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Nearly all estrogen receptor (ER)-positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER-POS breast cancer remain largely unexplored. Whole-blood (WB) specimens were collected at serial time points from patients with advanced ER-POS/HER2-negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch ® /DEPArray™ technologies and genomically profiled by targeted single-cell DNA next-generation sequencing (scNGS). High-quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC-based framework for precision medicine actionability reporting (MI-CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto-oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter-CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real-time tracking of tumor evolution during progression, permitting more combination precision medicine interventions., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: The activating mutation AKT1 E17K occurs in approximately 7% of estrogen receptor-positive (ER + ) metastatic breast cancer (MBC). We report, from a multipart, first-in-human, phase I study (NCT01226316), tolerability and activity of capivasertib, an oral AKT inhibitor, as monotherapy or combined with fulvestrant in expansion cohorts of patients with AKT1 E17K -mutant ER + MBC., Patients and Methods: Patients with an AKT1 E17K mutation, detected by local (next-generation sequencing) or central (plasma-based BEAMing) testing, received capivasertib 480 mg twice daily, 4 days on, 3 days off, weekly or 400 mg twice daily combined with fulvestrant at the labeled dose. Study endpoints included safety, objective response rate (ORR; RECIST v1.1), progression-free survival (PFS), and clinical benefit rate at 24 weeks (CBR 24 ). Biomarker analyses were conducted in the combination cohort., Results: From October 2013 to August 2018, 63 heavily pretreated patients received capivasertib (20 monotherapy, 43 combination). ORR was 20% with monotherapy, and within the combination cohort was 36% in fulvestrant-pretreated and 20% in fulvestrant-naïve patients, although the latter group may have had more aggressive disease at baseline. AKT1 E17K mutations were detectable in plasma by BEAMing (95%, 41/43), droplet digital PCR (80%, 33/41), and next-generation sequencing (76%, 31/41). A ≥50% decrease in AKT1 E17K at cycle 2 day 1 was associated with improved PFS. Combination therapy appeared more tolerable than monotherapy [most frequent grade ≥3 adverse events: rash (9% vs. 20%), hyperglycemia (5% vs. 30%), diarrhea (5% vs. 10%)]., Conclusions: Capivasertib demonstrated clinically meaningful activity in heavily pretreated patients with AKT1 E17K -mutant ER + MBC, including those with prior disease progression on fulvestrant. Tolerability and activity appeared improved by the combination., (©2020 American Association for Cancer Research.)

Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation - lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.

Purpose: Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)-positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene ( ESR1 LBD m ). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy., Experimental Design: CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1 LBD m by droplet digital PCR (BioRad)., Results: Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to <5 CTC/7.5 mL WB on C1D15. Five of 11 patients had baseline CTC-ER + , two of whom had CTC-ER + reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with <5 CTC ( P = 0.0003). Fourteen of 45 (31%) patients had ESR1 LBD m + ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline ESR1 LBD m status was not prognostic. Patients with persistently elevated CTC and/or ESR1 LBD m + ctDNA at C1D15 had worse PFS than patients who did not ( P = 0.0007)., Conclusions: Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER + and ESR1 LBD m + ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker., (©2018 American Association for Cancer Research.)

Multidrug-resistant organisms (MDROs) are increasingly implicated in both acute and chronic wound infections. The limited therapeutic options are further compromised by the fact that wound bacteria often co-exist within a biofilm community which enhances bacterial tolerance to antibiotics. As a consequence, topical antiseptics may be an important consideration for minimising the opportunity for wound infections involving MDROs. The objective of this research was to investigate the antimicrobial activity of a silver-containing gelling fibre dressing against a variety of MDROs in free-living and biofilm states, using stringent in vitro models designed to simulate a variety of wound conditions. MDROs included Acinetobacter baumannii, community-associated methicillin-resistant Staphylococcus aureus, and extended-spectrum beta-lactamase-producing bacteria. Clostridium difficile was also included in the study because it carries many of the characteristics seen in MDROs and evidence of multidrug resistance is emerging. Sustained in vitro antimicrobial activity of the silver-containing dressing was shown against 10 MDROs in a simulated wound fluid over 7 days, and inhibitory and bactericidal effects against both free-living and biofilm phenotypes were also consistently shown in simulated colonised wound surface models. The in vitro data support consideration of the silver-containing gelling fibre dressing as part of a protocol of care in the management of wounds colonised or infected with MDROs., (© 2012 The Authors. International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc.)