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Supplementary Figure 4. Individual ESR1 mutation detection and polyclonality in SoFEA and in EFECT

Supplementary Figure 2. Consort diagram of patients analysed from EFECT

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 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.

Supplementary Figure 6. Progression free survival in monoclonal and polyclonal ESR1 mutations

Supplementary Data

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.

Supplementary Figure 1. Impact of DNA input amount on ESR1 mutation detection in EFECT

Supplementary Data from Biomarkers of Response and Resistance to Palbociclib Plus Letrozole in Patients With ER+/HER2− Breast Cancer

Supplementary Figure 5. Progression free survival by ESR1 mutation detected in ctDNA

Supplementary Figure 3. Time To Progression in EFECT by detection of ESR1 mutations in baseline serum

Background Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage triple negative breast cancer (TNBC) is associated with a very high risk of future relapse. Identifiying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. The c-TRAK TN trial assessed the utility of prospective ctDNA surveillance in pts treated for TNBC and the activity of pembrolizumab (P) in pts with ctDNA detected.. Methods c-TRAK TN, a multi-centre phase II trial with integrated prospective screening component, enrolled pts with early-stage TNBC and either residual disease following neoadjuvant chemotherapy, or tumour size >20mm and/or axillary lymph node involvement if adjuvant chemotherapy was given. Tumour tissue was sequenced to identify somatic mutations suitable for tracking using personalised digital PCR ctDNA assays (BioRad QX200). Pts had “active” ctDNA surveillance via blood sample testing every 3 months to 12 months (potential up to 18 months if samples missed due to COVID) during which time if ctDNA was detected (ctDNA+) pts could be randomised 2:1 to P (200mg i.v. q 3 weeks for 1 year) or observation (Obs). Pts and clinicians were blinded to ctDNA+ results unless they were allocated P, when staging scans were done and those free of clinical recurrence started treatment. Following advice from the Independent Data Monitoring Committee, the Obs arm closed on 16/06/2020 with all subsequent ctDNA+ pts allocated P. Following the completion of active ctDNA surveillance, 3-monthly visits continued to 24 months to be analysed retrospectively. The aim was to recruit 150 pts to ctDNA surveillance, assuming 30% would be ctDNA+ within 12 months, allowing ctDNA+ rate to be estimated with a 2-sided 95%CI of +/- 7.3%. Co-primary endpoints are i) rates of ctDNA detection by 12 and 24 months from start of ctDNA surveillance; ii) rates of sustained ctDNA clearance on P defined as absence of detectable ctDNA, or disease recurrence 6 months after starting P.. Results 208 pts were registered between 30/01/18 and 06/12/19, 185 had tumour sequenced, 171 (92.4%) had trackable mutations, and 161 entered ctDNA surveillance. The rate of ctDNA detection by 12 months after start of surveillance was 27.3% (44/161, 95% CI 20.6-34.9). ctDNA+ rates from baseline, 3, 6, 9 and 12 month ctDNA samples were 23/161 (14.3%), 6/115 (5.2%), 6/99 (5.1%), 7/84 (8.3%), and 2/84 (2.4%) respectively. An additional 2 pts were ctDNA+ on COVID extended active surveillance at 15 (1/51, 2%) or 18 months (1/11, 9%). 7 pts relapsed without prior ctDNA detection. 45 pts entered the therapeutic component of the trial (initially 31 to P and 14 to Obs). 1 Obs pt was re-allocated to P. Of pts allocated to P, 72% (23/32) had metastatic disease at time of ctDNA detection on staging scans (75% (12/16) who were ctDNA+ at baseline and 69% (11/16) at other timepoints). 4 pts declined to start P, largely due to COVID concerns. Of the 5 pts who commenced P, at the time of analysis none achieved sustained ctDNA clearance and 4 had recurred. In pts allocated to Obs, median time to recurrence was 4.1 months (95% CI: 3.2-not-defined).. Conclusion The c-TRAK TN trial is to our knowledge the first study to assess the proof-of-principle of whether ctDNA assays have clinical utility in guiding further therapy in TNBC. Relatively few pts commenced P treatment precluding assessment of potential activity. At enrollment, patients had a relatively high of rate of undiagnosed metastatic disease when imaged. Our findings have implications for future trial design, emphasizing the importance of early start of ctDNA testing, and more sensitive and/or more frequent ctDNA testing regimes. Citation Format: Nicholas Turner, Claire Swift, Ben Jenkins, Lucy Kilburn, Maria Coakley, Matthew Beaney, Lisa Fox, Katie Goddard, Isaac Garcia-Murillas, Peter Hall, Catherine Harper-Wynne, Tamas Hickish, Sarah Kernaghan, Iain Macpherson, Alicia Okines, Carlo Palmieri, Sophie Perry, Katrina Randle, Claire Snowdon, Hilary Stobart, Andrew Wardley, Duncan Wheatley, Simon Waters, Matthew Winter, Judith Bliss. Primary results of the cTRAK TN trial: A clinical trial utilising ctDNA mutation tracking to detect minimal residual disease and trigger intervention in patients with moderate and high risk early stage triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-06.

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 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.

Background CDK4/6 inhibitors may trigger anti-tumour immunity, both through tumour cell intrinsic and extrinsic mechanisms, which can be enhanced by immune checkpoint blockade in pre-clinical studies. Although most triple negative breast cancers (TNBC) are resistant to CDK4/6 inhibition, palbociclib (palbo) has activity in pre-clinical models of the luminal androgen receptor (LAR) subtype. Here we report the dose finding phase Ib cohort A of the PAveMenT trial combining avelumab and palbociclib Methods The PAveMenT phase 1b trial consists of a dose finding phase (cohort A), to be followed by an expansion in androgen receptor positive (AR+) TNBC (Cohort B). The phase 1B phase investigated palbo dosing schedules (75mg intermittent dosing (ID), 100mg continuous dosing (CD) and 125mg ID) in combination with the anti-PD-L1 antibody, avelumab 10mg/kg 2-weekly (cohort A) to determine the optimal schedule for cohort B. The CD dose-level was pre-specified as the preferred schedule if tolerated, due to the proliferative nature of TNBC and potential for tumour re-growth with ID. Patients with previously treated advanced breast cancer (ABC) of any histology with measurable disease and adequate organ function, who had not received prior immunotherapy or CDK4/6 inhibitors or more than 2 lines of chemotherapy for ABC, were eligible for cohort A. Dose-limiting toxicities (DLTs) were defined as Grade(G) 4 neutropenia, complicated G3 neutropenia or G4 thrombocytopenia, or ≥G3 immune toxicity during cycle 1. AR staining of ≥10% using the SP107 antibody (Ventana) was considered AR+. PD-L1 was evaluated using CC23 Pharma Dx antibody (DAKO) and PD-L1 positive defined as CPS score of ≥10%. Results Fourteen patients were recruited to Part A, all female, 12 with TNBC, two with ER positive/HER2 negative ABC, median age 53.5 years. Three evaluable patients received palbo 75mg/day ID without DLTs, therefore two further dose levels were opened in parallel. Three evaluable patients received palbo 125mg/day ID without DLTs. Due to a DLT of fever with G3 neutropenia in one of 3 patients receiving palbo 100mg CD, 3 further evaluable patients received 100mg CD without DLTs. Two patients were not evaluable for DLTs having not received a full cycle of treatment so were replaced. One case of grade 3 colitis and one case of grade 2 hypothyroidism (both 100mg CD) occurred outside the DLT period. Amongst 12 patients with TNBC, metastatic tissue was available for 11, archival breast tissue for one. Four of 12 patients were AR+. Five of 10 TNBC patients with sufficient tissue were PD-L1 positive, all of whom had previously tested negative with the SP142 antibody (Ventana). One of the 5 PD-L1+ and 2/5 PD-L1 negative TNBC cases were also AR+. TNBC patients received a median of 2 cycles of treatment (range 1-17). Both ER+ patients were AR+ and PD-L1 negative; one progressed after 4 cycles, the other remains on treatment at 12 cycles. The 100mg CD schedule was selected for cohort B. Conclusions All schedules were tolerable, with activity observed in patients with both ER positive and TNBC cancers. Cohort B of the study is open to recruitment for patients with AR+ TNBC. NCT04360941. Citation Format: Alicia F. Okines, Houman Moghadam, Laura Sparks, Kabir Mohammed, Kathryn Dunne, Ashutosh Nerurkar, Peter Osin, Claire Swift, Ruth Sardinha, Nicholas Turner. Results from a dose escalation phase 1b study of palbociclib and avelumab in advanced breast cancer in the PAveMenT Trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-02.

