Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer.

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows.

Identification of a novel partner gene, KIAA1217, fused to RET: Functional characterization and inhibitor sensitivity of two isoforms in lung adenocarcinoma.

REarranged during Transfection (RET) fusion genes are detected in approximately 1% of lung adenocarcinomas and known primarily as oncogenic driver factors. Here, we found a novel RET fusion gene, KIAA1217-RET, and examined the functional differences of RET51 and RET9 protein, fused with KIAA1217 in cancer progression and drug response. KIAA1217-RET, resulting from the rearrangement of chromosome 10, was generated by the fusion of KIAA1217 exon 11 and RET exon 11 from a non-small cell lung cancer patient.

Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

Rationale: Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis.

Lung cancer in never-smoker Asian females is driven by oncogenic mutations, most often involving EGFR.

The aim of this study was to determine the distribution of known oncogenic driver mutations in female never-smoker Asian patients with lung adenocarcinoma. We analyzed 214 mutations across 26 lung cancer-associated genes and three fusion genes using the MassARRAY LungCarta Panel and the ALK, ROS1, and RET fusion assays in 198 consecutively resected lung adenocarcinomas from never-smoker females at a single institution. EGFR mutation, which was the most frequent driver gene mutation, was detected in 124 (63%) cases.

SEC31A-ALK Fusion Gene in Lung Adenocarcinoma.

Anaplastic lymphoma kinase (ALK) fusion is a common mechanism underlying pathogenesis of non-small cell lung carcinoma (NSCLC) where these rearrangements represent important diagnostic and therapeutic targets. In this study, we found a new ALK fusion gene, SEC31A-ALK, in lung carcinoma from a 53-year-old Korean man. The conjoined region in the fusion transcript was generated by the fusion of SEC31A exon 21 and ALK exon 20 by genomic rearrangement, which contributed to generation of an intact, in-frame open reading frame.

Noninvasive saliva-based EGFR gene mutation detection in patients with lung cancer.

Rationale: Constitutive activation of the epidermal growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small cell lung carcinoma (NSCLC). Mutations identified in EGFR predict the sensitivity to EGFR-targeted therapy. Detection of these mutations is mainly based on tissue biopsy, which is invasive, expensive, and time consuming.

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear.

A novel fusion of TPR and ALK in lung adenocarcinoma.

Introduction: Anaplastic lymphoma kinase (ALK) fusion is the most common mechanism for overexpression and activation in non-small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B, kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3, and huntingtin interacting protein 1 (HIP1).

Methods And Results: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

Introduction: The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3.

Methods: In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK.

A single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer.

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors.

Combined gemcitabine and CHK1 inhibitor treatment induces apoptosis resistance in cancer stem cell-like cells enriched with tumor spheroids from a non-small cell lung cancer cell line.

Evaluating the effects of novel drugs on appropriate tumor models has become crucial for developing more effective therapies that target highly tumorigenic and drug-resistant cancer stem cell (CSC) populations. In this study, we demonstrate that a subset of cancer cells with CSC properties may be enriched into tumor spheroids under stem cell conditions from a non-small cell lung cancer cell line. Treating these CSC-like cells with gemcitabine alone and a combination of gemcitabine and the novel CHK1 inhibitor PF-00477736 revealed that PF-00477736 enhances the anti-proliferative effect of gemcitabine against both the parental and the CSC-like cell populations.

Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer.

Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration-approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients.

A novel SND1-BRAF fusion confers resistance to c-Met inhibitor PF-04217903 in GTL16 cells through [corrected] MAPK activation.

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi).

An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735.

PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735.

Expression of CETP and of splice variants induces the same level of ER stress despite secretion efficiency differences.

The cholesteryl ester transfer protein (CETP) gene has been associated with a variety of phenotypes, including HDL-cholesterol levels and, more sporadically, with cardiovascular disease, obesity, and extreme longevity. Alterations of CETP activity levels can be caused by single-base polymorphisms as well as by alternative splicing. In addition to the previously characterized alternative splicing that skips exon 9, we found additional minor variants and characterized the activity of the resultant proteins.

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.

Novel variants in human and monkey CETP.

Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed.

CETP polymorphisms associated with HDL cholesterol may differ from those associated with cardiovascular disease.

To better understand the role of cholesteryl ester transfer protein (CETP) in cardiovascular disease, nine polymorphisms spanning the gene from the upstream promoter region to beyond the 3'UTR were genotyped in 2553 individuals from multiple ethnic groups and with different cardiovascular disease profiles. The frequency of four of these SNPs varied by 40-300% between Caucasians and African Americans. SNPs in each ethnic group fell into two haploblocks with significant linkage disequilibrium within each block.

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels.

Highly polymorphic repeat region in the CETP promoter induces unusual DNA structure.

Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter is associated with HDL cholesterol levels and cardiovascular disease with much of the genetic variation in CETP attributed to the promoter region. In this region, there are several single nucleotide polymorphisms as well as a variable length tandem repeat located 1946 base pairs upstream of the CETP transcription start that is highly polymorphic with respect to both length and sequence. There are more than 10 different long alleles and these vary in their repeat structure.