PONE-D-26-13956

Superior DNA- and sufficient RNA integrity in formalin-free paraffin-embedded tissues

PLOS One

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Dear Authors

Thank you for submitting your research for publication. This manuscript evaluates a formalin-free, supercritical CO₂–based tissue processing method (NFPE) versus FFPE, focusing on DNA/RNA integrity and RNA NGS performance. The topic is timely and relevant to molecular pathology, particularly given the increasing reliance on nucleic acid–based diagnostics and concerns regarding formalin-induced artifacts. However, there are substantial methodological and interpretational limitations that currently weaken the validity of the conclusions.

#Major Comments

1. Study Design and Cohort Heterogeneity

The study includes a relatively small number of samples with substantial heterogeneity in tissue types (e.g., colon, placenta, adrenal, myocardium, various tumors), without stratification or adjustment. Different tissue types have inherently DNA/RNA preservation characteristics. Additionally there was no stratification by tissue type, tumor vs normal status, or cellular composition.

Recommendation:Provide detailed breakdowns of sample numbers per tissue type and per group. Consider stratified analysis or justify pooling across heterogeneous tissues. Clarify whether matched comparisons are consistently applied.

2. Confounding Pre-analytical Variables

Key pre-analytical factors (Cold ischemia time, Time from excision to processing) are insufficiently controlled or described.

Recommendation:Clarify and standardize pre-analytical conditions where possible. Explicitly discuss this limitation and its potential impact on RNA and DNA integrity results.

3. Definition and Interpretation of False Positives

The classification of fusion calls in FFPE samples as “false positives” is not sufficiently supported by the data. In the Results section (lines 204–213), the authors report that several FFPE samples showed positive fusion calls, which were absent in matched NFPE samples and subsequently negative on an alternative assay. Based on this, the authors conclude that “indeed all positive results… were false-positive.” This interpretation is reiterated in the Discussion section (lines 273–278).

However, discordance between methods and negative results on a secondary assay do not definitively establish false positivity, particularly in the absence of an independent gold-standard validation (e.g., orthogonal sequencing, RT-PCR). Alternative explanations such as low-level true events, sampling variability/ tumor heterogeneity, or assay sensitivity differences are not excluded.

Recommendation: Define clear criteria for false-positive classification and provide supporting data. I suggest avoiding definitive claims without rigorous validation.

4. Statistical Analysis

The statistical approach is limited to t-tests, with no indication of:

* Adjustment for multiple comparisons

* Reporting of effect sizes or confidence intervals

Given the matched nature of samples and potential non-normal distributions, the current analysis may not be appropriate.

Recommendation:Reassess statistical methods. Consider non-parametric or paired analyses and report effect sizes alongside p-values.

5. Conflict of Interest and Potential Bias

The corresponding author is listed as an inventor on a patent related to the NFPE technology, and the study uses proprietary equipment (TISPA system). This may represent a significant potential conflict of interest.

Recommendation:Ensure that the role of the authors in developing the technology is transparently described. Consider including statements regarding measures taken to minimize bias. Independent validation would strengthen the manuscript.

6. Reproducibility and Generalizability

The study is conducted at a single center using a single platform (Ion Torrent), with no external validation.

Recommendation:Temper conclusions regarding broad applicability. Explicitly acknowledge limitations in reproducibility and generalizability.

7. Methodological Details and Reproducibility:

Important methodological details are missing or unclear:

* Input amounts for DNA/RNA extraction

* Extraction efficiency

* QC thresholds for NGS

*Library preparation success rates

* Potential batch effects

Recommendation:Expand the Methods section to ensure reproducibility and transparency where possible and expand on study limitations as required.

#Minor suggestions/comments

* The Discussion contains statements that are somewhat promotional. I recommend these toned down.

* Ensure consistent use of terms (e.g., NFPE vs “non-fixed”, FFPE vs “formalin-fixed”) in the manuscript.

* Figures:

Include sample sizes in figure legends.

Consider adding individual data points to boxplots.

* Data Availability

The statement that “all relevant data are within the manuscript and supporting files” is insufficient for a study involving NGS data.

Recommendation:Deposit sequencing data in a public repository (e.g., GEO, SRA) and provide accession numbers.

Kind regards

Reviewer #2: Abstract

• The phrase “falsely encourage personalized therapy” is overly strong/speculative for an abstract unless directly demonstrated by clinical validation. I recommend revising to more cautious wording.

Introduction

• Since the authors cite their own previous work demonstrating histologic and preliminary DNA quality benefits, the introduction should more clearly define how the current study advances beyond prior publications. The authors need to distinguish what was previously known, what remains unknown, and what new contribution this study provides.

Material and methods

• The authors need to specify the following: total number of tissue specimens included, number of samples per tissue type, whether samples were paired/matched between FFPE and NFPE conditions, and whether the same tissue specimen was split for comparative processing.

• Please justify the rationale for combining these diverse tissue types in the same analysis and discuss whether tissue-specific differences in cellularity/composition may influence DNA/RNA integrity outcomes.

• The authors used two different processors (TISPA I vs. TISPA II), thus introducing a potential technical confounder. Please explain the following: what differences exist between TISPA I and TISPA II systems, Whether protocol parameters differed between instruments, and why 2-year-old samples were processed differently than 3-day/7-year cohorts.

• The statement “stored under exactly the same conditions” is insufficiently specific. Please provide quantitative storage parameters: exact temperature range and whether storage was continuous and monitored.

• The authors need to specify the following: the total number of RNA samples subjected to NGS, how samples were selected from the larger cohort, and distribution of FFPE vs NFPE samples analyzed by NGS. This is essential for evaluating representativeness and study power.

Conclusion

• The sentence “RNA integrity in NFPE was lower than RNA integrity in young samples, but stable over time” is unclear and potentially confusing. It is not explicit whether “young samples” refers to young FFPE samples, young NFPE samples, or freshly processed tissues.

• The conclusion states NFPE is “quicker and cheaper” than FFPE. Unless cost/time analyses were formally performed in this study or cited robustly, these claims should be removed or qualified.

• Given that this is likely an early/technical validation study, the conclusion should acknowledge that: further multicenter validation, broader tissue-type testing, and workflow implementation studies may be needed before clinical adoption.

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Reviewer #1: No

Reviewer #2: Yes:  Hoda Y Abdallah

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Good DNA- and sufficient RNA integrity in formalin-free paraffin-embedded tissues

PONE-D-26-13956R1

Dear Dr. Hoogland,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Nasar Alwahaibi, PhD

Academic Editor

PLOS One

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