A Novel Single-Tube Next Generation Sequencing Assay for B-Cell Receptor Clonality Testing

Round 1

Reviewer 1 Report

In this paper, the authors assessed a new test for assessment of immunoglobulin rearrangements, all combined in one tube. They analyzed 27 neoplastic B-cell lines, and bone marrow samples of 45 patient with MM as well as related plasma neoplasms.

My major concern:

1)      This test is presented as a PAN-clonality test, however for clonality it is essential to discern polyclonal from clonal samples, and also to be able to show polyclonal results. However, no polyconal samples were examined or presented in this study. The authors should include many reactive cases in order to show applicability for testing clonality.

2)      How do the authors define a clone? They refer to existing publication, however, since this is another technology it is important to establish cut-offs with an own set of samples such as reactive polyclonal samples. Can the authors provide these data.

3)      Clonality testing occurs primarily in the pathology setting. The authors did not provide clonality data from FFPE tissues, although they describe this in the discussion. Also, the cases in pathology that need clonality detection mostly do not have high percentage of suspect malignant B cells. This is relevant information for the paper; so the performance in FFPE cases, and in FFPE cases with low percentage of neoplastic B-cells.

4)      The description of this assay merely described clone detection in fresh material samples with high tumorload; this should be stated clearly, it should not be presented as a clonality assay.

5)      Table 1. How do the authors explain that the PAN-clonality IGH clonotype result differs from the OncoMine IGH FR2 and FR3 clonotypes, in  U266B1, GM14952, Ramos, Pfeiffer. With each of these test the same clonotype is expected so this results is very strange and questions the accuracy of either technology and requires explanation.

6)      Table 1. Does this test provide IGKV-KDe rearrangements? How do the authors explain the lack of detection of an -KDE rearrangement while a clonal Lambda rearrangement is detected. This is found in a couple of cases and is an unexpected result that should be explained.

7)      Table 2 Why is there lack of detection of KDE-R while there is a lambda clonal rearrangement found. This is an unexpected result especially since the IGK-DE rearrangements do not undergo somatic hypermutation. The authors should explain why the (IGK-IGL) results of this test are reliable.

Minor:

8)      The reference to existing technologies and guidelines is incomplete. The authors should evaluate the literature more thoroughly and include the papers of  M. van den Brand et al. 2021 and 2023

9)      The authors should explain why it is expected that gDNA of PBL would provide highly matching results, because I do not think this is needed.

10)   I would like to see more specific information describing the minimal number of reads or clonotypes that are detected in the low cell line dilutions, what are the minimal specs for detection.

11)   Under lineage determination; the number of reads needed should be related to the input of DNA

12)   The tables in Fig 2 and 5 are very difficult to read; this should be improved

13)   The Spectratyping plots are also difficult to read, and need improvement and a better description of the plot.

Author Response

Please see the attachment, thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript focuses on  A Novel Single-Tube Next Generation Sequencing Assay for B- 2 Cell Receptor Clonality Testing represents a technically correct and timely relevant manuscript. Accordingly, few minor suggestions should be implemented to improve the readibility of the manuscript on this journal

- In the introduction section, please, could the authors overview the role of predictive molecular pathology in hematological malignancies patients?

- In the text, please, could the authors review figure 2 improving readibility ?

- In the discussion section, please, could the authors highlight technical and clinical limitations of  this approach? Could they also propose a diagnostic alghoritm where this approach may be integrated?

Minor english editing should be approached

Author Response

Please see the attachment, thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Reviewer reply to 1)The additional data, supplementary tables 2 and 3 are an improvement.

Please note that in these suppl tables, as well as in Table1 in the manuscript; different rearrangement targets such as; IGK-VJ, IGKV/intron KDE, IGL-VJ are lumped up in one column. This is not correct for the identification of clonality. Can the authors change that.

Can the authors confirm that by IgK-Kde they mean IGKV-KDE? Or did I misunderstood? (see also line 170 in the text). Please can this be altered consistingly throughout the text. 

Reviewer reply to 2) I thank the authors for clarification. For usage of clinical practice, measures for improvement considering data analysis (especially light chain genes, see point 1) are essential in my opinion.

Reviewer reply to 3) and 4) I thank the authors this addition. 

Reviewer reply to 5) The authors explain this correctly. However, it is important and educational for the readership that this is also explained in the legend of the paper.

As an additional point: May I conclude that the software does not include a description of the junctional region? The analysis of the junctional region is very helpful, in fact essential for the determination of a clonal relationship in patients with multiple lymphoma’s, a frequently occurring diagnostic question.   For the readership it should be clear that using this assay/ this software; diagnostic questions for determining the clonal relationship in patients with multiple lymphoma is not possible.

Reviewer reply to 6) This explanation is not entirely correct;  since the IGK-DE does not undergo somatic hypermutation.The explanation may be lack of the amplification due to the presence of SNPs under the primer-binding site. I suggest to explain this in the legend of the Table. Again, this will add educational value to the manuscript.

Reviewer reply to 7) see response to 6.

Reviewer reply to 8),  9) and 11). Thanks for this improvement.

Reviewer reply to 10).  It should be clear in the paper that the LOD of 10-6 is only possible at a specified high input of DNA. I still miss this in the paper.

Reviewer reply to 12).  The way it’s represented in the paper is still not clear to me, but this can be further addressed if the paper is going to be published.

Reviewer reply to 13).  Same as 12 for the plots. 

Author Response

Please see the attachment for inline responses in purple.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript may be accepted in the present form

No other comments

Author Response

The reviewer did not request any revisions.