OncomineWorld Autumn: A virtual NGS education meeting - We are back!

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