Integrated RNA-seq and RT-qPCR Workflow Identifies Non-IGH Fusion Transcripts as Individualized Molecular Markers for Monitoring Multiple Myeloma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present an integrated research framework based on RNA-seq and RT-qPCR aimed at identifying non-immunoglobulin (non-IGH) fusion transcripts as individualized molecular markers for monitoring minimal residual disease (MRD) in patients with multiple myeloma (MM), including non-secretory MM. Analysis of 22 patients revealed a high frequency of unique gene fusions, some of which were validated using RT-PCR and Sanger sequencing. The authors demonstrate that MRD monitoring based on these fusions can detect disease relapse earlier than flow cytometry.

I have a few concerns and would like to request the authors a minor revision:

 

1. The manuscript is generally well written, but there are grammatical and stylistic errors (e.g., English language errors, verb tense, sentence structure). Professional language editing would further improve clarity and readability.
2. Was the RNA used for the study isolated from all bone marrow cells or only from plasma cells?
3. In the RNA-seq analysis, the mm10 (mouse) genome reference was used. Could this be an error and require correction to hg38?
4. The study includes only 22 patients, and MRD analysis was performed in detail in only 4 cases. This limits the statistical strength of the conclusions and the general ability of the results.
5. Most of the detected fusions are unique to individual patients, which may be related to the cost-time efficiency of routine RNA-seq and the limited possibility of standardizing tests between centers.
6. The "Discussion" section should focus on comparing your findings with existing literature rather than restating the results. Avoid over-describing your data, which is already covered in the Results section; instead, focus on how your findings align with or diverge from the current state of knowledge. The Discussion section requires a better explanation of the findings and their comparison with similar results published by other authors. Some sentences in the Discussion are lengthy and could be streamlined.
7. The figures are factually correct, but some descriptions (legends) are imprecise or contain typos.
8. Please check if all abbreviations are defined at their first use in the main text.
9. Reference 10 (Nash, W., Treating diphenhydramine overdose. Nursing, 1994. 24(6): p. 33.) appears unrelated to MM and should be rechecked for relevance or replaced.
10. The manuscript requires standardization of the way of writing cited articles (e.g. Cleynen A [16], Cleynen A et ).

 

 

 

Author Response

Response to Reviewers Comments Manuscript ID: [biomedicines-4091119] Title: Integrated RNA‑seq and RT‑qPCR Workflow Identifies Non‑IGH Fusion Transcripts as Individualized Molecular Markers for Monitoring Multiple Myeloma Dear Editor and Reviewers, We would like to express our sincere gratitude to the reviewers for their constructive comments and insightful suggestions regarding our manuscript. These comments have been immensely helpful in improving the quality and rigor of our work. We have carefully addressed all the concerns raised. Major revisions include the correction of technical errors (e.g., the reference genome version), detailed clarification of our experimental methodology (e.g., plasma cell enrichment), and a more balanced discussion regarding the clinical utility versus technical feasibility of our approach. Point-by-point responses to the reviewers’ comments are provided below.

Reviewer 1

Comments 1: The manuscript is generally well written, but there are grammatical and stylistic errors (e.g., English language errors, verb tense, sentence structure). Professional language editing would further improve clarity and readability.

Response 1: We thank the reviewer for this suggestion. The revised manuscript has undergone comprehensive language editing by a professional academic service. And accordingly, we have corrected grammatical and stylistic errors including verb tense and sentence structure, with all changes marked in yellow, ensuring that the terminology conforms to international oncological standards.

Comments 2: Was the RNA isolated from all bone marrow cells or only from plasma cells?

