OncomineWorld Autumn: A virtual NGS education meeting

Precision oncology continues to evolve as additional clinically relevant biomarkers are identified. The challenges facing the clinical researchers include not only the ability to add additional relevant biomarkers to existing tests, but ensuring they are adequately validated and robust for translation into a clinical research setting. The Oncomine Comprehensive Assay v3 has been implemented for use in both translational and clinical research projects including the NCI MATCH trial and shown reproducible and robust results. The newest version of the Oncomine Comprehensive Assay, Oncomine Comprehensive Assay Plus extends the already demonstrated high quality of performance into a larger targeted panel encompassing additional relevant content. Moreover, the extension of the panel allows for the determination of mutational signatures such as Tumour Mutational Burden (TMB) and Microsatellite Instabililty (MSI). This presentation will review our current experience with the Oncomine Comprehensive Assay Plus in the profiling of solid tissues from both formalin fixed paraffin embedded and fresh frozen material for use in the translational research setting.

Learning Objectives:
1. Identifying key performance metrics for choosing the right assay for clinical research studies
2. Understanding the importance of a fluid workflow for integrating new content for precision medicine studies

To identify genetic variants in archival human samples, researchers need a powerful NGS platform that can accommodate input DNA and RNA that is often low quality and/or low quantity. This challenge is compounded the diversity of frequently co-existing complex variants underlying somatic changes. Consequently, robust NGS assays capable of identifying single nucleotide variants (SNV), small indels, fusions, and copy number variation (CNV) in a single workflow are required. Molecular pathology research lab are now adopting the Thermo Genexus NGS platform for routine analysis of archival, formalin-fixed, paraffin embedded (FFPE) human tissue samples.  Compared to earlier NGS-based assays, the Thermo Genexus offers several important advantages. The amount of input DNA and RNA is lower than other assays permitting testing of a wider range of samples. Sequencing costs are reduced due to lower reagent costs and less hands-on time. Turnaround times are significantly faster. The Genexus assays are sensitive for detecting challenging variants such indels and copy number variation, as well as sequencing G-C rich DNA.  Examples several classes of genetic variants detected in archival, human FFPE samples using the Genexus are provided to demonstrate the performance of the Genexus NGS system.

Learning Objectives:

1. Demonstrate detection of copy number variation and single nucleotide variants in archival human tissue research samples

2. Discuss germline EGFR T790M in hereditary non-small cell lung cancer

Myeloid neoplasms represent a highly heterogeneous group of diseases that encompass myeloproliferative, myelodysplastic (myeloproliferative/myelodysplastic) processes and acute myeloid leukemias. The diagnostic workup of patients with suspected myeloid neoplasms is labor-intensive and operationally complex requiring coordination of multiple clinical and laboratory personnel, resources and requires integration of highly fragmented results. In this presentation, we will discuss the hematopathologist’s perspective on the clinical impact of recent advancements in NGS testing.

Learning Objectives:
1. Understand the contribution of genetic abnormalities in the diagnostic workup of patients with suspected myeloid neoplasms
2. Recognize the limitations of current diagnostic workflow in providing timely integrated genomic results for patients with suspected myeloid neoplasms
3. Become familiar with clinical scenarios where rapid integrated NGS may facilitate diagnostic workup of patients with suspected hematologic malignancies

Molecular testing of NCSLC biopsies is extremely important in the care and management of these patients.  However, NCSLC biopsies are typically very small and require multiple types of analysis for complete workup including H&E for diagnosis, IHC for classification, and FISH, IHC and NGS for biomarker testing.  This talk will focus on overcoming some of the challenges laboratories face when working with such a limited amount of tissue and how NGS can help.

