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Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations. Poziotinib is a candidate inhibitor for a subset of EGFR or HER2 mutant non–small cell lung cancers that lack effective therapy.

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1 , NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations. Targeted cancer therapy requires knowledge of driver aberrations. Here the authors perform large-scale transcriptome analysis, and show that gene fusions in NRG1 , NF1 and Hippo pathway genes are recurrent mostly among lung cancers lacking known driver mutations.

The aim of this study was to identify differentially-expressed miRNAs in the serum of non-small cell lung cancer (NSCLC) patients that might be a clinically-useful tool for lung cancer early detection. We performed miRNA expression profile analysis using TaqMan OpenArray Human panel in a discovery set of 70 serum samples obtained at lung tumor resection and 22 non-cancer subjects (NC). Selected serum miRNAs were then validated by quantitative PCR using an independent validation set of serum samples from LC patients (n = 84) and NC (n = 23). Sixty miRNAs were significantly up-regulated and 31 were down-regulated in the serum from NSCLC patients versus NC (adjusted p  < 0.001). Four miRNAs (miR-193b, miR-301, miR-141 and miR-200b) were selected for validating their diagnostic value in an independent cohort. In the discovery set, the ROC plot derived from the combination of these miRNAs yielded an area under the curve (AUC) of 0.985 (95% CI 0.961–1.000, p  < 0.001). In the test set, this miRNA signature exhibited an AUC of 0.993 (95% CI 0.979–1.000, p  < 0.001). In conclusion, we identified a serum 4-miRNA signature that discriminated with high accuracy lung cancer patients from NC. Further prospective validation of this miRNA signature is warranted.

Malignant pleural mesothelioma (MPM) is an aggressive human cancer and miRNAs can play a key role for this disease. In order to broaden the knowledge in this field, the miRNA expression was investigated in a large series of MPM to discover new pathways helpful in diagnosis, prognosis and therapy. We employed nanoString nCounter system for miRNA profiling on 105 MPM samples and 10 healthy pleura. The analysis was followed by the validation of the most significantly deregulated miRNAs by RT-qPCR in an independent sample set. We identified 63 miRNAs deregulated in a statistically significant way. MiR-185, miR-197, and miR-299 were confirmed differentially expressed, after validation study. In addition, the results of the microarray analysis corroborated previous findings concerning miR-15b-5p, miR-126-3p, and miR-145-5p. Kaplan-Meier curves were used to explore the association between miRNA expression and overall survival (OS) and identified a 2-miRNA prognostic signature (Let-7c-5p and miR-151a-5p) related to hypoxia and energy metabolism respectively. In silico analyses with DIANA-microT-CDS highlighted 5 putative targets in common between two miRNAs. With the present work we showed that the pattern of miRNAs expression is highly deregulated in MPM and that a 2-miRNA signature can be a new useful tool for prognosis in MPM.

The emergence of drug resistance depends on the ability of the genome of cancer cells to constantly mutate and evolve under selective pressures. The generation of new mutations is accelerated when genes involved in DNA repair pathways are altered. Notably, although the emergence of new mutations fosters drug resistance, new variants can nevertheless become novel antigens that promote immune surveillance and even restrict cancer growth.

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) are promising prognostic and predictive biomarkers in non-small cell lung cancer (NSCLC). In this study, we examined the prognostic role of cfDNA and CTCs, in separate and joint analyses, in NSCLC patients receiving first line chemotherapy. Seventy-three patients with advanced NSCLC were enrolled in this study. CfDNA and CTC were analyzed at baseline and after two cycles of chemotherapy. Plasma cfDNA quantification was performed by quantitative PCR (qPCR) whereas CTCs were isolated by the ScreenCell Cyto (ScreenCell, Paris, France) device and enumerated according to malignant features. Patients with baseline cfDNA higher than the median value (96.3 hTERT copy number) had a significantly worse overall survival (OS) and double the risk of death (hazard ratio (HR): 2.14; 95% confidence limits (CL) = 1.24–3.68; p-value = 0.006). Conversely, an inverse relationship between CTC median baseline number (6 CTC/3 mL of blood) and OS was observed. In addition, we found that in patients reporting stable disease (SD), the baseline cfDNA and CTCs were able to discriminate patients at high risk of poor survival. cfDNA demonstrated a more reliable biomarker than CTCs in the overall population. In the subgroup of SD patients, both biomarkers identified patients at high risk of poor prognosis who might deserve additional/alternative therapeutic interventions.

Background Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. Methods To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v .3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v .5 (Agilent Technologies). Results Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. Conclusions High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

Metastatic colorectal cancer (mCRC) is frequently characterized by the presence of mutations of the KRAS oncogene, which are generally associated with a poor response to treatment with anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies. With the methods currently used, a case is classified as KRAS-mutated when approximately 20% of the cells bear an activating KRAS mutation. These considerations raise the question of whether cells with a mutated KRAS can be found in mCRC cases classified as KRAS wild-type when more sensitive methods are used. In addition, the issue arises of whether these mCRC cases with low proportion of KRAS -mutated cells could account at least in part for the therapeutic failure of anti-EGFR therapies that occur in 40–60% of cases classified as KRAS wild type. In this study, we compared the classical assays with a very sensitive test, a locked nucleic acid (LNA) polymerase chain reaction (PCR), capable of detecting KRAS-mutated alleles at extremely low frequency (detection sensitivity limit 0.25% mutated DNA/wild-type DNA). By analyzing a cohort of 213 mCRC patients for KRAS mutations, we found a 20.6% discordance between the sequencing/TheraScreen methods and the LNA-PCR. Indeed, 44 mCRC patients initially considered KRAS wild type were reclassified as KRAS mutated by using the LNA-PCR test. These patients were more numerous among individuals displaying a clinical failure to anti-EGFR therapies. Failure to respond to these biological treatments occurred even in the absence of mutations in other EGFR pathway components such as BRAF.