Myeloma immunoglobulin rearrangement and translocation detection through targeted capture sequencing

Targeted capture sequencing (CapIG-seq) is a novel, efficient, and effective approach to examine multiple genomic alterations in ctDNA and bone marrow aspirates of patients with multiple myeloma.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***

---------------------------------------------------------------------------
Reviewer #1 (Comments to the Authors (Required)): In the article "Immunoglobulin Rearrangement & Oncogenic Translocation detection in Multiple Myeloma through Targeted Capture Sequencing" [LSA-2022-01543-T], Chow, Pugh, and colleagues presented a novel hybrid-capture approach (CapIGseq) for the detection of immunoglobulin V(D)J rearrangements, oncogenic translocations, and gene mutations. The assay was tested on both model systems and patient specimens and was compared with PCR-based sequencing techniques as well as FISH. Longitudinal analyses for the same patients were performed and non-invasive disease monitoring was explored. Data presentation for each point was clear and thorough and demonstrated the utility of the assay. Genetic follow-up of plasma cell neoplasm (PCN) is often challenged by the limited number of cells available. The study presents a practical approach to workup PCN clinically that characterizes structural rearrangements and gene mutations in the same assay. 1. Although this is a method-focused paper, the reviewer suggests that the manuscript expands on exploring scenarios as to how this assay would be used in the context of routine tests such as FISH. What would be the added cost and turn-around-time (TAT) if applicable? Would the authors suggest this as an assay for all comers or would a selective and reflexive approach be more appropriate? 2. Minor comment: in table 1, MM1S IGHJ under LymphoTrack is missing a number.
Reviewer #2 (Comments to the Authors (Required)): Multiple myeloma is a hematooncological disease characterized by unstable genome, various translocations and clonal immunoglobulin variations. This paper describes a novel hybrid-capture approach targeting both ends of V and J segments of immunoglobulins to detect VDJ rearrangements as well as oncogenic translocations. Data are strongly supportive. The manuscript is clear. No additional experiments required in my opinion.
Reviewer #3 (Comments to the Authors (Required)): In this paper, Chow et al present their work on the development of a sensitive capture based method to detect V(D)J rearrangements, IGH translocations and mutations in MM samples. The work is mainly technical, and packed with details. This is to be praised, even if some issues arise from disconnects between tables and text in terms of numbers, and should be fixed because they make the results very hard to follow. Also, while their technical approach is somehow different from others, limitations in cfDNA have been extensively described (Oberle et al 2017, Manzoni et al 2020, and potential advantages of an "all in one" approaches have been published as well by several groups. Therefore, the novelty is relatively low, even if there is some confirmatory value • One important message is that shorter DNA fragments -150bp-may carry limited value to map IGH Tx breakpoints. The authors use cellular DNA to infer implications for cfDNA usage. However, aside from size, also quality and quantity of cfDNA can be a problem, and I would be careful translating findings here to the cfDNA field. It could be quite worse than this. • In

Reviewer #1
In the article "Immunoglobulin Rearrangement & Oncogenic Translocation detection in Multiple Myeloma through Targeted Capture Sequencing" [LSA-2022-01543-T], Chow, Pugh, and colleagues presented a novel hybrid-capture approach (CapIG-seq) for the detection of immunoglobulin V(D)J rearrangements, oncogenic translocations, and gene mutations. The assay was tested on both model systems and patient specimens and was compared with PCR-based sequencing techniques as well as FISH. Longitudinal analyses for the same patients were performed and non-invasive disease monitoring was explored. Data presentation for each point was clear and thorough and demonstrated the utility of the assay. Genetic follow-up of plasma cell neoplasm (PCN) is often challenged by the limited number of cells available. The study presents a practical approach to workup PCN clinically that characterizes structural rearrangements and gene mutations in the same assay.
1. Although this is a method-focused paper, the reviewer suggests that the manuscript expands on exploring scenarios as to how this assay would be used in the context of routine tests such as FISH. What would be the added cost and turn-around-time (TAT) if applicable? Would the authors suggest this as an assay for all comers or would a selective and reflexive approach be more appropriate?
Thank you for this suggestion. We think that there could multiple clinical and research applications for this assay. In its current form, CapIG-seq would be complementary to clinical FISH and cytogenetic assays. We would suggest using CapIG-Seq at diagnosis for all newly diagnosed patients and tailoring the sequencing panel to individuals at follow up time points if laboratory workflow allows. Overall, we would suggest a broader strategy at diagnosis and more targeted strategy at follow up sampling, with the ability to re-expand testing at the time progression or suspected therapeutic resistance. Due to the rapidly falling costs of high throughput sequencing as well as variations in adoption by different laboratories, it is difficult to comment precisely on the cost or turn around time (TAT) of this assay.
However, we anticipate that the cost would be similar to other sequencing panels of the same size used in clinical practice and it is currently equivalent to the cost of a single FISH assay. TAT may range from 1-4 weeks depending on institutional adaptation. In our laboratory workflow, the assay can be done in 1-2 weeks, including analysis and review, not accounting for wait times for sequencer availability. We have elaborated on some of these suggestions in the discussion, excerpted below: In its current form, CapIG-seq is complementary to clinical FISH, with the ability to detect additional rearrangements and mutations that are not present in standard of care testing. The addition of CapIG-seq to upfront testing in all comers may yield valuable information to guide clinical care. One advantage of CapIG-seq is its flexibility to modify testing in patient samples at follow-up timepoints. In clinical practice, this may take the form of testing for specific mutations to determine eligibility for clinical trials or novel agents, or testing for specific genomic alterations that give rise to therapeutic resistance. Although cfDNA testing is challenged by low concentrations of ctDNA at follow up time-points, assay sensitivity can be improved by increasing the number of genetic changes identified with a CapIG-seq approach. Thank you for this observation. This has been corrected.

