ERBB2 Mutation Testing in NSCLC: A Pan-European Real-World Evaluation of the Oncomine Precision Assay

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear colleagues, 
The idea of your manuscript is good, but not so innovative, and it lacks support from cases from other centers that confirm the method, as there are only 8.
At least 22 more samples.

Authors aimed to retrospectively assess the performance of the Oncomine™ Precision Assay* 30 (OPA) in combination with the Ion Torrent Genexus™ Integrated Sequencer*  to detect ERBB2 mutations.

It could be original if its aim was to compare much more mutations and not only ERBB2; as reported by Sara El Zaitouni in 2024 Oct 4;23:15330338241288907. doi: 10.1177/15330338241288907 et al has already reported a retrospective study of 76 formalin-fixed paraffin-embedded (FFPE) samples that have been profiled using the Oncomine™ Precision Assay on the Ion Torrent™ Genexus™ Integrated Sequencer across the panel of 50 key genes that are applicable for the selection of targeted therapy.

Authors propose to investigate the capabilities and limitations of the 86 OPA assay, to provide valuable insights into its potential use as a reliable tool for 87 accurate ERBB2 mutation detection. 

Methodology: Authors have considered that Phase II: Reproducibility Assessment Across the Six Centers, using only 8 samples, is not enough.

There are not enough samples to reproduce their data.

References lacks some recent papers:

Normanno et al Int. J. Mol. Sci. 2023, 24(18), 13788; https://doi.org/10.3390/ijms241813788et al; Zhenhao Qi et al February 2025 J Mol Diagn. 2025 Feb;27(2):119-129. doi: 10.1016/j.jmoldx.2024.11.006; Bozzi F Detecting gene copy number alterations by Oncomine Comprehensive genomic profiling in a comparative study on FFPE tumor samples in 2025.

 

Author Response

We sincerely thank you for your careful review of our manuscript and for your constructive feedback. Below, we provide our detailed responses to the points raised.

Reviewer Comment: The idea of your manuscript is good, but not so innovative, and it lacks support from cases from other centers that confirm the method, as there are only 8. At least 22 more samples.

Response: We appreciate the comment regarding the sample size. However, we would like to emphasize that the primary aim of this manuscript is not to present a comprehensive profiling study, but rather to provide focused insight into the performance of an automated rapid NGS solution in detecting ERBB2 (HER2) mutations specifically, in light of novel approved targeted therapies available for NSCLC.

We acknowledge the limited number of samples used in the reproducibility assessment (Phase II); however, these samples were carefully selected for their representativeness and relevance. Logistical constraints, including sample availability and the retrospective nature of the study, precluded the addition of further cases within a feasible timeframe. Nevertheless, our results were consistent across participating centers, supporting the robustness of the assay in this context. Our analysis demonstrate feasibility and reproducibility in a multicenter setting, which we believe is a necessary prerequisite for broader implementation.

Reviewer Comment: It could be original if its aim was to compare much more mutations and not only ERBB2; as reported by Sara El Zaitouni et al. (2024).

Response: While the cited study by El Zaitouni et al. offers valuable insights through broad mutational profiling across 50 genes, our work takes a complementary and focused approach. We specifically address the detection of ERBB2 mutations using the OPA assay, allowing for a more in-depth evaluation of assay performance in this clinically relevant target. Additionally, the novelty of our study lies in its targeted assessment of the assay’s strengths and limitations for HER2 mutation detection, which has not been previously explored in the same level of detail.

Reviewer Comment: Authors have considered that Phase II: Reproducibility Assessment Across the Six Centers, using only 8 samples, is not enough. There are not enough samples to reproduce their data.

Response: The consistent performance observed across six independent centers provides robust evidence of assay reproducibility, which in some cases can offer greater practical insight than larger, single-lab datasets. As mentioned, the number of samples was limited due to the retrospective nature of the study and the specific inclusion criteria (i.e., confirmed HER2 mutations). Despite the small number, the multicenter reproducibility was demonstrated across all participating laboratories, highlighting the assay’s consistent performance in real-world conditions. We believe these findings provide meaningful insights and pave the way for future, larger-scale studies.

Reviewer Comment: References lack some recent papers.

Response: We thank the reviewer for highlighting relevant recent literature. We have revised the manuscript to include and discuss the following references:

• Qi et al. Arch Pathol Lab Med . 2024 Sep 20. doi: 10.5858/arpa.2024-0014-OA
• P. Hofman, Transl Oncol. 2023 Sep:35:101735. doi: 10.1016/j.tranon.2023.101735.

