Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells.

Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.

Tumor mutational burden assessed by targeted NGS predicts clinical benefit from immune checkpoint inhibitors in non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), immune checkpoint inhibitors (ICIs) significantly improve overall survival (OS). Tumor mutational burden (TMB) has emerged as a predictive biomarker for patients treated with ICIs. Here, we evaluated the predictive power of TMB measured by the Oncomine™ Tumor Mutational Load targeted sequencing assay in 76 NSCLC patients treated with ICIs. TMB was assessed retrospectively in 76 NSCLC patients receiving ICI therapy. Clinical data (RECIST 1.1) were collected and patients were classified as having either durable clinical benefit (DCB) or no durable benefit (NDB). Additionally, genetic alterations and PD-L1 expression were assessed and compared with TMB and response rate. TMB was significantly higher in patients with DCB than in patients with NDB (median TMB = 8.5 versus 6.0 mutations/Mb, Mann-Whitney p = 0.0244). 64% of patients with high TMB (cut-off = third tertile, TMB ≥ 9) were responders (DCB) compared to 33% and 29% of patients with intermediate and low TMB, respectively (cut-off = second and first tertile, TMB = 5-9 and TMB ≤ 4, respectively). TMB-high patients showed significantly longer progression-free survival (PFS) and OS (log-rank test p = 0.0014 for PFS and 0.0197 for OS). While identifying different subgroups of patients, combining PD-L1 expression and TMB increased the predictive power (from AUC 0.63 to AUC 0.65). Our results show that the TML panel is an effective tool to stratify patients for ICI treatment. A combination of biomarkers might maximize the predictive precision for patient stratification. Our study supports TMB evaluation through targeted NGS in NSCLC patient samples as a tool to predict response to ICI therapy. We offer recommendations for a reliable and cost-effective assessment of TMB in a routine diagnostic setting. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study.

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1-10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.