Comparison of Circulating Tumor DNA Assays for Molecular Residual Disease Detection in Early-Stage Triple-Negative Breast Cancer.

PURPOSE: Detection of circulating tumor DNA (ctDNA) in patients who have completed treatment for early-stage breast cancer is associated with a high risk of relapse, yet the optimal assay for ctDNA detection is unknown. EXPERIMENTAL DESIGN: The cTRAK-TN clinical trial prospectively used tumor-informed digital PCR (dPCR) assays for ctDNA molecular residual disease (MRD) detection in early-stage triple-negative breast cancer. We compared tumor-informed dPCR assays with tumor-informed personalized multimutation sequencing assays in 141 patients from cTRAK-TN. RESULTS: MRD was first detected by personalized sequencing in 47.9% of patients, 0% first detected by dPCR, and 52.1% with both assays simultaneously (P < 0.001; Fisher exact test). The median lead time from ctDNA detection to relapse was 6.1 months with personalized sequencing and 3.9 months with dPCR (P = 0.004, mixed-effects Cox model). Detection of MRD at the first time point was associated with a shorter time to relapse compared with detection at subsequent time points (median lead time 4.2 vs. 7.1 months; P = 0.02). CONCLUSIONS: Personalized multimutation sequencing assays have potential clinically important improvements in clinical outcome in the early detection of MRD.

Baseline Mutations and ctDNA Dynamics as Prognostic and Predictive Factors in ER-Positive/HER2-Negative Metastatic Breast Cancer Patients.

PURPOSE: Prognostic and predictive biomarkers to cyclin-dependent kinases 4 and 6 inhibitors are lacking. Circulating tumor DNA (ctDNA) can be used to profile these patients and dynamic changes in ctDNA could be an early predictor of treatment efficacy. Here, we conducted plasma ctDNA profiling in patients from the PEARL trial comparing palbociclib+fulvestrant versus capecitabine to investigate associations between baseline genomic landscape and on-treatment ctDNA dynamics with treatment efficacy. EXPERIMENTAL DESIGN: Correlative blood samples were collected at baseline [cycle 1-day 1 (C1D1)] and prior to treatment [cycle 1-day 15 (C1D15)]. Plasma ctDNA was sequenced with a custom error-corrected capture panel, with both univariate and multivariate Cox models used for treatment efficacy associations. A prespecified methodology measuring ctDNA changes in clonal mutations between C1D1 and C1D15 was used for the on-treatment ctDNA dynamic model. RESULTS: 201 patients were profiled at baseline, with ctDNA detection associated with worse progression-free survival (PFS)/overall survival (OS). Detectable TP53 mutation showed worse PFS and OS in both treatment arms, even after restricting population to baseline ctDNA detection. ESR1 mutations were associated with worse OS overall, which was lost when restricting population to baseline ctDNA detection. PIK3CA mutations confer worse OS only to patients on the palbociclib+fulvestrant treatment arm. ctDNA dynamics analysis (n = 120) showed higher ctDNA suppression in the capecitabine arm. Patients without ctDNA suppression showed worse PFS in both treatment arms. CONCLUSIONS: We show impaired survival irrespective of endocrine or chemotherapy-based treatments for patients with hormone receptor-positive/HER2-negative metastatic breast cancer harboring plasma TP53 mutations. Early ctDNA suppression may provide treatment efficacy predictions. Further validation to fully demonstrate clinical utility of ctDNA dynamics is warranted.

Biomarkers of Response and Resistance to Palbociclib Plus Letrozole in Patients With ER+/HER2- Breast Cancer.

PURPOSE: To determine (i) the relationship between candidate biomarkers of the antiproliferative (Ki67) response to letrozole and palbociclib alone and combined in ER+/HER2- breast cancer; and (ii) the pharmacodynamic effect of the agents on the biomarkers. EXPERIMENTAL DESIGN: 307 postmenopausal women with ER+/HER2- primary breast cancer were randomly assigned to neoadjuvant treatment with letrozole for 14 weeks; letrozole for 2 weeks, then letrozole+palbociclib to 14 weeks; palbociclib for 2 weeks, then letrozole+palbociclib to 14 weeks; or letrozole+palbociclib for 14 weeks. Biopsies were taken at baseline, 2 and 14 weeks and surgery at varying times after stopping palbociclib. Immunohistochemical analyses were conducted for Ki67, c-PARP, ER, PgR, RB1, CCNE1, and CCND1. RESULTS: Higher baselines ER and PgR were significantly associated with a greater chance of complete cell-cycle arrest (CCCA: Ki67 <2.7%) at 14 weeks and higher baseline Ki67, c-PARP, and CCNE1 with a lower chance. The interaction with treatment was significant only for c-PARP. CCND1 levels were decreased c.20% by letrozole at 2 and 14 weeks but showed a tendency to increase with palbociclib. CCNE1 levels fell 82% (median) in tumors showing CCCA but were unchanged in those with no CCCA. Only 2/9 tumors showed CCCA 3-9 days after stopping palbociclib. ESR1 mutations were found in 2/4 tumors for which surgery took place ≥6 months after starting treatment. CONCLUSIONS: High CCNE1 levels were confirmed as a biomarker of resistance to letrozole+palbociclib. Ki67 recovery within 3-9 days of discontinuing palbociclib indicates incomplete suppression of proliferation during the "off" week of its schedule.