Background: Most patients with early stage oestrogen receptor positive (ER+) and HER2 negative breast cancer will be cured of their cancer. However, up to 20% of patients may experience disease recurrence in the first 10 years. Molecular relapse of ER+ breast cancer can be detected with circulating tumour DNA (ctDNA) before clinical relapse occurs. Palbociclib, a CDK4/6 inhibitor, plus fulvestrant, a selective oestrogen receptor degrader, is a standard first line therapy for patients with ER+ breast cancer who have relapsed on standard endocrine therapy. We designed TRAK-ER to establish a surveillance system for ctDNA detection and then to assess whether treating patients, who have ctDNA detected molecular relapse, with palbociclib and fulvestrant may defer or prevent relapse. Design: TRAK-ER is a phase 2 multi-centre, randomised, open-label parallel superiority trial in patients with ER+ early breast cancer, recruiting at centres in the UK and France. In the surveillance phase patients will be monitored for molecular recurrence with ctDNA testing. To be eligible for the surveillance phase patients must be aged 18 or over, have ER+ (≥10% or Allred score 6/8 or greater) and HER2 negative breast cancer and have completed their primary surgery, chemotherapy and radiotherapy. Standard endocrine therapy (GnRH analogues, aromatase inhibitors and tamoxifen) must have been received for a minimum of 6 months and a maximum of 7 years and be planned to continue for at least another 3 years. Inclusion criteria in patients who did not receive neoadjuvant chemotherapy are at least one of: (a) four or more involved axillary or positive supraclavicular lymph node; (b) tumour size >5cm; (c) one to three involved axillary lymph nodes together with at least one of: tumour size >3cm, grade 3 or a high genomic risk score. Patients who received neoadjuvant chemotherapy require at least one lymph node positive or a tumour size >3cm after chemotherapy. Invitae Personalized Cancer Monitoring (PCM TM) will be used for ctDNA analysis, a pan-cancer, tumour-informed liquid biopsy test that uses next-generation sequencing to detect minimal or molecular residual disease (MRD) in solid tumours. ctDNA analysis will be every 3 months for up to 3 years. Detection of ctDNA will trigger staging imaging. If no overt metastatic disease is identified, patients will be able to enter the treatment phase of the study, and be 1:1 randomised using minimisation to either remain on standard endocrine therapy or switch to palbociclib plus fulvestrant. Those who are allocated to remain on endocrine therapy are allowed to continue on the same therapy or change standard endocrine therapy. Duration of palbociclib and fulvestrant will be 2 years, or until relapse. Up to 1300 patients will enrol for tissue screening to allow 1100 patients to enter into ctDNA surveillance. 132 patients will enter the treatment part of the study. The primary endpoint of the surveillance phase is ctDNA detection rate. The primary endpoint of the treatment phase is relapse free survival (RFS). RFS will be calculated in the intention to treat population using Kaplan Meier methods from the date of randomisation to the date of recurrence or death from any cause. Secondary endpoints include relapse free interval, invasive disease free survival, distant recurrence free survival, overall survival and ctDNA clearance. (NCT04985266) Citation Format: Nicholas Turner, Edward R. Phillips, Catey Bunce, Marie Robert, Caroline Bailleux, Isaac Garcia-Murillas, Komel Khabra, Iain Macpherson, Ciara S. O’Brien, Alicia F. Okines, Carlo Palmieri, Peter Schmid, Claire Swift, Sabrina Yara, Simon Connolly, Jérôme Lemonnier, Dymphna Lee, Fabrice Andre. A randomised phase II trial of palbociclib and fulvestrant vs standard endocrine therapy in patients with ER positive HER2 negative breast cancer and ctDNA detected molecular relapse during adjuvant endocrine therapy (TRAK-ER) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-01-01.

