OncomineWorld Autumn: A virtual NGS education meeting

B-cell lymphomas and leukemias originate from the malignant transformation and proliferation of one or a small number of B cells. Since each B-cell expresses distinct B-cell receptors (BCRs) on its surface, potential malignant clones of interest can be identified and measured by the unique BCR sequences from the original cell. These B-cell transformations are an important focus area for ongoing translational and clinical research.

Today, next-generation sequencing (NGS) of the BCR repertoire is being increasingly adopted for hemato-oncology research due to the significant advantages it offers over traditional methods like Sanger Sequencing, PCR and flow cytometry. Comparatively, NGS provides ultra-high sensitivity, lower limits of detection (LOD), and greater flexibility to multiplex. In this webinar, we’ll explore a range of hemato-oncology applications for sequencing of the BCR repertoire. NGS-based clonality testing is becoming an essential method of analyzing hematolymphoid proliferations because it allows researchers to simultaneously sequence the malignant clone and evaluate the evolving clonal landscape. The sensitivity of NGS is also well suited for minimal residual disease research, enabling rare clone detection from low frequencies within the cell population. Moreover, NGS can be used to asses somatic hypermutation within the rearranged IGH/BCR gene, an important biomarker associated with Chronic Lymphocytic Leukemia samples.

Learning Objectives:

1. Understand the practical & technical advantages of NGS compared to traditional methodologies for BCR IGH analysis 

2. Evaluate NGS hemato-oncology research applications, including clonality assessment, somatic hypermutaion analysis, and minimal residual disease research 

3. Define how NGS can aid in resolution of challenging polyclonal samples

Operational barriers like complicated workflows and long turnaround times have hindered broad adoption of next-generation sequencing (NGS) in clinical oncology research. The new Ion Torrent Genexus System promises to address many of the current limitations of NGS. During this presentation, Dr. Kojo Elenitoba-Johnson from the University of Pennsylvania will discuss his early experience with the new Ion Torrent Genexus Sequencer and how the workflow could potentially simplify and speed up NGS-based oncologic testing in his laboratory. This will include preliminary studies evaluating the ease of use of the Genexus System as a sample-to-result platform for the identification of somatic variants in cancer, as well as determining performance characteristics of the system compared to the Ion Torrent Personal Genome Machine (PGM).  

Learning Objectives:

1. Participants will become familiar with basic aspects of the user experience with the Genexus system

2. Participants will learn about preliminary studies using a targeted panel and the Genexus system for the detection of somatic variants in cancer

Comprehensive genomic profiling of myeloid and, increasingly more, lymphoid malignancies is becoming an essential part of clinical diagnostics to improve diagnosis, assist therapeutic decisions, and provide better information about prognosis. Introduction of next-generation sequencing (NGS) into the for the profiling can be a solution for future. However it can bring challenges as well as benefits. Our institutions’ journey to introduce NGS into an integrated laboratory is discussed. The practical issues around panel content and data quality are also summarized.

Learning Objectives:

1. Understand the benefits of next-generation sequencing in the profiling  of haematological malignancies 

2. Develop an awareness of the practicalities of introducing new technologies to a medical laboratory

The evolution of clinical trial design in oncology reflects the current clinical paradigm of personalized medicine, weighing both histology and molecularly-defined biomarkers as the pillars for therapy selection. The number of predictive biomarkers in oncology is rapidly increasing, creating new treatment opportunities for cancer patients but also requiring additional costs and multiple tests. Complex profiling demanding extensive genomic analysis can be optimized by implementing comprehensive Next-Generation Sequencing (NGS) assays. In particular, broad but targeted NGS panels represent a viable option for diagnostics. Combining a large number of biomarkers in only one assay allows a reduction of turn-around-time at reasonable costs. Nevertheless, broad molecular profiling comes at the price of additional analysis time and the need for allocating supplementary resources. Benefits and drawbacks need to be carefully evaluated based on the clinical history of the patient. We address the benefits of comprehensive genomic profiling, particularly in the context of immuno-oncology, and assess the performance of the Oncomine™ Comprehensive Assay Plus for clinical sample profiling. 

Learning Objectives:

1. Identify the opportunities of comprehensive genomic profiling

2. Define biomarkers in immunooncology in the context of broad molecular profiling

NGS is still perceived as a complex, hard to implement and validate method that requires highly experienced people. Outsourcing next-generation sequencing (NGS) testing could be an option to overcome these limitations but it might end up being very expensive, and stringent sample requirements often lead to results being of poor quality and inconclusive. Recent clinical research studies show this issue effects approximately 20% of patients, excluding samples not submitted because they failed to meet sample quantity criteria.  A Multi-centric evaluation of performance of the Genexus Integrated Sequencer and Oncomine Precision Assay in detecting variants from FFPE and plasma samples of different type of tumors across four external customer laboratory sites will be discussed and concordance, success rate and reproducibility of results on different class of biomarkers will be presented. The results highlight how is possible to adopt in any molecular laboratory a complete and reliable molecular profiling solution for the testing of variants relevant for cancer management in an automated and fast manner.

Learning Objectives:

1. Discover how it is possible to get a comprehensive genomic profile of tumor samples with the speed of IHC

2. Identify how to enable 1 day results for FFPE and Liquid Biopsy testing with same assay

Increasing evidence suggests that targeted sequencing of cell free DNA (cfDNA) can provide a comprehensive molecular portrait of solid tumors. However, the concordance between tissue and cfDNA next generation sequencing (NGS) testing is usually low. This phenomenon might be due to different factors, including sequencing artifacts, clonal hematopoiesis and/or tumor heterogeneity. Therefore, appropriate internal and external validation of NGS methods are required before their use in the clinical scenario. Nevertheless, cfDNA testing can provide information that is complementary to tissue testing and that can aid to personalize treatments in patients with metastatic tumors. In addition, cfDNA testing might allow monitoring response to treatments and identifying mechanisms of acquired resistance that offer opportunity for therapeutic intervention. Results of liquid biopsy testing with NGS should be discussed in the Molecular Tumor Board to guarantee their appropriate interpretation and integration in the clinical scenario.

Learning Objectives:

1. Acquire knowledge on the limits and potential of cfDNA testing in solid tumors

2. Understand how to use cfDNA testing in clinical research

Molecular profiling is key in precision oncology research and whilst the tissue testing has become a routine, liquid biopsy might provide a non-invasive alternative when tissue biopsy is inaccessible. In future, detection of ctDNA could also provide a ‘real-time’ overview of disease burden owing to its short half-life and hence, useful in disease monitoring purposes.

To overcome the current obstacles of NGS systems, the Genexus Integrated Sequencer automates all steps of the targeted NGS workflow starting from nucleic acid of formalin-fixed paraffin-embedded tissues or plasma that significantly reduce laborious procedures. Importantly, the whole specimen-to-report workflow delivers results in a single day.

In this presentation, I will demonstrate the detection rate of alteration using Oncomine Precision Assay (OPA) on Genexus system using positive control samples, tissue and cell-free DNA. Nucleic acid was subjected to automated Genexus Integrated Sequencer for library construction using OPA panel, templating and sequencing. OPA covers key hotspot mutations, copy number gains or loss, fusion drivers. Particularly, among 44 of 48 (92%) cfDNA samples from non-small cell lung cancer were detected to carry at least one somatic mutation. Sensitizing EGFR mutations detected from 25 of 33 cfDNA of EGFR-positive plasma samples  were in complete concordance with tissue samples. Known resistance mutations of targeted therapies were also detected.

Learning Objectives:

1. Understand importance of NGS in molecular profiling of cancer

2. Define various NGS parameters and interpretation

In the past years, next-generation sequencing has revolutionized the way  testing is done in the field of clinical oncology research. Parallel testing of genomic markers in tissue biopsies as well as cytological and liquid biopsy specimens by NGS is now a key tool. This presentation will cover the role of NGS in a molecular pathology laboratory for the identification of relevant mutations in solid tumors. In particular, the workflow of the new Genexus sequencer and how it may improve on current limitations of NGS in will be discussed in detail.

Learning Objectives:

1. Understand NGS workflow in molecular profiling of tumor samples

2. Be able to identify current and future technologies used in molecular profiling

My laboratory at the Garvan Institute operates an ISO 15189 and NATA accredited laboratory for testing of patients using whole genome and whole exome sequencing, as well as gene panel testing of tumour tissue. In this presentation, I will outline my approach to test validation in medical laboratories.

Learning Objectives:

1. Summarize test validation

2. Explain genetic testing