In this study, we evaluated the concordance of targeted sequencing results between paired ctDNA and tumor samples from early breast cancers scheduled for curative surgery with or without adjuvant therapy. If high concordance was observed, pre-operative ctDNA testing could serve as a non-invasive surrogate for variants meant to be revealed after definite surgery. On the other hand, poor concordance may hamper wide clinical absorption of liquid biopsy early breast cancer. The study VGH-TAYLOR: Comprehensive precision medicine research on the heterogeneity of Taiwanese breast cancer Patients, consisted of three years enrollment and approximately four years follow-up after enrollment. Individual subject was assigned into 3 groups based on the staging and therapy before and/or after surgery. Molecular profiling and potential biomarkers were determined using Oncomine Comprehensive Assay v3 from FFPE tissues and Oncomine Breast cfDNA Assay v2 from plasma. Common genes interrogated by both ctDNA and TMO comprehensive assay were identified, and concordance-between paired targeted sequencing results from the same individuals were reported. The mutation landscape of initial 612 patients interrogated with the Oncomine Breast cfDNA assay v2; 239 out of 612 patients reported at least one mutation (39%). Among 246 patients assayed for both ctDNA and tumor tissue, cfDNA assay detected 73 (29.6%) and TMO comprehensive assay detected 201 (81 .7%) breast cancers with at least one variant. Sixty-seven (25.6%) were tested positive for both liquid and tissue assays. Although only one-quarter of patients were positive for both cfDNA and TMO comprehensive assays from the same subject, the most prevalent mutant genes from both platforms were TP53 (68.3%) and KRAS (53.5%), both were well-known drivers in cancers. Our study showed that tumor should be the preferred source and targeted sequencing and 4% variants wound be revealed from liquid biopsy.

Dr. Ritterhouse's laboratory has implemented rapid and ultra rapid protocol for NSCLC genomic profiling, based on NGS. This has led to significant shortening of turn around time (TAT) and improvements in cancer patient care. Dr. Ritterhouse is ging to descrive the workflow and share their routine reslts as well as a case study report.

Next-generation sequencing can detect multiple driver oncogenes simultaneously, enabling the analysis of limited amounts of biopsied tissue samples. . Druggable driver mutations cover more than 50% of the NSCLC patients. However, treatment options other than molecular targeting drugs have been desired for the remaining half of the populations (driver mutation-negative).The ctDNA dynamics can be evaluated by monitoring changes in ctDNA level in patient follow-up, which is one of the promising biomarkers of liquid biopsy. Many studies of liquid biopsy monitoring and quantifying ctDNA have been conducted to establish a novel approach for prediction of treatment response and monitoring of disease progression, and prediction of prognosis. The Oncomine Lung cfTNA Research Assay is part of a complete solution to detect lung tumor-derived cell-free DNA and RNA (cell-free total nucleic acid; cfTNA) isolated from the plasma fraction of whole blood.

The circulating T-cell receptor (TCR) repertoire is a dynamic representation of overall immune responses in an individual. Here we aimed to correlate pre-treatment circulating TCR repertoire metrics and clinical outcome in advanced non-small cell lung cancer (NSCLC). We prospectively collected baseline blood from patients treated with first-line pembrolizumab monotherapy (n=53) or in combination with chemotherapy (n=48). TCR-β repertoire was assessed using the Oncomine TCR Beta-SR Assay and correlated with clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS) and immune-related adverse events (irAEs). We observed an association between reduced number of unique clones and improved CBR among patients in treated with pembrolizumab (RR=2.86, 95%CI 1.04-8.73, P=0.039). However, there was no statistically significant correlation between TCR repertoire and PFS or OS. In patients in treated with pembrolizumab plus chemotherapy, increased number of unique clones (HR=2.96, 95%CI 1.28-6.88, P=0.012) and Shannon Diversity (HR=2.73, 95%CI 1.08-6.87, P=0.033); and reduced evenness (HR=0.43, 95%CI 0.21-0.90, P= 0.025) and convergence (HR= 0.41, 95%CI 0.19-0.90, P=0.027) were associated with improved PFS. Only increased number of unique clones (HR= 4.62, 95%CI 1.52-14.02, P=0.007) was associated with improved OS. A logistic regression model score combining the TCR repertoire metrics improved the prediction of CBR (RR=0.52,95%CI 0.24-0.84, P=0.006) and was strongly associated with OS (HR=0.20, 95%CI 0.05-0.76, P<0.0001). Reduced TCR conversion was associated with increased frequency of irAEs needing systemic steroid treatment. Overall, pre-treatment TCR repertoire might serve as a predictive biomarker for clinical outcomes among patients with advanced NSCLC treated with anti-PD1, in particular for those receiving combination therapy.

Liquid biopsy tests are an integral part of the molecular diagnostic workup of cancer specimens. We have performed extensive validation studies of the GeneStrat NGS test (GSNGS), that uses an NGS panel developed on semiconductor-based sequencing technology. The workflow requires very low input (20 ng) of cell-free nucleic acids and yields results in an average of three business days. GSNGS was validated for the detection of copy number amplifications (CNAs), fusions/exon skipping, somatic single nucleotide variants (SNV) and insertions/deletions (INDELs). Validation specimens included contrived and clinical samples. Dependent on the variant class we used ultrasensitive ddPCR, amplicon-based, and non-amplicon-based panels on multiple NGS platforms as orthogonal methods. All orthogonal technologies utilized the native workflows and analyses pipelines as intended by their manufacturers. We observed concordance with the GSNGS test for SNV and indels of greater than 97.7% with the orthogonal droplet digital PCR (ddPCR) test, GeneStrat ddPCR. Studies of the tested CNA and fusions/exon-skipping showed 100% concordance between GSNGS and TSO500. Accuracy studies with the two amplicon-based NGS systems (manual and automated workflows), used clinical specimens and here we observed 100% concordance in variant calls for SNVs, INDELs and fusions/exon-skipping. One CNA variant instance was observed on the manual workflow that was not called by the automated workflow (97% concordance). The high concordance of the independent workflows for the detection of nucleic acid variants in circulation demonstrates the capability of GSNGS test to be used for testing of clinical specimens. Specifically, actionable variants in the four major mutation classes were successfully detected in the reference and test specimen sets using orthogonal test methods. The validated test panel meets the criteria for being rapid, highly sensitive and specific, and has utility for the reporting of clinically re...

Clinical laboratories worldwide are implementing various targeted panels to identify cancer genomic variants and ultimately improve patient outcomes. As most of the panels and NGS platforms are research use only, their application in the clinical practice has to be regulated. Comprehensive validation process to examine the test sensitivity and specificity is required prior to their clinical use. Our lab performed validation work using Oncomine Comprehensive Assay V3 GX on Genexus system. Reference standard and clinical specimen were tested across fourteen instrument runs. Key instrument performance parameters reach satisfactory level. Sensitivity was 97% in SNVs, 100% in MNVs and 100% in indels. OCAv3 is able to detected CNV of >5 copies and all 23 fusion events were detected successfully. The overall specificity of SNVs, MNVs and indels was 100%. The specificity of CNVs was 100% while 100% specificity was detected in fusion events.

In her presentation Prof. Staebler will share data generated by new Genomic Assessment Metrics  (GIM) software for HRD assessment,  and Oncomine Comprehensive Assay Plus. The samples tested were previously tested by standard legacy method for HRD assessment and a comparison will be shared.

Prof. Hofman will present results of a multi-centric study evaluating performance of new Oncomine Dx Express Test, CE IVD for genomic profiling of cancer patient samples. The data cover performance characteristic as well as turn around time and sequencing success rate.