Progesterone receptor modulates ERα action in breast cancer.

Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling.

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

Whole blood mRNA in prostate cancer reveals a four-gene androgen regulated panel.

Due to increased sensitivity, the expression of circulating nucleotides is rapidly gaining popularity in cancer diagnosis. Whole blood mRNA has been used in studies on a number of cancers, most notably two separate studies that used whole blood mRNA to define non-overlapping signatures of prostate cancer that has become castration independent. Prostate cancer is known to rely on androgens for initial growth, and there is increasing evidence on the importance of the androgen axis in advanced disease. Using whole blood mRNA samples from patients with prostate cancer, we have identified the four-gene panel of FAM129A, MME, KRT7 and SOD2 in circulating mRNA that are differentially expressed in a discovery cohort of metastatic samples. Validation of these genes at the mRNA and protein level was undertaken in additional cohorts defined by risk of relapse following surgery and hormone status. All the four genes were downregulated at the mRNA level in the circulation and in primary tissue, but this was not always reflected in tissue protein expression. MME demonstrated significant differences in the hormone cohorts, whereas FAM129A is downregulated at the mRNA level but is raised at the protein level in tumours. Using published ChIP-seq data, we have demonstrated that this may be due to AR binding at the FAM129A and MME loci in multiple cell lines. These data suggest that whole blood mRNA of androgen-regulated genes has the potential to be used for diagnosis and monitoring of prostate cancer.

The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes.

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13-14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13-14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies.