Comparison of two targeted ultra-deep sequencing technologies for analysis of plasma circulating tumour DNA in endocrine-therapy-resistant breast cancer patients

Purpose There is growing interest in the application of circulating tumour DNA (ctDNA) as a sensitive tool for monitoring tumour evolution and guiding targeted therapy in patients with cancer. However, robust comparisons of different platform technologies are still required. Here we compared the InVisionSeq™ ctDNA Assay with the Oncomine™ Breast cfDNA Assay to assess their concordance and feasibility for the detection of mutations in plasma at low (< 0.5%) variant allele fraction (VAF). Methods Ninety-six plasma samples from 50 patients with estrogen receptor (ER)-positive metastatic breast cancer (mBC) were profiled using the InVision Assay. Results were compared to the Oncomine assay in 30 samples from 26 patients, where there was sufficient material and variants were covered by both assays. Longitudinal samples were analysed for 8 patients with endocrine resistance. Results We detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affecting ESR1, PIK3CA and TP53. Complete or partial concordance was found in 28/30 samples analysed by both assays, and VAF values were highly correlated. Excellent concordance was found for most genes, and most discordant calls occurred at VAF < 1%. In longitudinal samples from progressing patients with endocrine resistance, we detected consistent alterations in sequential samples, most commonly in ESR1 and PIK3CA. Conclusion This study shows that both ultra-deep next-generation sequencing (NGS) technologies can detect genomic alternations even at low VAFs in plasma samples of mBC patients. The strong agreement of the technologies indicates sufficient reproducibility for clinical use as prognosic and predictive biomarker

One-to-one midwifery care in Singapore - the first 100 births

The Enhanced Midwifery Maternity Care (EMMa Care) programme at the National University Hospital in Singapore was implemented in 2011 to provide women with primary obstetric care in collaboration with one-to-one midwifery support. The continuity of care women are able to access from both an obstetrician and a midwife provides a unique opportunity for women and babies to benefit from both obstetric expertise and holistic midwifery care. This article describes the development of the programme, and the outcomes of the births of the first 100 women who booked. Sixty women were primiparous and 40 were multiparous. Birth outcome data demonstrated a caesarean rate of 20% and seven out of nine vaginal birth after caesarean (VBAC) attempts were successful (77.77% success rate). Of the women birthing vaginally, 81.33% did so without any pharmacological pain management; 58 women used water immersion in labour and 36 birthed their babies in water. There were no third or fourth degree perineal tears, no postpartum haemorrhages and the episiotomy rate was 4%. Apgar scores were above 7 at 5 minutes in all but one baby; five babies required phototherapy for hyperbilirubinemia and eight babies (including one set of twins) were admitted to the neonatal intensive care unit (NICU) for hypoglycaemia and/or prematurity. EMMa Care is associated with good maternal and neonatal outcomes. Caesarean section rates were lower than overall rates in the same hospital setting and use of pharmacological pain management was minimal. © 2013 MA Healthcare Ltd

Circulating tumor DNA profiling from breast cancer screening through to metastatic disease

PURPOSE: We investigated the utility of the Oncomine Breast cfDNA Assay for detecting circulating tumor DNA (ctDNA) in women from a breast screening population, including healthy women with no abnormality detected by mammogram, and women on follow-up through to advanced breast cancer. MATERIALS AND METHODS: Blood samples were taken from 373 women (127 healthy controls recruited through breast screening, 28 ductal carcinoma in situ, 60 primary breast cancers, 47 primary breast cancer on follow-up, and 111 metastatic breast cancers [MBC]) to recover plasma and germline DNA for analysis with the Oncomine Breast cfDNA Assay on the Ion S5 platform. RESULTS: One hundred sixteen of 373 plasma samples had one or more somatic variants detected across eight of the 10 genes and were called ctDNA-positive; MBC had the highest proportion of ctDNA-positive samples (61; 55%) and healthy controls the lowest (20; 15.7%). ESR1, TP53, and PIK3CA mutations account for 93% of all variants detected and predict poor overall survival in MBC (hazard ratio = 3.461; 95% CI, 1.866 to 6.42; P = .001). Patients with MBC had higher plasma cell-free DNA levels, higher variant allele frequencies, and more polyclonal variants, notably in ESR1 than in all other groups. Only 15 individuals had evidence of potential clonal hematopoiesis of indeterminate potential mutations. CONCLUSION: We were able detect ctDNA across the breast cancer spectrum, notably in MBC where variants in ESR1, TP53, and PIK3CA predicted poor overall survival. The assay could be used to monitor emergence of resistance mutations such as in ESR1 that herald resistance to aromatase inhibitors to tailor adjuvant therapies. However, we suggest caution is needed when interpreting results from a single plasma sample as variants were also detected in a small proportion of HCs

Prevalence of ctDNA in early screen-detected breast cancers using highly sensitive and specific dual molecular barcoded personalised mutation assays

Breast screening has low sensitivity in many patients, and circulating biomarkers may be useful adjuncts to mammography. Circulating tumour DNA (ctDNA) has been studied as a potential biomarker in primary breast cancer (BC)1 and patient-specific mutation panels for detection of cancer recurrence and monitoring of residual disease via ctDNA has potential.2 The role of ctDNA in the detection and monitoring of asymptomatic, screen-detected BC remains unclear, since these patients generally have earlier-stage disease. Here, we report the first study to investigate the prevalence of ctDNA in an unbiased breast screening setting comparing women with early stage 1 and stage 2 BC, detected on incidental routine mammograms. We used a newly developed sequencing technology (Ion Ampliseq™ HD; Thermo Fisher Scientific, Waltham, MA) that uses dual unique molecular identifiers or barcodes to cluster ‘families’ of the same molecule for ctDNA detection. This provides a sensitivity equivalent to digital droplet polymerase chain reaction (PCR) (confident detection of three molecules of ctDNA in 10 000 human haploid genome equivalents) with >99% specificity for single nucleotide variants, hotspots, indels, copy number variations, and fusions.3 With the exception of one study of 12 hepatobiliary cancers in advanced patients,4 this is also the first report of the use of this technology.</p