Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients.

INTRODUCTION: Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system. METHODS: Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel. RESULTS: Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified. CONCLUSIONS: Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients.

Mutational profiling of familial male breast cancers reveals similarities with luminal A female breast cancer with rare TP53 mutations.

BACKGROUND: Male breast cancer (MBC) is still poorly understood with a large proportion arising in families with a history of breast cancer. Genomic studies have focused on germline determinants of MBC risk, with minimal knowledge of somatic changes in these cancers. METHODS: Using a TruSeq amplicon cancer panel, this study evaluated 48 familial MBCs (3 BRCA1 germline mutant, 17 BRCA2 germline mutant and 28 BRCAX) for hotspot somatic mutations and copy number changes in 48 common cancer genes. RESULTS: Twelve missense mutations included nine PIK3CA mutations (seven in BRCAX patients), two TP53 mutations (both in BRCA2 patients) and one PTEN mutation. Common gains were seen in GNAS (34.1%) and losses were seen in GNAQ (36.4%), ABL1 (47.7%) and ATM (34.1%). Gains of HRAS (37.5% vs 3%, P=0.006), STK11 (25.0% vs 0%, P=0.01) and SMARCB1 (18.8% vs 0%, P=0.04) and the loss of RB1 (43.8% vs 13%, P=0.03) were specific to BRCA2 tumours. CONCLUSIONS: This study is the first to perform high-throughput somatic sequencing on familial MBCs. Overall, PIK3CA mutations are most commonly seen, with fewer TP53 and PTEN mutations, similar to the profile seen in luminal A female breast cancers. Differences in mutation profiles and patterns of gene gains/losses are seen between BRCA2 (associated with TP53/PTEN mutations, loss of RB1 and gain of HRAS, STK11 and SMARCB1) and BRCAX (associated with PIK3CA mutations) tumours, suggesting that BRCA2 and BRCAX MBCs may be distinct and arise from different tumour pathways. This has implications on potential therapies, depending on the BRCA status of MBC patients.

Albumin banks peninsula: a new termination variant characterised by electrospray mass spectrometry.

Albumin Banks Peninsula is an electrophoretically fast variant that is expressed at only 2% of the total serum albumin. Electrospray ionisation analysis indicated a mass decrease of 755 Da relative to normal albumin and carboxypeptidase A digestion, together with CNBr peptide mapping, indicated a C-terminal truncation. This was confirmed by PCR and DNA sequence analysis which showed the introduction of a new AG acceptor splice site near the 3' end of intron 13. Predictably this results in the replacement of the C-terminal GKKLVAASQAALGL sequence by SLCSG and would be associated with an 861 Da decrease in molecular mass. We surmised that the new Cys was most probably cysteinylated as this albumin species would have a mass decrease of 742 Da and be very close to the measured value of 755 Da. Cysteinylation was confirmed when a mass decrease of 863 Da was measured between the proteins after reduction of their disulfide bonds.

A novel variant of transthyretin (prealbumin), Thr119 to Met, associated with increased thyroxine binding.

A group of patients with prealbumin associated hyperthyroxinemia possess a common single base substitution in the fourth exon of their transthyretin gene. This cytosine to thymine substitution occurs in the codon for residue 119 and results in the predicted replacement of a threonine residue with a methionine at this position. A new NcoI restriction endonuclease cleavage site is created by the point mutation and can be detected by a rapid and simple assay based on the polymerase chain reaction. This variant transthyretin is inherited in an autosomal dominant manner and is apparently not amyloidogenic but is associated with increased thyroxine binding. As healthy heterozygous individuals have normal serum thyroxine concentrations, the hyperthyroxinemia sometimes found may not be primarily due to the variant.

Molecular screening of cystic fibrosis patients.

A method for the direct direction of the delta F508 mutation in the cystic fibrosis gene has been developed and applied to the analysis of over 280 individuals including 104 individuals with cystic fibrosis. This technique allows the rapid analysis of DNA from whole blood, Guthrie card blood spots, and the antenatal diagnosis of cystic fibrosis from chorionic villus biopsy samples. Based on these analyses, the delta F508 mutation is found in 77.88% of cystic fibrosis chromosomes in New Zealand cystic fibrosis patients. Thus this test can be used to establish a direct DNA diagnosis in over 60% of cystic fibrosis patients. Approximately a further 30% are heterozygous for this mutation.