Background: Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage breast cancer is associated with a high risk of future relapse. Identifying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. Previous analysis of this cohort showed that tools capable of detecting ctDNA at lower concentrations are needed to increase sensitivity and lengthen the lead time between ctDNA detection and relapse. We compared ctDNA detection via a personalised sequencing assay to dPCR in patients from the cTRAK TN clinical trial. Methods: The cTRAK-TN trial recruited 161 pts into prospective ctDNA surveillance with dPCR, with ctDNA positive pts randomised to 1) CT staging plus pembrolizumab therapy for patients without relapse or 2) observation. Pts had serial post-treatment surveillance plasma samples collected every 3 months for up to 2 years. Whole exome sequencing (WES) was performed on tumor DNA from FFPE samples to design personalised Residual Disease and Recurrence (RaDaR®) multiplex PCR based NGS assays. Retrospectively, plasma DNA extracted from a minimum of 2mls banked plasma, was sequenced with personalised RaDaR assays, and ctDNA detection identified with a proprietary algorithm. dPCR assays tracked 1-2 mutations, as previously described. Primary endpoint was rate of positive ctDNA detection by 12 months from start of surveillance in both assays. Secondary endpoints were agreement in ctDNA detection between RaDaR and dPCR assays and lead-time between ctDNA detection and disease recurrence. Results: Overall, 147 pts and 241 tissue samples were subject to WES, and RaDaR assays were developed for 142 pts with sufficient plasma for testing. RaDaR assays tracked a median of 47 variants (range 33-56) per patient, and a total of 907 timepoints were analysed (median 6 timepoints per pt, range 1-11). With RaDaR, 39.4% (56/142) patients tested ctDNA positive during follow-up, with a median ctDNA detected level of 0.081% estimated variant allele fraction (eVAF). With dPCR, 35.2% (50/142) pts tested ctDNA positive. The ctDNA detection rate by 12 months from the start of ctDNA surveillance was 36.2% (95% CI; 27.6% – 43.7%) with RaDaR and 29.9% (95%CI; 21.6% – 37.3%) with dPCR. The overall test agreement between RaDaR and dPCR assays was 92.7% (95%CI; 90.7% – 94.4%). From a patient perspective, 58.7% pts were ctDNA negative for both assays, 32.9% ctDNA were positive for both assays and 8.6% presented discrepancies. ctDNA was detected by RaDaR but not by dPCR in 9 pts and it was detected by dPCR but not by RaDaR in 3 pts. Among ctDNA positive pts, 55.2% were first detected positive by RaDaR, 5.2% by dPCR, and 39.6% were detected at the same time-point (test of proportions, p< 0.001). The median lead time from ctDNA detection to relapse was 7.1 months (95% CI 5.9 – 15.9%) with RaDaR and 5.7 months (95% CI 3.2% – 7.4%) with dPCR. Conclusion: The RaDaR personalised multi-mutation sequencing assay detected MRD with a longer median lead time prior to relapse, and with higher sensitivity, than dPCR mutation tracking assays. These findings have implications for the choice of ctDNA assay in clinical trials designed to treat patients at the point of MRD detection. Citation Format: Maria Coakley, Prithika Sritharan, Guillermo Villacampa, Claire Swift, Kathryn Dunne, Lucy Kilburn, Katie Goddard, Patricia Rojas, Andy Joad, Warren Emmett, Charlene Knape, Karen Howarth, Peter S. Hall, Catherine Harper-Wynne, Tamas Hickish, Iain Macpherson, Alicia F. Okines, Andrew M. Wardley, Duncan Wheatley, Simon Waters, Rosalind Cutts, Isaac Garcia-Murillas, Judith Bliss, Nicholas Turner. PD5-03 Comparison of a personalized sequencing assay and digital PCR for circulating tumor DNA based Molecular Residual Disease detection in early-stage triple negative breast cancer in the cTRAK-TN trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-03.

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.