These additions further contextualize our findings within the evolving landscape of NGS-based diagnostics, in particular in the context of liquid biopsy.

Once again, we thank you for your thoughtful comments and hope our clarifications sufficiently address your concerns. We believe this work contributes valuable information to the field and respectfully request your consideration of the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Alborelli et al describes the testing of the Oncomine Precision Assay (OPA) in combination with the Genexus IonTorrent massively parallele sequencer to identify pathogenic variants in the HER2 (ERBB2) gene in samples from patients with non-squamous non-small cell lung cancer (nsNSCLC). The objective of the study is to determine whether this assay provides rapid, robust, accurate molecular diagnosis suceptible of use in a clinical setting. Results indicate excellent robustness and concordance of mutation detection across six European reference centres using the assay in the Genexus sequencer, for detection of both SNV and small insertions or deletions.

My concerns here are twofold: (1) The first one is the fact that most (92%) of the samples tested harbored just exon 20 alterations, and none of the tested alterations are CNVs in HER2, a type of variant that the kit is also designed to do. This should have been tested if possible, though it may seem that CNVs were not available for testing. This might have been discussed, in particular if CNVs are seldom (or not at all) identified in HER2. (2) The second concern is related to the fact that blind samples were just limited to 8, again with no CNVs tested. However, this limitation is clearly discussed (lines 224-226).

Author Response

We sincerely thank you for your thorough evaluation of our manuscript and for your thoughtful comments. We appreciate the opportunity to clarify our rationale and address the concerns raised.

Reviewer Concern 1: “Most (92%) of the samples tested harbored just exon 20 alterations, and none of the tested alterations are CNVs in HER2, a type of variant that the kit is also designed to do. This should have been tested if possible... This might have been discussed, in particular if CNVs are seldom (or not at all) identified in HER2.”

Response: We thank the reviewer for this important observation. As correctly pointed out, the majority of HER2 alterations in our cohort were located in exon 20, which reflects the current clinical landscape of HER2 mutations in non-squamous NSCLC, where exon 20 insertions are the most commonly reported activating events with therapeutic relevance. Our study was intentionally designed to focus on mutational events, particularly small insertions, deletions, and SNVs, which represent the clinically actionable alterations in this setting.

We acknowledge that copy number variations are detectable by the OPA assay; however, CNV-positive HER2 cases in NSCLC are exceedingly rare and not part of current therapeutic recommendations in this tumor type. Moreover, no such cases were available within the retrospective cohort assembled across participating centers. We now explicitly address this point in the revised manuscript, clarifying the biological rarity of HER2 CNVs in NSCLC and the resulting unavailability of such cases for this study. Nevertheless, we agree that exploring CNV detection is of interest and would be a valuable addition to future prospective studies as broader solid tumor cohorts become available.

Reviewer Concern 2: “The second concern is related to the fact that blind samples were just limited to 8, again with no CNVs tested. However, this limitation is clearly discussed (lines 224–226).”

Response: We appreciate the reviewer’s acknowledgment that the limitation regarding the number of blind samples was transparently discussed. As noted, only eight samples were available for blinded interlaboratory testing due to the retrospective nature of the study and the strict inclusion criteria requiring confirmed HER2 mutations. Nonetheless, we ensured that these samples were representative of the mutation types observed in routine diagnostics and that they were processed under real-world conditions across multiple European centers.

Despite the small number, the complete concordance observed for mutation detection and variant annotation provides strong support for the assay’s robustness and reproducibility in detecting clinically relevant HER2 alterations. Regarding CNVs, we reiterate that no such cases were available in our sample set and that HER2 amplification, while common in breast and gastric cancers, is not a typical or routinely targeted alteration in NSCLC. This point has now been more explicitly addressed in the discussion section.

We thank you once again for your constructive feedback and the opportunity to improve our manuscript. We hope that our responses provide clarity and justification for the scope and limitations of our study. We believe our findings offer a meaningful contribution to the evolving diagnostic strategies for NSCLC and help inform the integration of rapid, targeted NGS assays in clinical practice

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for the revised version of your manuscript and for the clear and thorough responses to the comments raised during the previous review round. I appreciate the improvements made to the article and the effort you have put into addressing the concerns.

It is good to publish even if 8 samples are a critical point.