Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture-Based Gene Panel.

Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.

Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study.

Introduction: Analyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). Methods: In 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. Results: A total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. Conclusion: In summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.