Learning Objectives:

1. Review clinical guidelines for molecular testing of testing of non-small cell lung cancer specimens

2. Become familiar with recommendations for biopsy tissue stewardship 

3. How NGS can be used for molecular testing of NSCLC biopsies

IIdentification of relevant mutations, determination of tumor mutational burden (TMB) and microsatellite (MS) status are paramount to precision oncology research and the Oncomine Comprehensive Assay Plus (OCAP) measures the three markers in one assay. To establish OCAP in our laboratory, we analyzed 21 formalin-fixed paraffin-embedded cancer specimens which we previously had subjected to Oncomine Focus (OFA), Comprehensive (OCA) and/or Tumor Mutation Load (TML) assays, respectively. In addition, we had assessed microsatellite status (MSI) by MSI-PCR and/or immunohistochemistry for mismatch repair proteins in 16 of these samples. MSI-scores determined by OCAP consistently matched immunohistochemistry and/or PCR results, showing the accuracy of MSI calling by OCAP. TMB was assessed by TML assay and OCAP in 8 samples and in all cases comparable results were obtained. Two cases displayed high TMB scores (mutations/megabase) of 54.27/69.71 and 44.64/76.78 (OCA/P/TML), respectively. Intermediate TMB scores were obtained in two samples (6.66/6.7; 12.69/9.22; OCAP/TML) and four samples showed matching low TMB scores. A total of 56 single nucleotide variants and small INDELS were called by OFA, OCA and/or TML, in the 21 samples analyzed. In 54/57 cases variants were called by OCAP at a similar allele frequency. In three cases variants were not called due to quality issues. Copy number variations (CNV) were called in 17/21 OCAP performed, in 4 samples high MAPD values (>0.5) prevented CNV calling. A total of 16 copy number gains had been called by OCAv3 or OFA. 13/16 gains were confirmed by OCAP at similar copy numbers. In three cases of low level amplifications no significant increase in copy numbers was detected by OCAP compared to OCAv3. In conclusion, we show that OCAP with its subsequent analyses can be established and carried out in a molecular pathology laboratory, thus avoiding sending out of tissues with its negative effects on turnaround times and costs.

Learning Objectives:
1. Identify biomarkers relevant for clinical research
2. Identify advantages/disadvantages of large panel genomic profiling

During the presentation, Dr. Hernandez-Losa will describe the experience in the Department of Pathology using NGS with S5-xl in different translational projects in solid tumors. Additionally, he will explain the key questions to implement Genexus in the Molecular Biology Laboratory. Finally, he will describe the experience of tumor samples analysis by Genexus and the comparative data vs different NGS panels and platforms in NSCLC samples.

Learning Objectives:
1. S5 evolution of service and benefits of NGS by Genexus
2. Set-up of Genexus in a Molecular Laboratory

Myeloid neoplasms are a group of heterogeneus disorders originating from the neoplastic transformation of a pluripotent hematopoietic progenitor cell. Following the acquisition of somatic mutations there is an abnormal proliferation of hematopoietic cells from one or more cell lineages with the predominance of the neoplastic clone over the normal cells. Myeloid disorders are characterized by a high molecular complexity and by the presence of subclonal mutations occurring in subclones of variable size. The “classic” molecular biology techniques present several limits in terms of sensibility, sensitivity and time consuming. Next generation sequencing (NGS) have become fundamental for the in-depth study of molecular complexity and translational research in these diseases. Moreover the access to semi-automated platform and dedicated myeloid gene panels, represent an efficient and reliable opportunity for researchers to improve the quality of molecular data analysis.

Learning Objectives:

1. Evolution of technology in molecular biology

2. Next generation utility in myeloid neoplasm

3. Impact of NGS on translational research

Precision oncology is revolutionizing the diagnostic and therapeutic approach to cancer. In fact, the availability of tumor-specific and tumor-agnostic biomarkers and matched therapies is rapidly increasing. For example, targets for therapeutic intervention have been found in over 50% lung adenocarcinoma and intra-hepatic cholangiocarcinoma. Therefore, diagnostic laboratories will face the problem to assess different types of genetic alterations (single nucleotide variants, indels, copy number variation and gene fusions) in multiple genes in order to allow the identification of the most active treatment for patients with metastatic cancer. Next generation sequencing (NGS) and in particular targeted sequencing allows sequencing dozen to hundred genes in a single analysis. The European Society of Medical Oncology (ESMO) recently released recommendations on the use of NGS for patients with metastatic solid tumors. According to ESMO recommendations, NGS is the preferred diagnostic approach to assess actionable genomic alterations ready for clinical practice in selected cancer types, including lung adenocarcinoma, prostate cancer and cholangiocarcinoma. However, the use of larger panels for comprehensive genomic profiling is suggested in academic centers. These recommendations for the first time provide a rationale for the use of NGS in the clinical practice. The increasing number of biomarkers and matched therapies that are being approved will likely lead to an extension of the recommendation to use NGS to other tumor types.

