Purpose We assessed the utility of blood cell-free DNA (cfDNA) whole genome sequencing (cfWGS) for minimal residual disease (MRD) monitoring in Waldenstrom’s macroglobulinemia (WM) by comparing this to 1) targeted panel sequencing of 27 genes of interest in WM and targeted capture of immunoglobulin gene rearrangements in blood and bone marrow 2) Multiplex-PCR of immunoglobulin loci followed by Illumina sequencing (clonoSEQ).

Experimental design Samples were collected from 7 patients on a clinical trial who were treated uniformly with chemoimmunotherapy and Bruton’s Tyrosine Kinase inhibitor (BTKi). Samples were collected prior to starting treatment and at clinical timepoints up to 18 months. MRD detection technologies were compared across all timepoints.

Results cfWGS was superior to both in-house targeted panel sequencing on cfDNA and clinical NGS in peripheral blood (PB) cells, using clinical bone marrow (BM) NGS as a standard. Tumor burden measured by cfWGS reflected MRD counts by clonoSEQ in BM.

Conclusions cfWGS may be a valuable non-invasive alternative to bone marrow testing in WM patients who require close follow up and provides greater sensitivity than targeted panel sequencing of cfDNA.

Statement of Translational Relevance Whole-genome sequencing in cell-free DNA (cfWGS) is a highly sensitive marker of minimal residual disease that has application as a biomarker in clinical trials. cfWGS more accurately reflects bone marrow tumor burden than other available non-invasive measures to date. Further exploration is warranted to determine its full potential for use in cancer diagnostics and research.

S. Chow has received honoraria from Sobi. N. Berinstein has received research support from AstraZeneca. C.I. Chen has received research support from Janssen, Beigene, Abbvie and Merck and honoraria for Astrazeneca. S. Trudel has received honoraria from Amgen Canada, Janssen Biotech, Pfizer, and Sanofi, grant funding from Amgen Inc and Bristol-Myers Squibb, research funding from Bristol-Myers Squibb, Genentech, GSK, Janssen, Pfizer and Roche and has acted as a consultant for Bristol-Myers Squibb, GSK, Roche, and Sanofi. T.J. Pugh has provided consultation for AstraZeneca, Chrysalis Biomedical Advisors, Roche, and Merck (compensated); and receives research support (institutional) from AstraZeneca and Roche/Genentech. T.J. Pugh is an inventor on patents of the CapIG-seq and CapTCR-seq methods held by the University Health Network. He is also a part of a technology licensing agreement between the University Health Network and Dynacare for technologies related to mismatch repair deficiency. The remaining authors have no conflicts to declare.

Funding for this research is provided by the Robert A. Kyle Career Development Award from the International Waldenstrom's Macroglobulinemia Foundation (IWMF), Waldenstrom's Macroglobulinemia Foundation of Canada and Poh Family Research Fund to S. Chow.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The BRAWM clinical trial was approved by the Ontario Cancer Research Ethics Board (OCREB, CTO project #3242). The biomarker substudy was approved by the Sunnybrook Research Ethics Board (REB#5500).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Primary whole-genome sequencing data are not available as the patients did not provide explicit written permission for their complete sequence to be shared. Secondary variant calls are available in the supplementary tables, as are the clinical data used for figures. The source code needed to reproduce the figures is available at https://github.com/pughlab/wm-cfwgs-mrd/.