Electroencephalographic evidence of unconscious and conscious attentional bias in people with functional gastrointestinal disorders: A pilot study.

Debate continues as to whether an attentional bias towards threat displayed by sufferers of functional gastrointestinal disorders (FGIDs) is conscious and, thus, more amenable to change through psychological therapy. We compared the amplitudes of early (unconscious) and later (conscious) electroencephalographic (EEG) event-related potentials following silent reading of symptom-related, emotionally neutral, and emotionally negative nouns across two participant groups: 30 female FGID-sufferers who met diagnostic criteria for irritable bowel syndrome or functional dyspepsia, and 30 female healthy controls. Analogous indices based on alpha desynchronization were also examined, as were correlations between the EEG-based indices and a range of psychosocial variables. FGID-sufferers displayed marginally significantly higher occipital EPN amplitudes for all nouns, indicating marginally higher levels of unconscious attention in the task. FGID-sufferers also displayed, for negative as compared to neutral nouns, significantly lower central N400 amplitudes indicative of higher conscious attention. The result was only apparent in post-hoc pairwise comparisons, however. Uniquely among FGID-sufferers, central N400 was strongly negatively correlated with a range of negative psychosocial traits and states. The findings provide preliminary evidence of hypervigilance to general (as opposed to symptom-specific) threat among FGID-sufferers. Amidst concerns over Type I error, recommendations are made for fine-tuning the operationalisation of unconscious and conscious attentional bias in this population.

Genomic profile of advanced breast cancer in circulating tumour DNA.

The genomics of advanced breast cancer (ABC) has been described through tumour tissue biopsy sequencing, although these approaches are limited by geographical and temporal heterogeneity. Here we use plasma circulating tumour DNA sequencing to interrogate the genomic profile of ABC in 800 patients in the plasmaMATCH trial. We demonstrate diverse subclonal resistance mutations, including enrichment of HER2 mutations in HER2 positive disease, co-occurring ESR1 and MAP kinase pathway mutations in HR + HER2- disease that associate with poor overall survival (p = 0.0092), and multiple PIK3CA mutations in HR + disease that associate with short progression free survival on fulvestrant (p = 0.0036). The fraction of cancer with a mutation, the clonal dominance of a mutation, varied between genes, and within hotspot mutations of ESR1 and PIK3CA. In ER-positive breast cancer subclonal mutations were enriched in an APOBEC mutational signature, with second hit PIK3CA mutations acquired subclonally and at sites characteristic of APOBEC mutagenesis. This study utilises circulating tumour DNA analysis in a large clinical trial to demonstrate the subclonal diversification of pre-treated advanced breast cancer, identifying distinct mutational processes in advanced ER-positive breast cancer, and novel therapeutic opportunities.

ESR1 Mutations and Overall Survival on Fulvestrant versus Exemestane in Advanced Hormone Receptor-Positive Breast Cancer: A Combined Analysis of the Phase III SoFEA and EFECT Trials.

PURPOSE: ESR1 mutations are acquired frequently in hormone receptor-positive metastatic breast cancer after prior aromatase inhibitors. We assessed the clinical utility of baseline ESR1 circulating tumor DNA (ctDNA) analysis in the two phase III randomized trials of fulvestrant versus exemestane. EXPERIMENTAL DESIGN: The phase III EFECT and SoFEA trials randomized patients with hormone receptor-positive metastatic breast cancer who had progressed on prior nonsteroidal aromatase inhibitor therapy, between fulvestrant 250 mg and exemestane. Baseline serum samples from 227 patients in EFECT, and baseline plasma from 161 patients in SoFEA, were analyzed for ESR1 mutations by digital PCR. The primary objectives were to assess the impact of ESR1 mutation status on progression-free (PFS) and overall survival (OS) in a combined analysis of both studies. RESULTS: ESR1 mutations were detected in 30% (151/383) baseline samples. In patients with ESR1 mutation detected, PFS was 2.4 months [95% confidence interval (CI), 2.0-2.6] on exemestane and 3.9 months (95% CI, 3.0-6.0) on fulvestrant [hazard ratio (HR), 0.59; 95% CI, 0.39-0.89; P = 0.01). In patients without ESR1 mutations detected, PFS was 4.8 months (95% CI, 3.7-6.2) on exemestane and 4.1 months (95% CI, 3.6-5.5) on fulvestrant (HR, 1.05; 95% CI, 0.81-1.37; P = 0.69). There was an interaction between ESR1 mutation and treatment (P = 0.02). Patients with ESR1 mutation detected had 1-year OS of 62% (95% CI, 45%-75%) on exemestane and 80% (95% CI, 68%-87%) on fulvestrant (P = 0.04; restricted mean survival analysis). Patients without ESR1 mutations detected had 1-year OS of 79% (95% CI, 71%-85%) on exemestane and 81% (95% CI, 74%-87%) on fulvestrant (P = 0.69). CONCLUSIONS: Detection of ESR1 mutations in baseline ctDNA is associated with inferior PFS and OS in patients treated with exemestane versus fulvestrant.

