The Clinically Actionable Molecular Profile of Early versus Late-Stage Non-Small Cell Lung Cancer, an Individual Age and Sex Propensity-Matched Pair Analysis

Round 1

Reviewer 1 Report

Please check the spelling: line 21, 57, 60, 61, 64, 66, 68, 69, 85, 100,110,116, 117,136, 248, 255, 257, 273, 274, 284, 291, 304, 307, 317, 319, 321, 327, 333

Why did the authors use different NGS assays to examine the early-stage and the late-stage cohort?

 

Author Response

Reviewer 1 Comment 1

Please check the spelling: line 21, 57, 60, 61, 64, 66, 68, 69, 85, 100,110,116, 117,136, 248, 255, 257, 273, 274, 284, 291, 304, 307, 317, 319, 321, 327, 333

Reviewer 1 Response 1

Thank-you for taking your valuable time to conduct this review. We have corrected the spelling errors noted in these lines in the paper. These errors appear to be related to manuscript transition in formatting from a word document to the journal template.

Reviewer 1 Comment 2

Why did the authors use different NGS assays to examine the early-stage and the late-stage cohort?

Reviewer 1 Response 2

Thank-you for this question. Different NGS assays were used for the early and late-stage cohort for two main reasons. The first is that reflexive molecular testing is not routinely conducted on early-stage resected surgical lung cancer specimens at our institution, so the “oncopanel” NGS assay was not available clinically for use in early stage NSCLC during the study period. Secondly, as part of a prospective clinical study in collaboration with Canexia Health Inc. ongoing at our institution, reflexive molecular testing of early-stage NSCLC was made available for the first time using the Find IT and Fusions Panels.

Reviewer 2 Report

To provide the evidence that reflexive molecular testing could benefit early stage NSCLC patients to guide treatment options when recurrent metastatic disease occurs, the authors compared the molecular profiles of genetic alterations in early and late NSCLC patients in a retrospective manner. The authors analyzed the targeted panel-based NGS data from a total of 635 NSCLC patients (59 early stage and 576 late stage) including several common and actionable gene mutations: EGFR, KRAS, ALK, MET, and ROS1. In unadjusted analysis, a higher frequency of EGFR mutations and lower frequency of KRAS mutations were observed in the early stage cohort, while all other frequencies were observed to be similar between the early and late stage subgroups. Switching to pair-matched multi variable analysis (all 59 early stage and 159/576 late stage), all the mutation frequencies were not significantly different between the early and late stage subgroups, indicating the similarities among these groups in terms of gene mutations. To the authors’ knowledge, this is the first report comparing the frequency of molecular alterations in early and late NSCLC in propensity matched cohort.

 

1. Introduction

 

The authors mostly described the current challenges in treating recurrent diseases where biopsies are needed but difficult to acquire, and raised the hypothesis that “both early and late-stage NSCLC display similar odds of driver mutation and fusion molecular alteration profiles in our population regardless of disease stage at presentation”. Although the hypothesis could be valid, not enough of previous studies were discussed here to support or lead to this hypothesis. The authors could list more supporting or contradicting studies before raising the hypothesis.

 

2. Methods

 

The methods were well described.

 

3. Results

 

The results were well described.

 

4. Discussion

 

The main discovery in this study was the similar frequency and odds of clinically actionable EGFR molecular alterations, along with other analyzed gene mutations, in early and late-stage NSCLC in propensity score matching analysis. However, the study was greatly limited by several factors including the sample size, the lack of intrapatient comparison over time, and the treatment outcome of recurrent disease guided by the molecular analysis in early stage. Without long-term follow-up of the patients to demonstrate the similarities in molecular alterations in early stage and recurrent phase, it is difficult to conclude that patients could benefit from the predetermined molecular profile. In fact, there are more evidence demonstrating the heterogeneity in time and space that tumor cells would greatly change over time and the genomic profile of them could also be biased by the sampling site. (e.g. Dagogo-Jack, I., Shaw, A. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol 15, 81–94 (2018). https://doi.org/10.1038/nrclinonc.2017.166). All in all, the connections between the statistical discoveries and the conclusions in this study are on the weaker side.

Author Response

Reviewer 2 Comment 1

Introduction. The authors mostly described the current challenges in treating recurrent diseases where biopsies are needed but difficult to acquire, and raised the hypothesis that “both early and late-stage NSCLC display similar odds of driver mutation and fusion molecular alteration profiles in our population regardless of disease stage at presentation”.

Although the hypothesis could be valid, not enough of previous studies were discussed here to support or lead to this hypothesis. The authors could list more supporting or contradicting studies before raising the hypothesis.

Reviewer 2 Response 1

Thank-you for taking the time to review our manuscript. We have added to the introduction section of the manuscript that demonstrate the validity of our study hypothesis.^1,2 Thank-you for this constructive feedback that has improved the introduction section of our R1 manuscript lines 64 to 69.

 

Reviewer 2 Comment 2

Methods. The methods were well described.

Reviewer 2 Response 2

Thank-you kindly for your review.

Reviewer 2 Comment 3

Results. The results were well described.

Reviewer 2 Response 3

Thank-you for your review.

Reviewer 2 Comment 4

Discussion. The main discovery in this study was the similar frequency and odds of clinically actionable EGFR molecular alterations, along with other analyzed gene mutations, in early and late-stage NSCLC in propensity score matching analysis.

The study was greatly limited by several factors including the a.) sample size, b.) lack of intra-patient comparison over time, and c.) the treatment outcome of recurrent disease guided by the molecular analysis in early stage.

Reviewer 2 Response 4.

Thank-you for your review.

