NSCLC Digital PCR Panel Returns Low-Input Sample Results Where Sequencing Fails

Round 1

Reviewer 1 Report

The authors report the performance and concordance of a next-generation sequencing assay (Illumina Trusight Oncology 500, TSO500) and a digital PCR assay (ChromaCode HDPCR™ NSCLC Panel) when using limited amounts of input material (DNA, RNA). The current study complements a study recently published in Diagnostics (reference 11) where the analytical sensitivity of the dPCR assay was described. The current study was well planned and conducted. All recommendations for testing the performance of an analytical test were followed and results were presented conclusively. As the amount of input material is a critical point in cancer testing, the results of this study are important for patient care. The data complements the previous study (reference 11) and should therefore be published in Diagnostics.

I would still like to suggest some minor revisions:

(1)   Improving diagnostic methods for minimal input is important. I understand that the TSO500 was just used as a reference for the HDPCR. However, one might get the impression that dPCR could replace NGS in all patients. The 10 targets (5 targets in each well, line 258) analyzed using the HDPCR panel may just “rescue” samples where the TSO500, which can be used to screen for genetic aberrations much more broadly, is not successful. Although this comparison was not the aim of this study, I suggest mentioning the different number of targets that can be analyzed with these two tests.

(2)   Methods: Is the TSO500 validated with just one sequencer (probably the NextSeq 500) or should the sequencer be mentioned as in line 81 the QIAcuity is mentioned to be used for dPCR? The sequencer and the amount of library put on the sequencer may influence coverage.

(3)   Abbreviations for which I am missing the explanation:
DIN - DNA Integrity Number (first mentioned in line 127, I am not using the Bioanlyzer and I am therefore not familiar with these abbreviations, what is the acceptable range of the DIN?)
RIN - RNA Integrity Number (see DIN)
FN – is it false negative? This explanation is missing in the headings of all  tables
tgt – line 163

(4)   Are the authors referring to table S1 or figure S1 in line 217 and line 256?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Author,

 I have carefully reviewed your manuscript titled "NSCLC Digital PCR Panel Returns Low Input Sample Results Where Sequencing Fails" and am pleased to provide feedback. Your study addresses a critical issue in the field of NSCLC diagnostics, highlighting the limitations of current standard-of-care tests and proposing a novel solution with the HDPCR NSCLC Panel.

The comprehensive discussion on the challenges associated with next-generation sequencing (NGS) and the impact of sample quality and quantity on test performance is well-articulated. Your findings, particularly the comparison between the HDPCR NSCLC Panel and the TSO500 assay, shed light on the potential of your approach to rescue samples with low mass inputs and improve sensitivity, which is a significant advancement.

The inclusion of supplemental material, such as Table S1 and Figures S1-S3, adds valuable detail and transparency to your study. The cut-off values provided in Table S2 and the concordance data in Table S3 and S4 contribute to the robustness of your methodology and support the reliability of the HDPCR NSCLC Panel.

Furthermore, the discussion on the advantages of the HDPCR NSCLC Panel, such as the ability to report partial DNA results and its cost-effectiveness compared to NGS testing, adds depth to the potential clinical utility of your assay. The emphasis on its role in providing immediate, clinically relevant information for patient management is a strong point.

I appreciate the clarity with which you acknowledge the limitations of your study, particularly highlighting the need for further clinical validation and testing in a clinical setting. This demonstrates a responsible approach to the translation of your research into clinical practice.

Overall, your manuscript is well-organized, and the results are presented with clarity. I believe that your study makes a valuable contribution to the field, and I recommend its publication with minor revisions. Please find specific comments and suggestions below.

Abstract: Consider providing a brief mention of the sample size in the abstract to give readers an immediate understanding of the scale of your study.

Introduction: In the introduction, consider expanding on the current challenges in NSCLC diagnostics using NGS, providing a bit more context for readers unfamiliar with the field.

Methods: Clarify the incubation conditions for the HDPCR NSCLC Panel. Provide details such as temperature and duration to enhance the reproducibility of your study.

   - Include the primer sequences for the HDPCR NSCLC Panel in the Methods section or in supplementary materials for the benefit of researchers who may want to replicate your work.

Results: Ensure consistency in the representation of data units throughout the results section (e.g., ng/µl, copies/µl) for clarity.

   - Consider providing a brief explanation or definition for any abbreviations or technical terms used in the results section to aid readers.

Discussion: When discussing limitations, briefly address any potential sources of bias or confounding factors that could have influenced the study outcomes.

   - Add a sentence to discuss the generalizability of your findings to other NSCLC populations or patient groups.

Conclusion: In the conclusion, reiterate the key findings and emphasize the potential clinical implications of your results.

References: Ensure that all references are complete and consistent in their formatting.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf