Molecular characterisation of aromatase inhibitor-resistant advanced breast cancer: the phenotypic effect of ESR1 mutations.

BACKGROUND: Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS: The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS: Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS: Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours.

Comparison of BEAMing and Droplet Digital PCR for Circulating Tumor DNA Analysis.

BACKGROUND: Circulating tumor DNA (ctDNA) assays are increasingly used for clinical decision-making, but it is unknown how well different assays agree. We aimed to assess the agreement in ctDNA mutation calling between BEAMing (beads, emulsion, amplification, and magnetics) and droplet digital PCR (ddPCR), 2 of the most commonly used digital PCR techniques for detecting mutations in ctDNA. METHODS: Baseline plasma samples from patients with advanced breast cancer enrolled in the phase 3 PALOMA-3 trial were assessed for ESR1 and PIK3CA mutations in ctDNA with both BEAMing and ddPCR. Concordance between the 2 approaches was assessed, with exploratory analyses to estimate the importance of sampling effects. RESULTS: Of the 521 patients enrolled, 363 had paired baseline ctDNA analysis. ESR1 mutation detection was 24.2% (88/363) for BEAMing and 25.3% (92/363) for ddPCR, with good agreement between the 2 techniques (κ = 0.9l; 95% CI, 0.85-0.95). PIK3CA mutation detection rates were 26.2% (95/363) for BEAMing and 22.9% (83/363) for ddPCR, with good agreement (κ = 0.87; 95% CI, 0.81-0.93). Discordancy was observed for 3.9% patients with ESR1 mutations and 5.0% with PIK3CA mutations. Assessment of individual mutations suggested higher rates of discordancy for less common mutations (P = 0.019). The majority of discordant calls occurred at allele frequency <1%, predominantly resulting from stochastic sampling effects. CONCLUSIONS: This large, clinically relevant comparison showed good agreement between BEAMing and ddPCR, suggesting sufficient reproducibility for clinical use. Much of the observed discordancy may be related to sampling effects, potentially explaining many of the differences in the currently available ctDNA literature.

Predicting response to radical (chemo)radiotherapy with circulating HPV DNA in locally advanced head and neck squamous carcinoma.

BACKGROUND: Following chemo-radiotherapy (CCRT) for human papilloma virus positive (HPV+) locally advanced head and neck cancer, patients frequently undergo unnecessary neck dissection (ND) and/or repeated biopsies for abnormal PET-CT, which causes significant morbidity. We assessed the role of circulating HPV DNA in identifying 'true' residual disease. METHODS: We prospectively recruited test (n=55) and validation (n=33) cohorts. HPV status was confirmed by E7 RT-PCR. We developed a novel amplicon-based next generation sequencing assay (HPV16-detect) to detect circulating HPV DNA. Circulating HPV DNA levels post-CCRT were correlated to disease response (PET-CT). RESULTS: In pre-CCRT plasma, HPV-detect demonstrated 100% sensitivity and 93% specificity, and 90% sensitivity and 100% specificity for the test (27 HPV+) and validation (20 HPV+) cohorts, respectively. Thirty-six out of 37 patients (test and validation cohort) with complete samples-set had negative HPV-detect at end of treatment. Six patients underwent ND (3) and repeat primary site biopsies (3) for positive PET-CT but had no viable tumour. One patient had positive HPV-detect and positive PET-CT and liver biopsy, indicating 100% agreement for HPV-detect and residual cancer. CONCLUSIONS: We demonstrate that HPV16-detect is a highly sensitive and specific test for identification of HPV DNA in plasma at diagnosis. HPV DNA post-treatment correlates with clinical response.

Relationship of PIK3CA mutation and pathway activity with antiproliferative response to aromatase inhibition.

INTRODUCTION: PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α) somatic mutations are the most common genetic alteration in breast cancer (BC). Their prognostic value and that of the phosphatidylinositol 3-kinase (PI3K) pathway in BC remains only partly defined. The effect of PIK3CA mutations and alterations of the PI3K pathway on the antiproliferative response to aromatase inhibitor treatment was determined. METHODS: The Sequenom MassARRAY System was used to determine the presence of 20 somatic mutations across the PIK3CA gene in 85 oestrogen receptor-positive (ER+) BC patients treated with 2 weeks of anastrozole before surgery. Whole-genome expression profiles were used to interrogate gene signatures (GSs) associated with the PI3K pathway. Antiproliferative activity was assessed by the change in Ki67 staining between baseline and surgery. Three GSs representing the PI3K pathway were assessed (PIK3CA-GS (Loi), PI3K-GS (Creighton) and PTEN-loss-GS (Saal)). RESULTS: In our study sample, 29% of tumours presented with either a hotspot (HS, 71%) or a nonhotspot (non-HS, 29%) PIK3CA mutation. Mutations were associated with markers of good prognosis such as progesterone receptor positivity (PgR+) (P=0.006), low grade (P=0.028) and luminal A subtype (P=0.039), with a trend towards significance with degree of ER positivity (P=0.051) and low levels of Ki67 (P=0.051). Non-HS mutations were associated with higher PgR (P=0.014) and ER (P<0.001) expression than both wild-type (WT) and HS-mutated samples, whereas neither biomarker differed significantly between WT and HS mutations or between HS and non-HS mutations. An inverse correlation was found between the Loi signature and both the Creighton and Saal signatures, and a positive correlation was found between the latter signatures. Lower pretreatment Ki67 levels were observed in mutation compared with WT samples (P=0.051), which was confirmed in an independent data set. Mutation status did not predict change in Ki67 in response to 2 weeks of anastrozole treatment; there was no significant difference between HS and non-HS mutations in this regard. CONCLUSIONS: PIK3CA mutations are associated with classical markers of good prognosis and signatures of PI3K pathway activity. The presence of a PIK3CA mutation does not preclude a response to neoadjuvant anastrozole treatment.

The genomic landscape of oesophagogastric junctional adenocarcinoma.

The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets.

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.

Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene is a key regulator of apoptosis, migration and invasion in colorectal cancer.

Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene maps to chromosomal region 20q13.13, a region frequently amplified in solid tumours. In this study, we investigated the roles played by CSE1L in colorectal cancer by examining CSE1L expression and clinico-pathological parameters in colorectal cancer and investigating the effect of CSE1L on the viability, adhesion and migration of colorectal cancer cells. RT-PCR showed that CSE1L mRNA was over-expressed in colorectal cancer. CSE1L depletion by knock-down with CSE1L-specific siRNA significantly reduced viability in HCT116 cells (p = 0.004) and SW480 cells (p = 0.003) whilst significantly increasing the proportion of apoptotic HCT116 cells (p < 0.001) and SW480 cells (p < 0.001). Furthermore, CSE1L depletion significantly reduced the adhesive capacity of HCT116 (p = 0.003) and SW480 cells (p = 0.004). Analysis by qRT-PCR following CSE1L siRNA treatment of HCT116 and SW480 cells showed significant modulation of key apoptotic (p53, p73 and BAK) and adhesive (E-cadherin, Ep-CAM and ICAM-1) molecules. Immunohistochemistry of a colorectal cancer tissue microarray showed that CSE1L had a significantly increased level in colorectal cancer compared to normal colorectal epithelium (p < 0.001). There were significant decreases in both nuclear (p = 0.006) and cytoplasmic (p = 0.003) staining of CSE1L in tumours with lymph node metastasis (stage 3 tumours) compared with lymph node-negative tumours (stage 1 and 2 tumours). In lymph node-negative patients, poor survival was associated with increased CSE1L cytoplasmic expression (p = 0.042). These results indicate that CSE1L is associated with viability and apoptosis, cellular adhesion and invasion, thus implicating CSE1L in the progression of colorectal cancer.

Circulating HPV DNA as a Biomarker for Pre-Invasive and Early Invasive Cervical Cancer: A Feasibility Study.