Background: Circulating tumor DNA (ctDNA) is found in the plasma of over 90% of patients with advanced breast cancer (BC). ctDNA analysis can establish the current genomic profile of an individual’s cancer and identify potentially targetable mutations. The genomic landscape of advanced BC and clinical associations has yet to be fully defined. This analysis describes the genomic landscape of ctDNA in patients screened for the UK plasmaMATCH study. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC. The trial consists of parallel treatment cohorts (Cohorts A-E) with therapies matched to mutations identified in ctDNA testing. The genomic landscape of ctDNA in advanced BC was prospectively analysed for hotspot PIK3CA,AKT1, HER2 and ESR1 mutations using digital droplet PCR (ddPCR, Bio-Rad), and using an error-corrected 73-gene targeted panel (Guardant360, Guardant Health) prospectively from part-way through the trial, and retrospectively for the remaining patients. Results: Entry into ctDNA testing for Cohorts A-D was closed on 26/Apr/2019, at which time 1044 patients had been registered and 1025 tested with ddPCR. 800 patients had targeted panel ctDNA sequencing (364 prospective, 436 retrospective) (Table 1). Targeted sequencing identified a somatic alteration in 92.9% of patients (743/800). The most frequent mutations were TP53 (44.1%), PIK3CA (34.9%), ESR1 (33.1%), GATA3 (11.0%) and ARID1A (7.8%). ESR1 (33.1%) and KRAS (4.1%) alterations occurred at higher frequency in ctDNA compared to a metastatic tissue sequencing series (q Genomic AlterationddPCR*Targeted Panel**All % n = 1025HR+ HER2-% n = 658HR+ HER2+ % n = 62HR- HER2+ % n = 36TNBC % n = 172All % n = 800HR+ HER2- % n = 515HR+ HER2+ % n = 48HR- HER2+ % n = 26TNBC % n = 136PIK3CA mutation25.829.933.919.49.334.939.041.734.614.7ESR1 mutation27.737.819.40033.143.522.900.7AKT1 mutation4.24.01.604.75.05.22.104.4ERBB2 mutation2.73.208.31.26.46.612.515.42.2PTEN mutationNANANANANA6.98.54.23.84.4ERBB2 amplificationNANANANANA5.81.931.357.71.5MSI highNANANANANA1.10.42.101.5* Phenotype data based on 928 patients with known phenotype tested by ddPCR for targetable hotspot mutations.** Phenotype data based on 725 patients with known phenotype sequenced by targeted panel. All pathogenic mutations are shown, including mutations not tested by ddPCR. Citation Format: Belinda Kingston, Hannah Bye, Michael Hubank, Giselle Walsh, Claire Swift, Matthew Beaney, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Rebecca Roylance, Katie Wilkinson, Isaac Garcia-Murillas, Judith M Bliss, Nicholas Turner, Alistair Ring, on behalf of the plasmaMATCH Trial Management Group. The genomic landscape of breast cancer based on ctDNA analysis: Data from the plasmaMATCH trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-07.

To determine (i) the relationship between candidate biomarkers of the antiproliferative (Ki67) response to letrozole and palbociclib alone and combined in ER307 postmenopausal women with ERHigher baselines ER and PgR were significantly associated with a greater chance of complete cell-cycle arrest (CCCA: Ki672.7%) 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.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.

1019 Background: The randomized PEARL trial found no superiority of PAL plus endocrine therapy over CAPE in patients (pts) with metastatic HR-positive, HER2-negative breast cancer resistant to aromatase inhibitors (Martin M, Ann Oncol 2020). We investigated associations between baseline genomic landscape and on-treatment plasma ctDNA dynamics with progression free survival, in pts from cohort 2 of the trial. Methods: Plasma was collected for ctDNA analysis from -7 days to cycle 1-day 1 (C1D1) for baseline prognostic analysis and cycle 1-day 15 (C1D15) when available, and sequenced with an in-house error-corrected targeted capture panel encompassing 21 genes commonly altered in breast cancer. For predictive ctDNA dynamics analysis, a pre-specified criteria of 14 minimum days of treatment in first cycle was required and variants with VAF

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., Circulating tumour DNA can provide useful information on disease burden. Here, the authors analysed circulating tumour DNA from 800 patients from a breast cancer clinical trial and investigate the subclonal nature of the disease, and identify DNA mutations associated with resistance and poor survival.