Response 2: We appreciate the opportunity to clarify this. In this study, RNA was extracted from plasma cells which were isolated using commercial CD138 positive magnetic-activated cell sorting (MACS) kit. This step was crucial to ensure that the sequencing data primarily reflects the genomic landscape of the malignant clone, thereby increasing the detection sensitivity for low-abundance fusion transcripts. We have added this detail to Section 2.2 of the revised manuscript (page 3, line 103).   Comment 3: In the RNA-seq analysis, the mm10 (mouse) genome reference was used. Could this be an error and require correction to hg38? Response: We sincerely apologize for this clerical error. All RNA-seq data in this study were indeed aligned to the human reference genome hg38 (GRCh38) using GENCODE Release 27. The mention of "mm10" was an inadvertent error during manuscript preparation. We have corrected this in Section 2.3 (page 3, line 112).   Comment 4: The study includes only 22 patients, and MRD analysis was performed in detail in only 4 cases. This limits the statistical strength.   Response: We agree that the small sample size is a limitation. However, this study was designed as a proof-of-concept investigation to demonstrate the feasibility of a "Discovery-to-Validation" pipeline. While the cohort is modest, the longitudinal, high-resolution MRD tracking of the 4 cases provided depth that cross-sectional studies often lack, revealing early relapse signals that conventional FCM missed. We have acknowledged this limitation in the Discussion and emphasized the need for larger, multi-center prospective trials.   Comment 5: Most of the detected fusions are unique to individual patients, which may be related to the cost-time efficiency of routine RNA-seq and the limited possibility of standardizing tests between centers.   Response: The reviewer is correct regarding the heterogeneity of non-IGH fusions. However, our study emphasizes Personalized Medicine—the pipeline itself is standardized, even if the markers are patient-specific. Furthermore, we identified 5 recurrent fusions (e.g., DDX5::EEF1A1) across different centers, suggesting that a hybrid approach—combining a panel of recurrent markers with individualized ones—may be the optimal path for clinical implementation.   Comment 6: The "Discussion" section should focus on comparing your findings with existing literature rather than restating the results. Avoid over-describing your data, which is already covered in the Results section; instead, focus on how your findings align with or diverge from the current state of knowledge. The Discussion section requires a better explanation of the findings and their comparison with similar results published by other authors. Some sentences in the Discussion are lengthy and could be streamlined.   Response: Following the reviewer’s guidance, we have significantly restructured the Discussion. We now focus on how our findings regarding OAZ1 and KLF2 align with known MM pathogenesis and how our RNA-based detectability compares with DNA-based NGS methods (e.g., targeted IGH sequencing). The revisions have been highlighted in green on page 10, line 305, and page 11, line 349 of the updated version.

Comment 7: The figures are factually correct, but some descriptions (legends) are imprecise or contain typos.   Response: We have replaced the errors with the followed sentence on page 7, line 198-200 colored in green. “C-E: Gene Ontology (GO) analysis was performed to annotate the biological processes (C), molecular functions (D), and cellular components (E) associated with these genes that underwent breakage events.”   Comment 8: Please check if all abbreviations are defined at their first use in the main text.   Response: Thanks for your instruction. We have replaced the errors with the followed sentence on page 1, line 27 colored in green “RNA sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT‑qPCR)”, and page 4, line 135 colored in green “Durie-Salmon”.   Comment 9: Reference 10 (Nash, W., Treating diphenhydramine overdose. Nursing, 1994. 24(6): p. 33.) appears unrelated to MM and should be rechecked for relevance or replaced.   Response: We thank the reviewer for catching this. This reference was incorrect and has been replaced with: Coriu, D., et al., A molecular basis for nonsecretory myeloma. Blood, 2004. 104(3): p. 829-31. (page 13, line 404)   Comment 10: The manuscript requires standardization of the way of writing cited articles (e.g. Cleynen A [16], Cleynen A et).   Response: Thanks for your instruction. We have replaced the errors with the followed sentence on page 10, line 302 colored in green “Cleynen A et al”.

 

We believe that these revisions have significantly strengthened the manuscript and addressed all concerns. We look forward to your further assessment.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The study proposes an integrated RNA-seq and RT-qPCR workflow to identify non-IGH fusion transcripts as molecular markers for multiple myeloma monitoring. The technical pipeline is clearly described, and the concept of expanding MRD markers beyond IGH rearrangements is clinically relevant. However, the current interpretation occasionally exceeds the direct evidentiary support. In particular, the transition from fusion detection to claims of sensitivity, specificity, and clinical applicability requires more explicit quantitative benchmarking, longitudinal validation, and clearer distinction between technical feasibility and clinical utility. In addition, several figures contain text and labels that are too small to be clearly legible at standard print or screen resolution, which hampers independent assessment of the data. 
1.The manuscript positions the two identified non-IGH fusion transcripts as “sensitive” molecular markers for disease monitoring, yet sensitivity is primarily inferred from detectability rather than formally quantified performance. Could the authors clarify how sensitivity is defined in this study, and whether limit of detection, false-negative rate, or comparison with established MRD assays were systematically evaluated?
2.RNA-seq–based fusion discovery is followed by RT-qPCR validation, but the dependency between these two steps is not fully discussed. How robust is the workflow across samples with lower RNA quality or lower tumor burden, and to what extent does the success of RT-qPCR rely on the initial RNA-seq depth and fusion read support?
3.The manuscript implies potential clinical applicability for longitudinal disease monitoring, yet most analyses appear cross-sectional. Could the authors clarify whether serial samples from the same patients were analyzed, and if not, how conclusions regarding monitoring and dynamic disease tracking are justified?
4.Non-IGH fusion transcripts are proposed as broadly useful markers, but the prevalence and inter-patient heterogeneity of these fusions are not deeply explored. How generalizable are the identified fusion transcripts across different multiple myeloma subtypes, and what proportion of patients would realistically benefit from this approach?
5.While the workflow demonstrates technical feasibility, the manuscript sometimes conflates analytical detection with clinical relevance. Could the authors more clearly separate proof-of-concept methodology from claims of clinical sensitivity and utility, and discuss practical limitations such as assay standardization, reproducibility across laboratories, and integration into existing MRD frameworks?