BACKGROUND: High-risk HPV infection is responsible for >99% of cervix cancers (CC). In persistent infections that lead to cancer, the tumour breaches the basement membrane, releasing HPV-DNA into the bloodstream (cHPV-DNA). A next-generation sequencing assay (NGS) for detection of plasma HPV circulating DNA (cHPV-DNA) has demonstrated high sensitivity and specificity in patients with locally advanced cervix cancers. We hypothesised that cHPV-DNA is detectable in early invasive cervical cancers but not in pre-invasive lesions (CIN). METHODS: Blood samples were collected from patients with CIN (n = 52) and FIGO stage 1A-1B CC (n = 12) prior to treatment and at follow-up. DNA extraction from plasma, followed by NGS, was used for the detection of cHPV-DNA. RESULTS: None of the patients with pre-invasive lesions were positive for CHPV-DNA. In invasive tumours, plasma from one patient (10%) reached the threshold of positivity for cHPV-DNA in plasma. CONCLUSION: Low detection of cHPV-DNA in early CC may be explained by small tumour size, poorer access to lymphatics and circulation, and therefore little shedding of cHPV-DNA in plasma at detectable levels. The detection rate of cHPV-DNA in patients with early invasive cervix cancer using even the most sensitive of currently available technologies lacks adequate sensitivity for clinical utility.

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.

Predicting Response to Radical Chemoradiotherapy with Circulating HPV DNA (cHPV-DNA) in Locally Advanced Uterine Cervix Cancer.

BACKGROUND: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay-panHPV-detect-in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. METHOD: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. RESULTS: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70-99%) and 100% (95% CI-30-100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. CONCLUSIONS: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort.

Assessing CSF ctDNA to Improve Diagnostic Accuracy and Therapeutic Monitoring in Breast Cancer Leptomeningeal Metastasis.

PURPOSE: Cerebrospinal fluid (CSF) cytology is the gold standard diagnostic test for breast cancer leptomeningeal metastasis (BCLM), but has impaired sensitivity, often necessitating repeated lumbar puncture to confirm or refute diagnosis. Further, there is no quantitative response tool to assess response or progression during BCLM treatment. EXPERIMENTAL DESIGN: Facing the challenge of working with small-volume samples and the lack of common recurrent mutations in breast cancers, cell-free DNA was extracted from the CSF and plasma of patients undergoing investigation for BCLM (n = 30). ctDNA fraction was assessed by ultra-low-pass whole genome sequencing (ulpWGS), which does not require prior tumor sequencing. RESULTS: In this proof-of-concept study, ctDNA was detected (fraction ≥0.10) in the CSF of all 24 patients with BCLM+ (median ctDNA fraction, 0.57), regardless of negative cytology or borderline MRI imaging, whereas CSF ctDNA was not detected in the six patients with BCLM- (median ctDNA fraction 0.03, P < 0.0001). Plasma ctDNA was only detected in patients with extracranial disease progression or who had previously received whole brain radiotherapy. ctDNA fraction was highly concordant with mutant allele fraction measured by tumor mutation-specific ddPCR assays (r = 0.852; P < 0.0001). During intrathecal treatment, serial monitoring (n = 12 patients) showed that suppression of CSF ctDNA fraction was associated with longer BCLM survival (P = 0.034), and rising ctDNA fraction was detectable up to 12 weeks before clinical progression. CONCLUSIONS: Measuring ctDNA fraction by ulpWGS is a quantitative marker demonstrating potential for timely and accurate BCLM diagnosis and therapy response monitoring, with the ultimate aim to improve management of this poor-prognosis patient group.

Circulating Tumor DNA Markers for Early Progression on Fulvestrant With or Without Palbociclib in ER+ Advanced Breast Cancer.

BACKGROUND: There are no established molecular biomarkers for patients with breast cancer receiving combination endocrine and CDK4/6 inhibitor (CDK4/6i). We aimed to determine whether genomic markers in circulating tumor DNA (ctDNA) can identify patients at higher risk of early progression on fulvestrant therapy with or without palbociclib, a CDK4/6i. METHODS: PALOMA-3 was a phase III, multicenter, double-blind randomized controlled trial of palbociclib plus fulvestrant (n = 347) vs placebo plus fulvestrant (n = 174) in patients with endocrine-pretreated estrogen receptor-positive (ER+) breast cancer. Pretreatment plasma samples from 459 patients were analyzed for mutations in 17 genes, copy number in 14 genes, and circulating tumor fraction. Progression-free survival (PFS) was compared in patients with circulating tumor fraction above or below a prespecified cutoff of 10% and with or without a specific genomic alteration. All statistical tests were 2-sided. RESULTS: Patients with high ctDNA fraction had worse PFS on both palbociclib plus fulvestrant (hazard ratio [HR] = 1.62, 95% confidence interval [CI] = 1.17 to 2.24; P = .004) and placebo plus fulvestrant (HR = 1.77, 95% CI = 1.21 to 2.59; P = .004). In multivariable analysis, high-circulating tumor fraction was associated with worse PFS (HR = 1.20 per 10% increase in tumor fraction, 95% CI = 1.09 to 1.32; P < .001), as was TP53 mutation (HR = 1.84, 95% CI = 1.27 to 2.65; P = .001) and FGFR1 amplification (HR = 2.91, 95% CI = 1.61 to 5.25; P < .001). No interaction with treatment randomization was observed. CONCLUSIONS: Pretreatment ctDNA identified a group of high-risk patients with poor clinical outcome despite the addition of CDK4/6 inhibition. These patients might benefit from inclusion in future trials of escalating treatment, with therapies that may be active in these genomic contexts.