Assessment of Molecular Relapse Detection in Early-Stage Breast Cancer.

Importance: Current treatment cures most cases of early-stage, primary breast cancer. However, better techniques are required to identify which patients are at risk of relapse. Objective: To assess the clinical validity of molecular relapse detection with circulating tumor DNA (ctDNA) analysis in early-stage breast cancer. Design, Setting, and Participants: This prospective, multicenter, sample collection, validation study conducted at 5 United Kingdom medical centers from November 24, 2011, to October 18, 2016, assessed patients with early-stage breast cancer irrespective of hormone receptor and ERBB2 (formerly HER2 or HER2/neu) status who were receiving neoadjuvant chemotherapy followed by surgery or surgery before adjuvant chemotherapy. The study recruited 170 women, with mutations identified in 101 patients forming the main cohort. Secondary analyses were conducted on a combined cohort of 144 patients, including 43 patients previously analyzed in a proof of principle study. Interventions: Primary tumor was sequenced to identify somatic mutations, and personalized tumor-specific digital polymerase chain reaction assays were used to monitor these mutations in serial plasma samples taken every 3 months for the first year of follow-up and subsequently every 6 months. Main Outcomes and Measures: The primary end point was relapse-free survival analyzed with Cox proportional hazards regression models. Results: In the main cohort of 101 female patients (mean [SD] age, 54 [11] years) with a median follow-up of 35.5 months (interquartile range, 27.9-43.0 months), detection of ctDNA during follow-up was associated with relapse (hazard ratio, 25.2; 95% CI, 6.7-95.6; P < .001). Detection of ctDNA at diagnosis, before any treatment, was also associated with relapse-free survival (hazard ratio, 5.8; 95% CI, 1.2-27.1; P = .01). In the combined cohort, ctDNA detection had a median lead time of 10.7 months (95% CI, 8.1-19.1 months) compared with clinical relapse and was associated with relapse in all breast cancer subtypes. Distant extracranial metastatic relapse was detected by ctDNA in 22 of 23 patients (96%). Brain-only metastasis was less commonly detected by ctDNA (1 of 6 patients [17%]), suggesting relapse sites less readily detectable by ctDNA analysis. Conclusions and Relevance: The findings suggest that detection of ctDNA during follow-up is associated with a high risk of future relapse of early-stage breast cancer. Prospective studies are needed to assess the potential of molecular relapse detection to guide adjuvant therapy.

Microscopic gastrointestinal stromal tumours: a clinical and molecular study of 13 cases.

AIMS: Recent literature suggests that clinically silent, microscopic gastrointestinal stromal tumours (micro-GISTs) are common incidental findings. The aim of this study was to examine the histological, immunohistochemical and molecular characteristics of these tumours, which we have defined as measuring ≤20 mm, in order to determine whether the rate and spectrum of mutations are similar to those of clinically symptomatic gastrointestinal stromal tumours (GISTs). METHODS AND RESULTS: Thirteen micro-GISTs identified as incidental findings in patients undergoing management of concomitant disease were tested for KIT/PDGFRA mutations. Ten micro-GISTs (77%) were located in the stomach, two (15%) in the duodenum, and one (8%) in the rectum. The mean tumour size was 9.3 mm (range 2-19 mm). All tumours were well-circumscribed lesions showing a predominantly spindle-cell morphology and a very low mitotic rate. Twelve of 13 (92%) tumours carried mutations in either KIT (83%) or PDGFRA (17%), a rate higher than in other published series. A high mutation rate (80%) was also seen in lesions measuring ≤5 mm. CONCLUSIONS: Our results suggest that KIT/PDGFRA mutation is a very common early event in GIST development, that tumour size does not reliably predict the presence of mutation, and that one or more subsequent mutations are required for clinical manifestation.