thank the reviewer for valuable feedback regarding the alignment between the stated aim and the obtained data in our study. We accept your suggestion to reformulate the study objective to more accurately reflect the scope of our data and its preliminary nature. The new text is:

“The aim of this preliminary study was to evaluate the potential benefit of a clinically-based anti-EGFR re-challenge approach in selected patients with RAS wild-type mCRC and to conduct a retrospective liquid biopsy analysis for an initial exploration of resistance mechanisms in a small cohort of patients.”

2) Table 2 should be placed immediately after the mention.

Author’s Reply:

The correction has been made.

3) Could the authors please provide the most visual results in the form of a diagram in addition to the tables?

Author’s Reply:

We thank the reviewer for this reasonable comment. After careful consideration, we believe our limited dataset may not lend itself well to additional graphical representation beyond the tables already provided. This is also why we have designated our manuscript as a Communication, reflecting its preliminary nature and limited scope.

4) The efficacy and applicability of Cetuximab should be mentioned in the introduction.

Author’s Reply:

We thank the reviewer for this helpful suggestion, which we have addressed in the revised manuscript's introduction.

5) For all reagents, Cat, country of origin, etc. should be provided

Author’s Reply:

We thank the reviewer for this important point. We included information on manufacturer details and the country of origin of kits and reagents used in our study in Materials and Methods section (4.1. Patients Selection and Molecular Profiling)

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am generally satisfied with authors response. They clarified the major problems and I suggest acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have taken into account most of my comments. Their explanations are satisfactory. I recommend accepting the article in its current form.