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.

BACKGROUND: Palbociclib plus endocrine therapy (ET) is the standard treatment for hormone receptor-positive and human epidermal growth factor receptor 2-negative, metastatic breast cancer (MBC). However, its efficacy has not been compared with that of chemotherapy in a phase III trial. PATIENTS AND METHODS: PEARL is a multicentre, phase III randomised study in which patients with aromatase inhibitors (AIs)-resistant MBC were included in two consecutive cohorts. In cohort 1 (C1), patients were randomised 1:1 to palbociclib plus exemestane or capecitabine. On discovering new evidence about oestrogen receptor-1 (ESR1) mutations inducing resistance to AIs, the trial was amended to include cohort 2 (C2), in which patients were randomised 1:1 between palbociclib plus fulvestrant and capecitabine. The stratification criteria were disease site, prior sensitivity to ET, prior chemotherapy for MBC, and country of origin. Co-primary endpoints were progression-free survival (PFS) in C2 and in wild-type ESR1 patients (C1+C2). ESR1 hotspot mutations were analysed in baseline circulating tumour DNA. RESULTS: From March-2014 to July-2018, 296 and 305 patients were included in C1 and C2, respectively. Palbociclib plus ET was not superior to capecitabine in both C2 (median PFS: 7.5 vs. 10.0 months; adjusted hazard ratio [aHR]: 1.13; 95% confidence Interval [CI]: 0.85-1.50) and wild-type ESR1 patients (median PFS: 8.0 vs. 10.6 months; aHR: 1.11; 95% CI: 0.87-1.41). The most frequent grade 3-4 toxicities with palbociclib plus exemestane, palbociclib plus fulvestrant, and capecitabine were neutropenia (57.4%, 55.7% and 5.5%), hand/foot syndrome (0%, 0% and 23.5%), and diarrhoea (1.3%, 1.3% and 7.6%). Palbociclib plus ET offered better quality of life (aHR for time to deterioration of global health status: 0.67; 95% CI: 0.53-0.85). CONCLUSIONS: There was no statistical superiority of palbociclib plus ET over capecitabine with respect to PFS in MBC patients resistant to AIs. Palbociclib plus ET showed a better safety profile and improved quality of life.

Background. ESR1 mutations are acquired frequently in hormone receptor positive breast cancer after treatment with aromatase inhibitors (AI) in the metastatic setting. In prior analysis of the SoFEA phase III randomised trial, we demonstrated the detection of ESR1 mutations in circulating tumor DNA (ctDNA) predicted for greater benefit of fulvestrant compared to exemestane (Fribbens et al JCO 2016). Methods. The phase III EFECT study randomised 693 patients with ER+ metastatic breast cancer who had progressed on a prior non-steroidal AI, between fulvestrant loading dose and then 250mg q28 days and exemestane (Chia S, et al. J Clin Oncol 2008). Baseline serum samples were available from 227 patients in EFECT, and were analysed for the 8 most common ESR1 mutations by multiplex digital PCR. The association between baseline ESR1 status and time to progression (TTP) was analysed using Kaplan-Meier methods. Results. ESR1 mutations were successfully analysed in 98% (222/227) of patients with baseline serum samples, with ESR1 mutations detected in 23.4% (52/222) samples. Overall, detection of ESR1 mutations at baseline was associated with shorter TTP (hazard ratio [HR] 2.03, 95% CI 1.26-3.29, p=0.004). In patients with ESR1 mutation detected, TTP was 2.0 months (95%CI, 1.7-2.4) on exemestane and 3.5 months (95%CI, 1.9-5.0) on fulvestrant (HR 0.67, 95%CI 0.37-1.19, p=0.17). In patients without ESR1 mutations detected, TTP was 4.5 months (95%CI, 3.7-5.6) on exemestane and 3.7 months (95%CI, 3.3-5.2) on fulvestrant (HR 1.05, 95%CI 0.75-1.45, p=0.78). A meta-analysis of SoFEA and EFECT will be presented at the conference. Conclusions. Historical serum samples may be used for ctDNA analysis, illustrating the potential for future research of sample archives. Patients with ESR1 mutation detected in ctDNA have poor outcome when treated with exemestane, replicating prior results from SoFEA and demonstrating clinical utility for ESR1 mutation ctDNA analysis. Citation Format: Turner N, Swift C, Kilburn L, Garcia-Murillas I, Johnston S, Budzar A, Robertson J, Gradishar W, Piccart M, Schiavon G, Chia S. Baseline circulating ESR1 mutation analysis in the randomised phase III EFECT study of fulvestrant versus exemestane in advanced hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-04.

