Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

Background: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material.

Methods: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples.

The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review.

Background: The presence of circulating cell-free DNA from tumours in blood (ctDNA) is of major importance to those interested in early cancer detection, as well as to those wishing to monitor tumour progression or diagnose the presence of activating mutations to guide treatment. In 2014, the UK Early Cancer Detection Consortium undertook a systematic mapping review of the literature to identify blood-based biomarkers with potential for the development of a non-invasive blood test for cancer screening, and which identified this as a major area of interest. This review builds on the mapping review to expand the ctDNA dataset to examine the best options for the detection of multiple cancer types.

Validation of an NGS mutation detection panel for melanoma.

Background: Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy.

Methods: While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time.

Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays.

Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories.

Development of a semi-conductor sequencing-based panel for genotyping of colon and lung cancer by the Onconetwork consortium.

Background: The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges.

KRAS mutation analysis by PCR: a comparison of two methods.

Background: KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.

Gene expression and protein array studies of folliculin-regulated pathways.

The familial cancer syndrome Birt-Hogg-Dube syndrome is characterised by the development of skin (fibrofolliculomas) and renal tumours (and lung cysts) and is caused by mutations in the FLCN tumour suppressor gene. Though the FLCN gene product (folliculin) has been linked to the regulation of a variety of signalling pathways (e.g.

Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis.

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis.

Birt Hogg-Dubé syndrome-associated FLCN mutations disrupt protein stability.

Germline mutations in the FLCN gene cause Birt-Hogg-Dubé syndrome, familial spontaneous pneumothorax, or apparently nonsyndromic inherited RCC. The vast majority of reported FLCN mutations are predicted to result in a truncated/absent gene product and so infrequent missense and inframe-deletion (IFD) FLCN mutations might indicate critical functional domains. To investigate this hypothesis we (1) undertook an in silico evolutionary analysis of the FLCN sequence and (2) investigated in vitro the functional effects of naturally occurring FLCN missense/IFD mutations.

Therapeutic targeting the loss of the birt-hogg-dube suppressor gene.

Brit-Hogg-Dubé (BHD) syndrome, an autosomal dominant familial cancer, is associated with increased risk of kidney cancer. BHD syndrome is caused by loss-of-function mutations in the folliculin (FLCN) protein. To develop therapeutic approaches for renal cell carcinoma (RCC) in BHD syndrome, we adopted a strategy to identify tumor-selective growth inhibition in a RCC cell line with FLCN inactivation.

Modeling ATM mutant proteins from missense changes confirms retained kinase activity.

Ataxia-telangiectasia mutated (ATM) is the gene mutated in the cancer-predisposing disorder ataxia-telangiectasia (A-T). We modeled ATM sequence variants identified in UK A-T patients to determine the stability and kinase activity of the resulting proteins as well as the distribution of these mutations across the coding region. Of 20 missense changes modeled, 10 proteins showed ATM kinase activity and 10 showed none.

Pathogenic ATM mutations occur rarely in a subset of multiple myeloma patients.

Ataxia Telangiectasia (A-T) patients have biallelic inactivation of the ATM gene and exhibit a 200-fold-increased frequency of lymphoid tumours. ATM mutations have been found in a number of adult lymphoid malignancies but there is no data on the occurrence of ATM mutations in multiple myeloma tumours. The purpose of our work was to investigate the occurrence of ATM mutations in multiple myeloma and to this end we screened 45 sporadic cases for ATM mutations using denaturing high-performance liquid chromatography analysis and DNA sequencing.

Cancer risks and mortality in heterozygous ATM mutation carriers.

Background: Homozygous or compound heterozygous mutations in the ATM gene are the principal cause of ataxia telangiectasia (A-T). Several studies have suggested that heterozygous carriers of ATM mutations are at increased risk of breast cancer and perhaps of other cancers, but the precise risk is uncertain.

Methods: Cancer incidence and mortality information for 1160 relatives of 169 UK A-T patients (including 247 obligate carriers) was obtained through the National Health Service Central Registry.