Targeted Gene Next-Generation Sequencing Panel in Patients with Advanced Lung Adenocarcinoma: Paving the Way for Clinical Implementation

Abstract

Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (κ = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.

Keywords: lung cancer; molecular profiling; next-generation sequencing; targeted therapy.

Figure 1

Study design; 127 patients genomically profiled with sanger and anaplastic lymphoma kinase (ALK) FISH test (standard approach) were selected. Among those, in 117 the same tumor sample previously tested was submitted to Next-Generation Sequencing (NGS), comprising the experimental phase of the study (experimental cohort (EC)). Among the EC, * 22/117 were epidermal growth factor receptor (EGFR) mutated and 95 had indication for anaplastic lymphoma kinase (ALK) testing, ** 17/95 patients did not perform it due to insufficient sample. After the EC, a combined DNA + RNA panel was applied to characterize genomically patients with lung adenocarcinoma; 123 cases were included for the purpose of this study (clinical implementation phase).

Figure 2

(A) Number of patients with genomic alterations assessed by NGS findings, in each patient´s group assessed by the standard classification (EGFR, ALK, and unclassifiable (UC)); (B) number of hotspot alterations in each gene analyzed by the Ion AmpliSeq™ assay.

Figure 3

(A) Patient’s distribution by EGFR and ALK status (Standard Approach); (B) patient’s classification by Ion Ampliseq Lung and colon panel plus ALK FISH status (Investigational Approach); (C) distribution of hotspot DNA and RNA subgroups in the clinical cohort (Clinical application of the combined DNA + RNA Panel).

Figure 4

Kaplan-Meier overall survival (OS) by (A) NGS subgroups; (B) targeted treatment vs. non-targeted treatment.