Clinical framework for next generation sequencing based analysis of treatment predictive mutations and multiplexed gene fusion detection in non-small cell lung cancer

Abstract

Precision medicine requires accurate multi-gene clinical diagnostics. We describe the implementation of an Illumina TruSight Tumor (TST) clinical NGS diagnostic framework and parallel validation of a NanoString RNA-based ALK, RET, and ROS1 gene fusion assay for combined analysis of treatment predictive alterations in non-small cell lung cancer (NSCLC) in a regional healthcare region of Sweden (Scandinavia). The TST panel was clinically validated in 81 tumors (99% hotspot mutation concordance), after which 533 consecutive NSCLCs were collected during one-year of routine clinical analysis in the healthcare region (˜90% advanced stage patients). The NanoString assay was evaluated in 169 of 533 cases. In the 533-sample cohort 79% had 1-2 variants, 12% >2 variants and 9% no detected variants. Ten gene fusions (five ALK, three RET, two ROS1) were detected in 135 successfully analyzed cases (80% analysis success rate). No ALK or ROS1 FISH fusion positive case was missed by the NanoString assay. Stratification of the 533-sample cohort based on actionable alterations in 11 oncogenes revealed that 66% of adenocarcinomas, 13% of squamous carcinoma (SqCC) and 56% of NSCLC not otherwise specified harbored ≥1 alteration. In adenocarcinoma, 10.6% of patients (50.3% if including KRAS) could potentially be eligible for emerging therapeutics, in addition to the 15.3% of patients eligible for standard EGFR or ALK inhibitors. For squamous carcinoma corresponding proportions were 4.4% (11.1% with KRAS) vs 2.2%. In conclusion, multiplexed NGS and gene fusion analyses are feasible in NSCLC for clinical diagnostics, identifying notable proportions of patients potentially eligible for emerging molecular therapeutics.

Pre-operative plasma cell-free circulating tumor DNA and serum protein tumor markers as predictors of lung adenocarcinoma recurrence

“…TruSightTumor 26-gene panel which is an exon-focused panel, performed as described previously [12]. 77/157 tumor specimens harbored at least one mutation in either of the EGFR, BRAF or KRAS genes and 58 tumors were included in the study.…”

Minimizing inequality in access to precision medicine in breast cancer by real-time population-based molecular analysis in the SCAN-B initiative

“…To support this, an infrastructure for blood and tumour tissue collection and storage was established at all major hospitals in southern Sweden. This infrastructure was not limited to breast cancer research, and has also been implemented for other cancer diagnoses such as lung cancer. The decision and support of the regional governing body were instrumental in implementation of the same routines across a large geographical area, encompassing seven major hospitals and covering a population of 1.8 million ( Fig .…”

Gene rearrangement detection by next-generation sequencing in patients with non-small cell lung carcinoma

“…With the introduction of NGS to molecular genetics and molecular diagnostics by allowing formalin-fixed paraffin embedded (FFPE) tissues to be screened, there are new efficiency and time duration options available. This technology is being introduced at new laboratories requiring accurate and timely results which guide patient therapy decisions [20][21][22][55][56][57][58] . NGS for SNVs, INDELs and CNVs detection is based on DNA sequencing, in contrast to RNA-seq developed mainly for detection of gene fusions.…”