NGS method for parallel processing of high quality, damaged or fragmented input material using target enrichment.

Next-generation sequencing (NGS) has been increasingly popular in genomics studies over the last decade and is now commonly used in clinical applications for precision diagnostics. Many disease areas typically involve different kinds of sample specimens, sample qualities and quantities. The quality of the DNA can range from intact, high molecular weight molecules to degraded, damaged and very short molecules. The differences in quality and quantity pose challenges for downstream molecular analyses. To overcome the challenge with the need of different molecular methods for different types of samples, we have developed a joint procedure for preparing enriched DNA libraries from high molecular weight DNA and DNA from formalin-fixed, paraffin-embedded tissue, fresh frozen tissue material, as well as cell-free DNA.

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.

Sensitive Detection of Cell-Free Tumour DNA Using Optimised Targeted Sequencing Can Predict Prognosis in Gastro-Oesophageal Cancer.

In this longitudinal study, cell-free tumour DNA (a liquid biopsy) from plasma was explored as a prognostic biomarker for gastro-oesophageal cancer. Both tumour-informed and tumour-agnostic approaches for plasma variant filtering were evaluated in 47 participants. This was possible through sequencing of DNA from tissue biopsies from all participants and cell-free DNA from plasma sampled before and after surgery (n = 42), as well as DNA from white blood cells (n = 21) using a custom gene panel with and without unique molecular identifiers (UMIs). A subset of the plasma samples (n = 12) was also assayed with targeted droplet digital PCR (ddPCR). In 17/31 (55%) diagnostic plasma samples, tissue-verified cancer-associated variants could be detected by the gene panel. In the tumour-agnostic approach, 26 participants (59%) had cancer-associated variants, and UMIs were necessary to filter the true variants from the technical artefacts. Additionally, clonal haematopoietic variants could be excluded using the matched white blood cells or follow-up plasma samples. ddPCR detected its targets in 10/12 (83%) and provided an ultra-sensitive method for follow-up. Detectable cancer-associated variants in plasma correlated to a shorter overall survival and shorter time to progression, with a significant correlation for the tumour-informed approaches. In summary, liquid biopsy gene panel sequencing using a tumour-agnostic approach can be applied to all patients regardless of the presence of a tissue biopsy, although this requires UMIs and the exclusion of clonal haematopoietic variants. However, if sequencing data from tumour biopsies are available, a tumour-informed approach improves the value of cell-free tumour DNA as a negative prognostic biomarker in gastro-oesophageal cancer patients.

BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.