1014 Background: The randomized PEARL trial found no superiority of palbociclib plus endocrine therapy over capecitabine in patients (pts) with metastatic HR-positive, HER2-negative breast cancer resistant to prior aromatase inhibitors (Martin M, Ann Oncol 2020). Gene expression analysis showed high CCNE1 mRNA ( CCNE1) conferring relative resistance to palbociclib in the PALOMA-3 trial (Turner N, JCO 2019), but further validation is needed. Cyclin E1 protein (cyclin E1) expression in this context has not been studied in randomized trials. We explored CCNE1 and cyclin E1 as predictive biomarkers in tumor samples from the PEARL study. Methods: Formalin-fixed paraffin-embeded tumor samples were retrieved from pts enrolled in PEARL cohort 2 (palbociclib (PAL) + fulvestrant (FUL) vs capecitabine (CAPE)). We measured CCNE1 using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics). Cyclin E1 immunohistochemistry (IHC) staining was performed using specific mouse monoclonal antibody HE12 (Abcam) and scored as percentage of invasive nuclei stained (0-100%). CCNE1 and cyclin E1 correlations were explored using Pearson coefficients. Cox regression models were used for progression free-survival (PFS) analyses using expression levels split by median, to define high ( > median values) vs. low expression. Site of disease and prior chemotherapy were used as confounders in multivariate models. Results: Analyses were conducted in 219 pts (47% receiving PAL + FUL and 53% CAPE) with available tumors, with the analysed patients representative of the overall study. Most samples were from the archival primary (72%), obtained > 5 years before this analysisº (74%). CCNE1 and cyclin E1 were only moderately correlated (r = 0.5). Median CCNE1 was higher in metastatic vs primary (7.37 vs 6.94, p < 0.01), and in luminal B and non-luminal subtypes compared to luminal A (p < 0.001). In patients with high CCNE1 expression, median PFS on CAPE was 10.35 and PAL + FUL was 5.68 (HR = 1.63, 95% CI 1.02-2.59, p = 0.042). In patients with low CCNE1 expression, median PFS on CAPE was 9.43 and PAL + FUL was 8.97 (adjusted HR = 0.93, 95% CI 0.59-1.48, p = 0.762, interaction p = 0.072). Median cyclin E1 protein was higher in luminal B and non-luminal subtypes compared to luminal A (p < 0.01). Cyclin E1 protein expression was not predictive of treatment effect (high cyclin E1 expression CAPE vs PAL + FUL HR = 1.17, low cyclin E1 expression CAPE vs PAL + FUL HR = 1.21, interaction p = 0.977). Conclusions: High tumor CCNE1 mRNA expression identified patients with relative resistance to palbociclib plus fulvestrant, validating prior observations although without statistical significance for interaction. Assessment of Cyclin E1 protein expression did not show predictive value. Investigation treatments to enhance CDK4/6 inhibitor efficacy in tumors with high CCNE1 expression is warranted. Clinical trial information: NCT02028507 .

