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2021 EAHP Symposium Overview

As part of the 20th Meeting of the European Association for Haematopathology virtual event, Thermo Fisher Scientific hosted an educational symposium focused on the uses of next-generation sequencing (NGS) in lymphoid research.

NGS is increasingly being adopted for BCR and TCR sequence analysis, as well as targeted gene panels in lymphoma research. Low limits of detection (LOD), and a greater flexibility to multiplex are just a few of the advantages that NGS offers over traditional methods.

In this on-demand presentation, lab directors shared their insights and experience on the value of NGS in their research.

Lymphoma NGS Molecular Characterization - Assessing the Journey to Answers

Marianne Grantham - Head of Cytogenetics and Molecular Haematology | Barts Health NHS Trust

In this symposium session, Dr. Marianne Grantham provided a brief overview of testing for lymphoid malignancy samples. She discussed current research on profiling lymphoma samples, the different requirements for various diseases, the benefits of using NGS, and her lab’s experience with the ION Torrent, the Oncomine Lymphoma Research Core Panel, and developing the Pan-lymphoid Custom Ion AmpliseqTM Panel.

Overview

Historically, morphology and immune phenotyping were the mainstays of lymphoid malignancy testing. More recently, cytogenetic and molecular testing are playing an important role. Dr. Grantham cited the WHO Classification of lymphoid neoplasms. It documents that genetic markers are as important as clinical, morphologic, and immunophenotypic markers. She noted that since nearly every lymphoma subtype is now known, this expanding knowledge is enabling sequencing to become more targeted, relevant, and useful.

As an example, Dr. Grantham illustrated how NGS has increased our understanding of the genetic underpinnings of follicular lymphoma.

90% of cases had 1 or more mutations in an epigenetic regulator that persist through early driver events, the course of the disease, and final evolution

Sharing her lab’s NGS experience, Dr. Grantham highlighted the benefits of amplicon-based sequencing, reviewed the Oncomine Lymphoma Research Core Panel, and provided an overview of the Pan-lymphoid Custom Ion AmpliSeqTM Panel capabilities that she and other labs are developing with Thermo Fisher.

Benefits of amplicon-based sequencing

• Sequences shorter, more fragmented DNA (smaller tissue samples)
• Target specific areas of interest
• Ability to multiplex up to thousands of primers in a single PCR reaction
• Fully automated process with built-in bioinformatics and streamlined analysis

Overview of Pan-lymphoid Custom Ion AmpliSeqTM Panel

• 60 genes, 1400 amplicons, DNA only
• B- and T-cell lineage
• T-NHL, MM, ALL, and new targets: RHOA, STAT2=3, STAT5B, MAP2K1, CXCR4
• 24-hour turnaround time
• Full automation from sample to result

The Time is NOW for NGS-Based Clonality Testing

Michael Hummel - Head of Molecular Pathology | Charité – Universitätsmedizin Berlin

Overview

In this symposium session, Dr. Michael Hummel reviewed the background of clonality testing in lymphomas and highlighted some of the advantages of NGS over traditional methods. He addressed the limitations of current IG FR3 short read assays and shared how the Ion Torrent ™ Oncomine ™ Pan-clonality BCR assay overcomes them. Dr. Hummel concluded his session with a strong assertion that the time is now for labs to adopt NGS-based clonality testing.

Background

In the late 90s, the lack of standardization for molecular testing was a significant obstacle to progress in NGS clonality testing. Progressive scientists in the field developed a consortium to address this problem. Dr. Hummel and his Euroclonality consortium colleagues developed new primer sets for clonality detection–and they became the gold standard in the field.

NGS clonal detection today and into the future

Dr. Hummel noted the drawbacks of previous IG FR3 short read assays and discussed how extensive somatic hypermutations can prevent primer binding. A potential solution is to include IG-kappa and lambda (heavy and light chain in 1 tube—the Oncomine Pan-clonality BCR Assay does just that.

Dr. Hummel asserted that NGS-based clonality detection is preferable to fragment analysis on every metric: sensitivity, specificity, workflow, cost, multiplexing, resolution, and reproducibility. In addition, it provides much more robust, easy to interpret results compared to conventional methods. Thus, Dr. Hummel concluded that the time for NGS-based clonality testing is now.

Fragment Analysis vs. NGS-Based Clonality Detection

Next-Generation Sequencing for B Cell Receptor Sequencing in Multiple Myeloma: Uses and Impact

Artur Kowalik, MD, PhD - Head Department Molecular Diagnostics | Holy Cross Cancer Center

In this symposium session, Dr. Artur Kowalik presented his work on multiple myeloma and the value of NGS for detecting rare clones. He also shared his early access data evaluating the Ion Torrent ™ Oncomine ™ Pan-clonality BCR assay and shared his perspective on what the future may hold.

He began with a brief discussion comparing the molecular and immunophenotypic techniques used for MRD detection in multiple myeloma.

Comparison of the molecular and immunophenotypic techniques for MRD detection in Multiple Myeloma

Evaluating Ion TorrentTM Oncomine TM Assays for Rare Clone Detection
Dr. Kowalik conducted multiple myeloma rare clone detection utilizing the Ion TorrentTM Oncomine TM BCR IGH SR Assays (DNA + RNA) and the Oncomine TM BCR Pan-Clonality Assay (IGH, IGK, IGL chains in a single tube reaction). With 16 multiple myeloma bone marrow samples per S530 chip, clones were detected down to 10-6 LOD within 48 hours, yielding 15M reads.

Dr. Kowalik described how the ongoing somatic hypermutation of a marker clone can give rise to subclones that differ from the dominant clone at one or more bases in the IGH chain. Hence the importance of clonal lineage analysis in multiple myeloma research. He added that the Ion Reporter clonal lineage analysis tool offered the advantage of automatically detecting these subclones.

Early Access Evaluation: Ion Torrent^TM Oncomine^TM BCR Pan-Clonality (IGH/K/L) Assay and Ion Reporter^TM

Key Takeaways

• NGS-based clonality testing in multiple myeloma provides comprehensive, highly sensitive results
• NGS of rearranged IGH chains is a reproducible approach for the detection of B-cell malignancies / multiple myeloma
• Results obtained demonstrate the feasibility of both RNA and DNA based IGH sequencing as research methods for clonal detection

Future Perspective

Dr. Kowalik anticipates increasing demand for NGS in the clonal assessment of multiple myeloma samples. His experience validates the use of the Oncomine BCR Pan-Clonality Assay. He predicts a bright future for NGS testing for rare clone detection with sensitivity improving down to the LOD of 10-7 for RNA input and 24-hour turnaround times to result.

> Watch Entire EAHP Symposium Presentation