Reviewer 2 raises important valid points regarding the limitations of this study, including sample size impacting the power and generalizability of our findings to other institutions. We transparently discussed the limitation of sample size in lines 324 to 326 of the manuscript R1. It was additionally pre-emptively addressed in our study design and statistical analysis to the best of our ability using matching on the propensity score (lines 156 to 164 manuscript R1).

With respect to comment of lack of intra-patient comparison over time for recurrence and treatment outcome, Reviewer 2 proposes a different time-to-event study design wherein the early-stage patient has molecular profiling and is then followed forward in time to identify recurrence and then assess for similarity. Although we agree this would be very interesting and valuable, this is beyond the scope and objective of the current retrospective study. In the current study we simply wish to examine frequency of putative driver mutations in our population of patients in the clinical setting at their stage of presentation, be it early stage (surgical) or late stage (non-surgical).

Although unrelated to the current study, given Reviewer 2’s comment we believe it relevant to not that a prospective cohort time-to-event study in early-stage NSCLC similar to that proposed is in fact already underway at our institution. However, the follow-up data for recurrence after treatment in the early-stage cohort after molecular profiling is immature. 

Additionally, prior studies of NGS in early-stage lung cancer have been conducted in the manner proposed by Reviewer 2. The review paper from the Memorial Sloan Kettering Cancer Centre (MSKCC) experience by Lengel et. al. 2021, as well as the prospective cohort study by Kim et. al. demonstrates that certain driver mutation present at the time of surgical resection portend prognosis. Additionally, although subclones may be present at the time of recurrent lung cancer following initial curative intent surgery, the dominant clinically clonal “oncogenic driver” identified at the time of surgery was also present at the time of recurrence conferring sensitivity to targeted therapy.

Reviewer 2 Comment 5

Without long-term follow-up of the patients to demonstrate the similarities in molecular alterations in early stage and recurrent phase, it is difficult to conclude that patients could benefit from the predetermined molecular profile.

Reviewer 2 Response 5

Previous papers in surgical early-stage NSCLC have conducted molecular analysis, intra-patient comparison over time, and treatment outcome of recurrent disease guided by the molecular analysis in early stage.^1,2 Based on these papers we would argue that indeed early stage lung cancer patients could benefit from a predetermined molecular profile in the event of recurrence. We have added these references to the manuscript in the introduction and discussion sections.

As noted above in Reviewer 2 Response 4: Prior studies of NGS in early-stage lung cancer have been conducted in the manner proposed by Reviewer 2. The review paper from the Memorial Sloan Kettering Cancer Centre (MSKCC) experience by Lengel et. al. 2021, as well as the prospective cohort study by Kim et. al. demonstrates that certain driver mutation present at the time of surgical resection portend prognosis. Additionally, although subclones may be present at the time of recurrent lung cancer following initial curative intent surgery, the dominant clinically clonal “oncogenic driver” identified at the time of surgery was also present at the time of recurrence conferring sensitivity to targeted therapy.^1,2

We believe that the value of our current paper lies in this fundamental difference, as to our knowledge there are no studies comparing the early and late-stage NSCLC molecular profiles in the routine clinical setting for the population.

Reviewer 2 Comment 6

There are more evidence demonstrating the heterogeneity in time and space that tumor cells would greatly change over time and the genomic profile of them could also be biased by the sampling site. (e.g. Dagogo-Jack, I., Shaw, A. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol 15, 81–94 (2018). https://doi.org/10.1038/nrclinonc.2017.166).

Reviewer 2 Response 6

Thank-you for sharing this very interesting and relevant reference.³ We read the excellent review paper with great interest and have added this reference in the discussion of study limitations section, where we acknowledge that by using clinically available cancer panels to detect genetic alterations, we are not able to map the evolution clonal heterogeneity as one could with emerging techniques. Although extremely interesting, single cell sequencing, multi-region sequencing in the early stage lung cancer population in particular, along with whole genome sequencing techniques, are beyond the scope of this paper.

We agree that tumour heterogeneity and systemic therapy resistance noted by Reviewer 2 is a clinically important phenomenon as it invariably portends inferior patient prognosis. The clinically relevant problem of resistance described relates to that of tumour evolution in response to the unique tumour and clonal microenvironment over space and time, as well as enhancing genomic complexity in response to target systemic treatment over time. This is distinct from the clinical scenario of early-stage NSCLC at presentation in a systemic therapy naïve patient following curative intent surgical resection.

Although theoretically possible with all the known complexities of tumour heterogeneity, and de novo mutations such as EGFR T790M occasionally occurring, it is clinically unlikely that in the unfortunate event of recurrent cancer without prior systemic therapy, the patient would not possess the actionable driver mutational molecular profile identified at surgery (EGFR exon 19 deletion for example).^1,2

Response to Reviewer References

 

 

1. Kim IA, Hur JY, Kim HJ, et al. Targeted Next-Generation Sequencing Analysis for Recurrence in Early-Stage Lung Adenocarcinoma. Ann Surg Oncol. Jul 2021;28(7):3983-3993. doi:10.1245/s10434-020-09276-x
2. Lengel HB, Connolly JG, Jones GD, et al. The Emerging Importance of Tumor Genomics in Operable Non-Small Cell Lung Cancer. Cancers (Basel). Jul 21 2021;13(15)doi:10.3390/cancers13153656
3. Dagogo-Jack I, Shaw AT. Tumour heterogeneity and resistance to cancer therapies. Nat Rev Clin Oncol. 02 2018;15(2):81-94. doi:10.1038/nrclinonc.2017.166

Round 2

Reviewer 2 Report

Thank you for the responses. Although the scope of this study is limited, I do agree that your discovery is valuable. I have no further comments.