1022 Background: Prior retrospective data has shown ESR1 mutation is an acquired resistant mechanism to AI. However, little is known about ist prognostic and predictive value to endocrine-based therapy and chemotherapy. PEARL study compared PAL+ET vs CAP in AI resistant patients. Here we wxplored prospectively the ESR1 mutational status based on updated PFS and OS. Methods: PEARL is a phase 3 study with 2 subsequent cohorts: cohort 1 with 296 pts randomized to PAL+Exemestane (EXE) vs CAP and cohort 2 with 305 pts randomized to PAL+ Fulvestran (FUL) vs CAP. ESR1 hotspot mutations were analyzed in circulating free DNA at baseline by digital PCR. Cox regresion analysis for PFS and OS were performed. Adjusted hazard ratios (aHR) and interaction test between treatment and ESR1 status were calculated. Results: ESR1 mutational status was assessed in 557 pts, 91% and 94% treated with CAP and PAL+ET, respectively; 164 (29%) had ESR1 mutations. Characteristics between ESR1 wild-type (WT) and mutated pts were balanced except for ECOG, prior sensitivity to ET, treatment line, prior AI for metastatic disease and time from first metastatic diagnosis to randomization. Median follow-up was 22.5 months (m). Median PFS was 9.6 vs. 7.5 m (HR: 1.06 [0.88 - 1.28], p = 0.522) and median OS was 30.2 vs 30.3 (HR: 0.99 [0.78 - 1.26] p = 0.934), for CAP vs. PAL+ET, respectively. No interaction was seen between treatment arm and ESR1 status either for PFS (p = 0.538) or OS (p = 0.957). Conclusions: In luminal MBC, ESR1-mutated pts had poorer OS than ESR1-WT pts regardless of treatment received. Clinical trial information: NCT02028507 . [Table: see text]

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

Background. Detection of circulating tumor DNA (ctDNA) after treatment of early stage breast cancer may identify molecular residual disease. In a prior proof-of-principle study we demonstrated that detection of ctDNA predicted relapse with high accuracy (Garcia-Murillas et al Science Trans Med 2015). We conducted an independent, prospective, multi-centre validation study. Methods. In this validation study, a cohort of 170 early stage breast cancer patients were recruited from five hospitals into two prospective sample collection studies. Patients were scheduled to receive standard chemotherapy, surgery +/- radiotherapy, adjuvant endocrine therapy and HER2 antibodies as appropriate. Plasma samples were collected for ctDNA analysis at baseline, post-surgery, three monthly for the first year of follow-up, and six monthly thereafter and shipped to a central lab for processing. Using previously established criteria, tumor was sequenced to identify somatic mutations that were tracked by digital PCR in DNA extracted from 4mls of plasma at all available time points. Buffy coat DNA was analysed at all time-points to control for clonal haematopoesis of indeterminate potential (CHIP) detection. The primary endpoint was to compare invasive disease free survival between patients with and without detection of ctDNA after treatment. A combined analysis of this validation study, and the prior proof-of-principle study, was also conducted to analyse secondary endpoints. Results. After tumor sequencing, 101 patients from the validation study had at least one mutation to track. At median 35.5 months follow-up, ctDNA was detected in plasma of 15.8% (16/101) patients. Detection of ctDNA strongly predicted relapse, hazard ratio 24.5 (95% CI 6.5 to 93.2, P In the combined analysis (N=144), lead-time between ctDNA detection and relapse was 10.7 months (95% CI 7.7-17.0). Six patients had a clinical relapse that was not detected by ctDNA prior to relapse. These patients had a distinct pattern of oligo-metastatic relapse, 3 patients with brain-only metastases (P=0.0068), 1 ovarian oligo-metastasis and 2 local disease recurrence. The level of ctDNA in baseline plasma, prior to treatment, was associated with tumor subtype, highest in triple negative breast cancer (P=0.0036). Conclusion. Detection of ctDNA after treatment is associated with a high risk of future relapse in early-stage breast cancer. Prospective studies are required to assess the potential of molecular residual disease detection to guide adjuvant therapy. Citation Format: Turner N, Garcia-Murillas I, Chopra N, Comino-Mendez I, Beaney M, Tovey H, Cutts R, Swift C, Kriplani D, Afentakis M, Hrebien S, Walsh G, Johnston S, Ring A, Russell S, Evans A, Skene A, Wheatley D, Dowsett M, Smith I. Molecular residual disease detection with circulating tumor DNA analysis predicts relapse in patients with early stage breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-01.

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.

This abstract was withdrawn by the authors.