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Response to Reviewers Comments Manuscript ID: [biomedicines-4091119] Title: Integrated RNA‑seq and RT‑qPCR Workflow Identifies Non‑IGH Fusion Transcripts as Individualized Molecular Markers for Monitoring Multiple Myeloma Dear Editor and Reviewers, We would like to express our sincere gratitude to the reviewers for their constructive comments and insightful suggestions regarding our manuscript. These comments have been immensely helpful in improving the quality and rigor of our work. We have carefully addressed all the concerns raised. Major revisions include the correction of technical errors (e.g., the reference genome version), detailed clarification of our experimental methodology (e.g., plasma cell enrichment), and a more balanced discussion regarding the clinical utility versus technical feasibility of our approach. Point-by-point responses to the reviewers’ comments are provided below.

Reviewer 2

Comment 1: The manuscript positions the two identified non-IGH fusion transcripts as “sensitive” molecular markers for disease monitoring, yet sensitivity is primarily inferred from detectability rather than formally quantified performance. Could the authors clarify how sensitivity is defined in this study, and whether limit of detection, false-negative rate, or comparison with established MRD assays were systematically evaluated?

 

Response: We acknowledge that we did not perform a formal LOD titration (e.g., serial dilution experiments) to determine the absolute theoretical sensitivity.

In the revised text, we have clarified that our claim of "sensitivity" refers to analytical detectability in a clinical context. Specifically, in Case 1 and Case 2 (NSMM), the fusion transcripts were reliably detected when FCM (threshold 10⁻⁴) yielded negative results, and these molecular signals preceded clinical relapse. We have revised our terminology from "highly sensitive" to "potentially more sensitive than conventional FCM" to be more scientifically rigorous. We have revised the article title to: “Integrated RNA‑seq and RT‑qPCR Workflow Identifies Non‑IGH Fusion Transcripts as Individualized Molecular Markers for Monitoring Multiple Myeloma.” (page 1, line 3)

 

Comment 2: RNA-seq-based fusion discovery is followed by RT-qPCR validation, but the dependency between these two steps is not fully discussed. How robust is the workflow across samples with lower RNA quality or lower tumor burden, and to what extent does the success of RT-qPCR rely on the initial RNA-seq depth and fusion read support?

 

Response: Our pipeline includes strict quality control: 1) Only samples with sufficient sequencing depth (>50M reads) and split-read support (≥5) were selected for validation. 2) All qPCR assays included internal controls (ABL or ACTIN) to normalize for RNA degradation. We found that even in samples with lower tumor burdens, the high specificity of breakpoint-spanning primers allowed for successful amplification.

 

Comment 3: The manuscript implies potential clinical applicability for longitudinal disease monitoring, yet most analyses appear cross-sectional. Could the authors clarify whether serial samples from the same patients were analyzed, and if not, how conclusions regarding monitoring and dynamic disease tracking are justified?

 

Response: We confirm that for the 4 MRD cases, serial samples were collected and analyzed at multiple time points, including diagnosis, post-induction, post-transplant, and relapse. This longitudinal tracking is the basis for our conclusions regarding dynamic disease monitoring. We have clarified this in the Figure 4 legend.

 

Comment 4: Non-IGH fusion transcripts are proposed as broadly useful markers, but the prevalence and inter-patient heterogeneity of these fusions are not deeply explored. How generalizable are the identified fusion transcripts across different multiple myeloma subtypes, and what proportion of patients would realistically benefit from this approach?

 

Response: While MM is highly heterogeneous, the methodology is universally applicable. Patients with Non-Secretory MM (NSMM), who represent 1–3% of cases and often lack trackable markers, stand to benefit most. Given the global incidence of MM, providing a molecular tracking tool for even 3% of "untrackable" patients represents a significant clinical impact.

 

Comment 5: While the workflow demonstrates technical feasibility, the manuscript sometimes conflates analytical detection with clinical relevance. Could the authors more clearly separate proof-of-concept methodology from claims of clinical detectability and utility, and discuss practical limitations such as assay standardization, reproducibility across laboratories, and integration into existing MRD frameworks?

 

Response: This is a vital point. We have revised the manuscript to clearly distinguish between technical feasibility (the ability to detect fusions via RNA-seq/qPCR) and clinical utility (the proven ability to improve patient outcomes). We have added a dedicated "Limitations" section discussing the challenges of assay standardization, cross-center reproducibility, and the need for standardized "positivity" cut-offs before this can be integrated into routine clinical frameworks.

We believe that these revisions have significantly strengthened the manuscript and addressed all concerns. We look forward to your further assessment.