A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA.

BACKGROUND: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs). METHODS: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow. RESULTS: sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively). CONCLUSIONS: The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.

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.

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.

A Review of Circulating Tumour Cell Enrichment Technologies.

Circulating tumour cells (CTCs) are the precursor cells for the formation of metastatic disease. With a simple blood draw, liquid biopsies enable the non-invasive sampling of CTCs from the blood, which have the potential to provide important insights into cancer detection and monitoring. Since gaining FDA approval in 2004, the CellSearch system has been used to determine the prognosis of patients with metastatic breast, prostate and colorectal cancers. This utilises the cell surface marker Epithelial Cell Adhesion Molecule (EpCAM), to enrich CTCs, and many other technologies have adopted this approach. More recently, the role of mesenchymal-like CTCs in metastasis formation has come to light. It has been suggested that these cells are more aggressive metastatic precursors than their epithelial counterparts; however, mesenchymal CTCs remain undetected by EpCAM-based enrichment methods. This has prompted the development of a variety of 'label free' enrichment technologies, which exploit the unique physical properties of CTCs (such as size and deformability) compared to other blood components. Here, we review a wide range of both immunocapture and label free CTC enrichment technologies, summarising the most significant advantages and disadvantages of each. We also highlight the important characteristics that technologies should possess for routine clinical use, since future developments could have important clinical implications, with the potential to direct personalised therapies for patients with cancer.