Reviewer #2
Multiple myeloma is a hematooncological disease characterized by unstable genome, various translocations and clonal immunoglobulin variations. This paper describes a novel hybrid-capture approach targeting both ends of V and J segments of immunoglobulins to detect VDJ rearrangements as well as oncogenic translocations. Data are strongly supportive. The manuscript is clear. No additional experiments required in my opinion.
We very much appreciate your review. Thank you for the positive comments on our work.

Reviewer #3
In this paper, Chow et al present their work on the development of a sensitive capture based method to detect V(D)J rearrangements, IGH translocations and mutations in MM samples. The work is mainly technical, and packed with details. This is to be praised, even if some issues arise from disconnects between tables and text in terms of numbers, and should be fixed because they make the results very hard to follow.
Also, while their technical approach is somehow different from others, limitations in cfDNA have been extensively described (Oberle et al 2017, Manzoni et al 2020), and potential advantages of an "all in one" approaches have been published as well by several groups. Therefore, the novelty is relatively low, even if there is some confirmatory value One important message is that shorter DNA fragments -150bp-may carry limited value to map IGH Tx breakpoints. The authors use cellular DNA to infer implications for cfDNA usage. However, aside from size, also quality and quantity of cfDNA can be a problem, and I would be careful translating findings here to the cfDNA field. It could be quite worse than this.
We agree that there may be significant limitations in translation of this technique to cfDNA as outlined in the discussion. We have elaborated further on these limitations in this revision, excerpted below (bold indicates revised text)

These limit of detection findings were also not reproducible with shorter 150bp DNA fragments. A minimal length spanning the junction between structural rearrangements may be required to detect such structural changes. Previous work with T cell receptor V(D)J rearrangements has estimated >99% sensitivity to detect V-J rearrangements with a fragment length of 182bp (Mahé 2016).
In spite of this, cfDNA and bone marrow findings were highly concordant for V(D)J rearrangements in patients that had clinically detectable disease (Table 2), provided a minimum input of 83ng of cfDNA were input into DNA library preparation. We were not as successful in post-transplant patients with MRD (Suppl. Table 7). In addition to the structural limitations described, this may also be due to significantly decreased ctDNA shedding in cases where myeloma is relatively quiescent. Therefore detection of V(D)J rearrangements may be limited to use in either genomic DNA or where there is sufficient disease activity to generate the minimum required ctDNA.
In Thank you for submitting your revised manuscript entitled "Myeloma Immunoglobulin Rearrangement & Translocation detection through Targeted Capture Sequencing". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -please add ORCID ID for secondary corresponding author-they should have received instructions on how to do so -please add a category for your manuscript to our system -please consult our manuscript preparation guidelines https://www.life-science-alliance.org/manuscript-prep and make sure your manuscript sections are in the correct order -please add a figure legend for Supplementary Figure 5 to your figure legend section -please add figure callouts for Figure S6 and Figure S7 to your main manuscript text -please include the supp. Material file in the supplementary figures -please include the supp. Methods file into the main manuscript Material and Methods section -please add a separate approval/ethics statement for the human samples If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained. We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608). Corresponding or first-authors are welcome to submit the video. Please submit only one video per manuscript. The video can be emailed to contact@life-science-alliance.org To upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plex You will be guided to complete the submission of your revised manuscript and to fill in all necessary information. Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

A. FINAL FILES:
These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.
We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. **Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.** **It is Life Science Alliance policy that if requested, original data images must be made available to the editors. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original data images prior to final submission.** **The license to publish form must be signed before your manuscript can be sent to production. A link to the electronic license to publish form will be sent to the corresponding author only. Please take a moment to check your funder requirements.** **Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript. If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.** Thank you for your attention to these final processing requirements. Please revise and format the manuscript and upload materials within 7 days.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The reviewer is satisfied with the revisions. The manuscript is now ready for the next step in publication.
Reviewer #3 (Comments to the Authors (Required)): the authors have addressed my concerns