Crizotinib is an effective drug for patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC), but upon treatment, the tumors inevitably become crizotinib resistant in time. The resistance mechanisms are only partly understood. In this study, we aim to identify gene mutations associated with resistance in ALKpositive advanced non-squamous NSCLC treated with crizotinib. Four ALK positive patients with progressive disease following crizotinib treatment were identified with paired pre- and post-crizotinib tumor tissue from our previously published cohort. Somatic variants in these samples were detected by whole exome sequencing. In one of the four patients, an ALK-resistance associated mutation was identified. In the other three patients, no ALK-resistance associated mutations were present. In these patients we identified 89 relevant somatic mutations in 74 genes that were specific to the resistant tumors. These genes were enriched in 15 pathways. Four pathways, were related to epithelial-mesenchymal transition (EMT): proteoglycans in cancer, HIF-1 signaling, FoxO signaling pathway, and ECM-receptor interaction. Analysis of other EMT-related pathways revealed three additional genes with mutations specific to the crizotinib-resistant tumor samples. The enrichment of mutations in genes associated with EMT-related pathways indicates that loss of epithelial differentiation may represent a relevant resistance mechanism for crizotinib.

The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a disease biomarker in oesophageal squamous cell carcinoma (ESCC) that can be used for treatment response evaluation and early detection of tumour recurrence. Matched tumour tissue, pre- and post-surgery plasma and WBCs obtained from 17 ESCC patients were sequenced using a panel of 483 cancer-related genes. Somatic mutations were detected in 14 of 17 tumour tissues. Putative harmful mutations were observed in genes involved in well-known cancer-related pathways, including PI3K-Akt/mTOR signalling, Proteoglycans in cancer, FoxO signalling, Jak-STAT signalling, Chemokine signalling and Focal adhesion. Forty-six somatic mutations were found in pre-surgery cfDNA in 8 of 12 patients, with mutant allele frequencies (MAF) ranging from 0.24 to 4.91%. Three of the 8 patients with detectable circulating tumour DNA (ctDNA) had stage IIA disease, whereas the others had stage IIB-IIIB disease. Post-surgery cfDNA somatic mutations were detected in only 2 of 14 patients, with mutant allele frequencies of 0.28 and 0.36%. All other somatic mutations were undetectable in post-surgery cfDNA, even in samples collected within 3–4 h after surgery. Our study shows that somatic mutations can be detected in pre-surgery cfDNA in stage IIA to IIIB patients, and at a lower frequency in post-surgery cfDNA. This indicates that cfDNA could potentially be used to monitor disease load, even in low disease-stage patients.

Background: Small cell lung cancer (SCLC) is one of the most aggressive lung cancers. Treatment of SCLC has remained unchanged during the past decades. Preclinical studies have revealed ASCL1 as a transcription regulator in the neuroendocrine (NE) differentiation and carcinogenesis of SCLC. However, there are few studies on correlation of ASCL1 expression and clinicopathological factors in resected SCLCs. Here, we aimed to analyze the ASCL1 expression of SCLC and investigate its associations with clinicopathological factors and survival.Methods: A total of 247 surgically resected pure SCLC specimens were included in this retrospective study, all of which were processed using tissue microarrays for immunohistochemistry analysis of ASCL1. A total of 48 of 247 cases were tested by NanoString for mRNA expression analysis on 50 SCLC related genes. Statistical analysis was performed using R studio and SPSS software.Results: NE scores of 48 pure SCLC specimens were calculated by analyzing 50 preselected genes. A significant correlation between NE score with both ASCL1 mRNA expression and ASCL1 protein expression were observed. For the entire cohort of 247 patients, ASCL1 was highly expressed in 42.5% of pure SCLC patients according to IHC results. Significant differences were observed between ASCL1 high and low expression groups in variables including staging, lymph node metastasis, nerve invasion and overall survival.Conclusions: In limited staged pure SCLC, ASCL1 expression was positively correlated with NE signature, pTNM stage, nerve invasion and OS. ASCL1 may therefore serve as a potential biomarker to predict prognosis as well as in the selection of patients for therapies targeting ASCL1‐regulated downstream molecules.

The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with >50 K unique reads and a DV200 >30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive.