Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer.

Purpose: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing.

Experimental Design: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative.

Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.

Background: Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a "glass ceiling" in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations.

Methods: We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations.

Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.

Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR.

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

Background: The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns.

Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53.

Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect.

The breast cancer immunophenotype of TP53-p.R337H carriers is different from that observed among other pathogenic TP53 mutation carriers.

Germline TP53 mutations are associated with Li-Fraumeni syndrome, an autosomal dominant disorder characterized by a predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are clustered within the DNA-binding domain of the gene, disrupting the structure and function of the protein. A specific germline mutation in the tetramerization domain of p53, p.

Expression of cancer stem cell markers in basal and penta-negative breast carcinomas--a study of a series of triple-negative tumors.

Purpose: Breast cancer is a heterogeneous disease. Immunohistochemistry has given rise to triple-negative carcinoma (TNC). Concomitantly, biological origins of neoplasia and its heterogeneity has been strongly debated in cancer stem cells (CSC) theme.

A TP53 founder mutation, p.R337H, is associated with phyllodes breast tumors in Brazil.

A few studies have reported phyllodes tumors (PT) of the breast with germline TP53 mutations. Given this potential association and the high frequency of the TP53 p.R337H in southern and southeastern Brazil, the aim of this study was to assess whether p.