Learning Objectives:
1. To increase the knowledge on precision oncology for solid tumors.
2. To increase the knowledge on the utility of next generation sequencing for genomic profiling of solid tumors.

The treatment of cancer has remarkably improved because of increased knowledge of the molecular abnormalities that drive human cancer growth. This has led to the development of ever more effective targeted cancer therapies. Considering these advances, the testing of molecular biomarkers for cancer patient stratification has become mandatory. Such tests are performed routinely using biopsy/cytology material from primary tumors obtained at the time of diagnosis. Next generation sequencing solution presents several advantages and current guidelines suggest NGS as first line molecular test to be performed. An additional aspect to consider is time to result. Any test is only effective is it provides its information in time to be considered for medical intervention. In particular, cancer molecular testing where the time window to initiate treatment are days. Here we will discuss the Ion Torrent Genexus System, the first turnkey NGS solution that automates the specimen-to-report workflow and can deliver results in a single day. This talk will focus on different aspects surrounding the system, from technological developments to analytical validation and clinical implementation.

Learning Objectives:

1. The importance of biomarker testing for precision medicine

2. How to implement biomarker using next generation sequencing

Comprehensive genomic profiling in clinical research studies helps to detect clinically relevant cancer driver mutations using low amount of tumor tissues within several days. Recent studies have shown that Immune checkpoint inhibitors (ICIs) treatment is promising across cancer type and multiple gene biomarkers like MSI and TMB can be a predictor of response for ICIs. OCA Plus DNA is the most comprehensive panel with more than 500 genes which can detect TMB and MSI as well as emerging biomarkers like HRR (Homologous recombination repair) genes. To evaluate the analytical performance of OCA Plus DNA, we tested sensitivity, accuracy and specificity of SNV, Indel and CNV using reference materials. In addition, to assess the reliability of TMB and MSI results, we tested commercial reference cell lines and clinical research samples tested by commercial MSI qPCR assay, respectively. The assay showed excellent analytical and clinical performance and high reproducibility between operators.

Learning Objectives:
1. To evaluate the analytical performance of OCA Plus DNA
2. To assess the reliability of TMB and MSI results of OCA Plus DNA

High grade serous ovarian cancer is characterized by genomic instability, with ~50% of advanced tumors harboring homologous recombination repair (HRR) pathway deficiency (HRD). HRD results in irreparable DNA damage and increased sensitivity to platinum chemotherapy, as well as increased susceptibility to poly-ADP ribose polymerase (PARP) inhibitors. PARPis are able to induce synthetic lethality in cells with HRD by binding to and trapping PARP1 and PARP2 to single-stranded DNA breaks, which generates double-stranded breaks. The lack of high-fidelity HRR in these cells leads to reliance on error-prone DNA repair pathways (e.g.: non-homologous end joining), further genomic instablity and cell death. The presence of HRD leads to detectable signature or genomic scar which can be scored by different tools. Available tools have computed the following: loss of heterozygosity, telomeric allelic imbalance and large-scale state transitions to derive a summated HRD score. The HRD score has come to the fore as important individualised information in patients with advanced ovarian cancer, due to its correlation with response to maintenance PARPis in the first-line therapeutic setting. In this session, we discuss the role of PARPis in the treatment of advanced ovarian cancer, with particular focus on the use of HRD testing to better define optimal therapy for front-line treatment of these patients.

Learning Objectives:
1. To understand the role of PARPis in the treatment of advanced ovarian cancer
2. Understanding biomarkers to select patients for PARPi therapy

There is an expanding list of therapeutically relevant biomarkers for non-small cell lung cancer (NSCLC), and molecular profiling at diagnosis is paramount. Tissue attrition in scaling traditional single biomarker assays from small biopsies is an increasingly encountered problem. We compared the performance of targeted NGS panels with traditional assays and correlated the mutational landscape with PD-L1 status in Singaporean patients with the National Lung Profiling (NLP) study. A cost-effectiveness analysis of NGS compared to standard molecular testing was conducted. The study demonstrated that even in an EGFR mutant predominant population, upfront NGS represented a feasible, cost-effective method of diagnostic molecular profiling compared with sequential testing strategies. These results support the implementation of diagnostic NGS in non-squamous NSCLC in Asia to allow patients access to the most appropriate personalized therapy.

Learning Objectives:
1. To recognise key considerations for the routine clinical implementation of next-generation sequencing
2. To appraise the cost-effectiveness of diagnostic next-generation sequencing for metastatic lung cancer

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

Genomic profiling of liquid biopsies is no longer an emerging area of clinical research, but quickly becoming translated as part of contemporary clinical trials for potential future use in the diagnostic setting.  Comparatively less invasive than traditional solid tissue biopsy approaches, liquid biopsy specimens such as blood or urine can be used not only for early detection, but for the monitoring of therapeutic response and progression.  While custom circulating tumor DNA (ctDNA) assays are being academically developed for the profiling of specific biomarkers in a specific disease or therapeutic context, there are commercially available pan-cancer circulating nucleic acid (cNA) panels available. These commercially available panels can be readily used  to reveal the spectrum of  key genomic changes within liquid biopsies, determine the level of sensitivity that can be obtained and provide a means for validation. This presentation will summarize the evaluation of the Oncomine Pan-Cancer Cell Free Assay and together with the Oncomine Comprehensive Assay v3 we demonstrate the utility of this platform to map nucleic acids released to the blood or urine in the context of breast and prostate cancer.

Learning Objectives:

1. Define liquid biopsy profiling potential for improved cancer clinical management

2. Define liquid biopsy profiling provides insight on tumour progression

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

Next-generation sequencing (NGS) has become the most effective tool in the practice of clinical oncology. The technology allows for rapid detection of variants in DNA/RNA simultaneously in a single run within a few days. The results provide valuable information on diagnostic, prognostic and therapeutic targets, which help physicians determine the treatment strategy for cancer patients. This presentation includes two parts. Part 1 will cover NCCN/CAP/ASH guidelines and treatment options for hematologic malignancies. Part 2 will share the experience with the Oncomine Myeloid Research Assay including technical validation and summary of the clinical data from over 1300 patients. Overall, the abnormality rate was 56%. Among these patients, 3% were MPN, 34% were AML, 10% were MDS, and 53% were other myeloid disorders. The most common variants detected include TET2, ASXL1, DNMT3A, RUNX1, TP53, FLT3, IDH1, IDH2, WT1, JAK2, CALR, and CEBPA. The fusions include BCR/ABL1, CBFB-MYH11, KMT2A fusions, RUNX1 fusions, and PML-RARA. These variants are clinically actionable with therapeutic drugs or have prognostic implications.  In summary, this presentation demonstrates that the NGS test is a robust and powerful tool for detecting genetic alterations associated with various types of cancers. This advanced tool will help maximize the benefit of precision medicine in the clinical oncology practice for cancer patients.

Learning Objectives:

1. Explain the overview of the guidelines and treatment options for hematologic neoplasms

2. Describe the clinical utility of targeted next generation sequencing tests in the context of myeloid neoplasms

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

The prevalence of EGFR mutation rate positivity in NSCLC is high in Asia, and this is the case in Malaysia as well. This presentation will provide Malaysia’s approach in implementation of NGS in 1st line testing for NSCLC. The challenges posed by the discovery of an increasing number of driver mutations amenable to targeted therapy, the need to often work with small biopsy samples and a requirement to improve turnaround times were critical issues  that prompted a change in workflow from single to multigene panel testing. A small patient cohort study conducted with the participation of diagnostics and pharma partners  confirmed the integral part molecular testing plays in the theragnostic approach to patient care. This multigene testing approach also provides further insights into other concomitantly occurring molecular biomarkers which may have potential prognostic value. 

Learning Objectives:

1. Explain what factors drive NSCLC molecular testing towards Multigene Testing

2. Identify the barriers for adopting NGS Multigene Testing