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.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in PIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

lazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction.

An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate PI(4,5)P2 by phospholipase Cbeta (PLCbeta) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates its levels, mediating both amplification and response termination. In this study, we demonstrate that lazaro (laza) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. We demonstrate that the synergistic activity of laza and rdgA regulates response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in phosphatidic acid (PA) levels and an associated reduction in phosphatidylinositol (PI) levels. Together our results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo.

Next Generation Sequencing Assay for Detection of Circulating HPV DNA (cHPV-DNA) in Patients Undergoing Radical (Chemo)Radiotherapy in Anal Squamous Cell Carcinoma (ASCC).

Background: Following chemo-radiotherapy (CRT) for human papilloma virus positive (HPV+) anal squamous cell carcinoma (ASCC), detection of residual/recurrent disease is challenging. Patients frequently undergo unnecessary repeated biopsies for abnormal MRI/clinical findings. In a pilot study we assessed the role of circulating HPV-DNA in identifying "true" residual disease. Methods: We prospectively collected plasma samples at baseline (n = 21) and 12 weeks post-CRT (n = 17). Circulating HPV-DNA (cHPV DNA) was measured using a novel next generation sequencing (NGS) assay, panHPV-detect, comprising of two primer pools covering distinct regions of eight high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52, and 58) to detect circulating HPV-DNA (cHPV DNA). cHPV-DNA levels post-CRT were correlated to disease response. Results: In pre-CRT samples, panHPV-detect demonstrated 100% sensitivity and specificity for HPV associated ASCC. PanHPV-detect was able to demonstrate cHPV-DNA in 100% (9/9) patients with T1/T2N0 cancers. cHPV-DNA was detectable 12 weeks post CRT in just 2/17 patients, both of whom relapsed. 1/16 patients who had a clinical complete response (CR) at 3 months post-CRT but relapsed at 9 months and 1/1 patient with a partial response (PR). PanHPV-detect demonstrated 100% sensitivity and specificity in predicting response to CRT. Conclusion: We demonstrate that panHPV-detect, an NSG assay is a highly sensitive and specific test for the identification of cHPV-DNA in plasma at diagnosis. cHPV-DNA post-treatment may predict clinical response to CRT.

PIK3CA mutation enrichment and quantitation from blood and tissue.

PIK3CA is one of the two most frequently mutated genes in breast cancers, occurring in 30-40% of cases. Four frequent 'hotspot' PIK3CA mutations (E542K, E545K, H1047R and H1047L) account for 80-90% of all PIK3CA mutations in human malignancies and represent predictive biomarkers. Here we describe a PIK3CA mutation specific nuclease-based enrichment assay, which combined with a low-cost real-time qPCR detection method, enhances assay detection sensitivity from 5% for E542K and 10% for E545K to 0.6%, and from 5% for H1047R to 0.3%. Moreover, we present a novel flexible prediction method to calculate initial mutant allele frequency in tissue biopsy and blood samples with low mutant fraction. These advancements demonstrated a quick, accurate and simple detection and quantitation of PIK3CA mutations in two breast cancer cohorts (first cohort n = 22, second cohort n = 25). Hence this simple, versatile and informative workflow could be applicable for routine diagnostic testing where quantitative results are essential, e.g. disease monitoring subject to validation in a substantial future study.

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.

Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.