Comparison of phenomics and cfDNA in a large breast screening population: the Breast Screening and Monitoring Study (BSMS).

To assess their roles in breast cancer diagnostics, we aimed to compare plasma cell-free DNA (cfDNA) levels with the circulating metabolome in a large breast screening cohort of women recalled for mammography, including healthy women and women with mammographically detected breast diseases, ductal carcinoma in situ and invasive breast cancer: the Breast Screening and Monitoring Study (BSMS). In 999 women, plasma was analyzed by nuclear magnetic resonance (NMR) and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and then processed to isolate and quantify total cfDNA. NMR and UPLC-MS results were compared with data for 186 healthy women derived from the AIRWAVE cohort. Results showed no significant differences between groups for all metabolites, whereas invasive cancers had significantly higher plasma cfDNA levels than all other groups. When stratified the supervised OPLS-DA analysis and total cfDNA concentration showed high discrimination accuracy between invasive cancers and the disease/medication-free subjects. Furthermore, comparison of OPLS-DA data for invasive breast cancers with the AIRWAVE cohort showed similar discrimination between breast cancers and healthy controls. This is the first report of agreement between metabolomics and plasma cfDNA levels for discriminating breast cancer from healthy subjects in a true screening population. It also emphasizes the importance of sample standardization. Follow on studies will involve analysis of candidate features in a larger validation series as well as comparing results with serial plasma samples taken at the next routine screening mammography appointment. The findings here help establish the role of plasma analysis in the diagnosis of breast cancer in a large real-world cohort.

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.

Personalized Detection of Circulating Tumor DNA Antedates Breast Cancer Metastatic Recurrence.

PURPOSE: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer. EXPERIMENTAL DESIGN: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n = 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X). RESULTS: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling. CONCLUSIONS: This study demonstrates that patient-specific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention.

The pioneer factor PBX1 is a novel driver of metastatic progression in ERα-positive breast cancer.

Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.

Influence of plasma processing on recovery and analysis of circulating nucleic acids.

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.