Biological and technical factors in the assessment of blood-based tumor mutational burden (bTMB) in patients with NSCLC.

BACKGROUND: Patients treated with immunotherapy are at risk of considerable adverse events, and the ongoing struggle is to accurately identify the subset of patients who will benefit. Tumor mutational burden (TMB) has emerged as a promising predictive biomarker but requires tumor tissue which is not always available. Blood-based TMB (bTMB) may provide a minimally invasive assessment of mutational load. However, because of the required sequencing depth, bTMB analysis is costly and prone to false negative results. This study attempted to design a minimally sized bTMB panel, examined a counting-based method for bTMB in patients with stage I to IV non-small cell lung cancer (NSCLC) and evaluated both technical factors such as bTMB and tissue-based TMB (tTMB) cut-off, as well as sample-related factors such as cell-free DNA input mass which influence the correlation between bTMB and tTMB. METHODS: Tissue, plasma, and whole blood samples collected as part of the LEMA trial (NCT02894853) were used in this study. Samples of 185 treatment naïve patients with stage I to IV NSCLC were sequenced at the Roche Sequencing Solutions with a custom panel designed for TMB, using reagents and workflows derived from the AVENIO Tumor Tissue and circulating tumor DNA Analysis Kits. RESULTS: A TMB panel of 1.1 Mb demonstrated highly accurate TMB high calls with a positive predictive value of 95% when using a tTMB cut-off of 16 mut/Mb, corresponding with 42 mut/Mb for bTMB. The positive per cent agreement (PPA) of bTMB was relatively low at 32%. In stage IV samples with at least 20 ng of cfDNA input, PPA of bTMB improved to 63% and minimizing the panel to a subset of 577 kb was possible while maintaining 63% PPA. CONCLUSION: Plasma samples with high bTMB values are highly correspondent with tTMB, whereas bTMB low results may also be the result of low tumor burden at earlier stages of disease as well as poorly shedding tumors. For advanced stages of disease, PPA (sensitivity) of bTMB is satisfactory in comparison to tTMB, even when using a panel of less than 600 kb, warranting consideration of bTMB as a predictive biomarker for patients with NSCLC eligible for immunotherapy in the future.

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&E) annotations are used to target small tumor areas using a blade that is 250 µm2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 µm in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Innovative Tumor Tissue Dissection Tool for Molecular Oncology Diagnostics.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-µm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.

Novel Circulating Tumor Cell Assay for Detection of Colorectal Adenomas and Cancer.

OBJECTIVES: There is a significant unmet need for a blood test with adequate sensitivity to detect colorectal cancer (CRC) and adenomas. We describe a novel circulating tumor cell (CTC) platform to capture colorectal epithelial cells associated with CRC and adenomas. METHODS: Blood was collected from 667 Taiwanese adults from 2012 to 2018 before a colonoscopy. The study population included healthy control subjects, patients with adenomas, and those with stage I-IV CRC. CTCs were isolated from the blood using the CellMax platform. The isolated cells were enumerated, and an algorithm was used to determine the likelihood of detecting adenoma or CRC. Nominal and ordinal logistic regression demonstrated that CTC counts could identify adenomas and CRC, including CRC stage. RESULTS: The CellMax test demonstrated a significant association between CTC counts and worsening disease status (Cuzick's P value < 0.0001) with respect to the adenoma-carcinoma sequence. The test showed high specificity (86%) and sensitivity across all CRC stages (95%) and adenomatous lesions (79%). The area under the curve was 0.940 and 0.868 for the detection of CRC and adenomas, respectively. DISCUSSION: The blood-based CTC platform demonstrated high sensitivity in detecting adenomas and CRC, as well as reasonable specificity in an enriched symptomatic patient population. TRANSLATIONAL IMPACT: If these results are reproduced in an average risk population, this test has the potential to prevent CRC by improving patient compliance and detecting precancerous adenomas, eventually reducing CRC mortality.

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

INTRODUCTION: Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges. METHODS: The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm. RESULTS: We have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.

Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I-IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

CSF-ctDNA SMSEQ Analysis to Tailor the Treatment of a Patient with Brain Metastases: A Case Report.

Brain metastases are the most common neurological complications of adult cancers, accounting for more than half of brain tumors. The incidence of brain metastases may be increasing due to improved detection of small lesions by advanced imaging technologies. Given the fast evolution of targeted and immunotherapy regimens, it is essential to serially assess brain malignancies during the disease course for disease monitoring and tailoring of the therapeutic management. For such serial and repetitive assessment, cerebrospinal fluid (CSF) could be the biological fluid of choice to supplement cytology examination for the presence or absence of CNS malignancy, as well as provide extensive information on tumor mutational profile for personalization of treatment. The case described here emphasizes the importance of CSF-ctDNA analysis with the CellMax SMSEQ technology that led to treatment adjustment resulting in clinical remission of the patient.

Liquid Biopsy Prevents Inaccurate Her2 Status Determination by in situ Hybridization in a Patient with Invasive Ductal Adenocarcinoma of the Breast: Case Report.

Utilization of circulating tumor DNA as a novel and noninvasive test for diagnosis confirmation, therapy selection, and cancer surveillance is a rapidly growing area of interest. In the wake of FDA approval of a liquid biopsy test, it is important for clinicians to acknowledge the obvious clinical utility of liquid biopsy for cancer management throughout the course of the disease. This case report describes a female with invasive ductal adenocarcinoma of the breast, where liquid biopsy was instrumental for her cancer characterization and personalized therapy selection.