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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.

BACKGROUND: Circulating tumour DNA (ctDNA) testing might provide a current assessment of the genomic profile of advanced cancer, without the need to repeat tumour biopsy. We aimed to assess the accuracy of ctDNA testing in advanced breast cancer and the ability of ctDNA testing to select patients for mutation-directed therapy.METHODS: We did an open-label, multicohort, phase 2a, platform trial of ctDNA testing in 18 UK hospitals. Participants were women (aged ≥18 years) with histologically confirmed advanced breast cancer and an Eastern Cooperative Oncology Group performance status 0–2. Patients had completed at least one previous line of treatment for advanced breast cancer or relapsed within 12 months of neoadjuvant or adjuvant chemotherapy. Patients were recruited into four parallel treatment cohorts matched to mutations identified in ctDNA: cohort A comprised patients with ESR1 mutations (treated with intramuscular extended-dose fulvestrant 500 mg); cohort B comprised patients with HER2 mutations (treated with oral neratinib 240 mg, and if oestrogen receptor-positive with intramuscular standard-dose fulvestrant); cohort C comprised patients with AKT1 mutations and oestrogen receptor-positive cancer (treated with oral capivasertib 400 mg plus intramuscular standard-dose fulvestrant); and cohort D comprised patients with AKT1 mutations and oestrogen receptor-negative cancer or PTEN mutation (treated with oral capivasertib 480 mg). Each cohort had a primary endpoint of confirmed objective response rate. For cohort A, 13 or more responses among 78 evaluable patients were required to infer activity and three or more among 16 were required for cohorts B, C, and D. Recruitment to all cohorts is complete and long-term follow-up is ongoing. This trial is registered with ClinicalTrials.gov, NCT03182634; the European Clinical Trials database, EudraCT2015-003735-36; and the ISRCTN registry, ISRCTN16945804.FINDINGS: Between Dec 21, 2016, and April 26, 2019, 1051 patients registered for the study, with ctDNA results available for 1034 patients. Agreement between ctDNA digital PCR and targeted sequencing was 96–99% (n=800, kappa 0·89–0·93). Sensitivity of digital PCR ctDNA testing for mutations identified in tissue sequencing was 93% (95% CI 83–98) overall and 98% (87–100) with contemporaneous biopsies. In all cohorts, combined median follow-up was 14·4 months (IQR 7·0–23·7). Cohorts B and C met or exceeded the target number of responses, with five (25% [95% CI 9–49]) of 20 patients in cohort B and four (22% [6–48]) of 18 patients in cohort C having a response. Cohorts A and D did not reach the target number of responses, with six (8% [95% CI 3–17]) of 74 in cohort A and two (11% [1–33]) of 19 patients in cohort D having a response. The most common grade 3–4 adverse events were raised gamma-glutamyltransferase (13 [16%] of 80 patients; cohort A); diarrhoea (four [25%] of 20; cohort B); fatigue (four [22%] of 18; cohort C); and rash (five [26%] of 19; cohort D). 17 serious adverse reactions occurred in 11 patients, and there was one treatment-related death caused by grade 4 dyspnoea (in cohort C).INTERPRETATION: ctDNA testing offers accurate, rapid genotyping that enables the selection of mutation-directed therapies for patients with breast cancer, with sufficient clinical validity for adoption into routine clinical practice. Our results demonstrate clinically relevant activity of targeted therapies against rare HER2 and AKT1 mutations, confirming these mutations could be targetable for breast cancer treatment.FUNDING: Cancer Research UK, AstraZeneca, and Puma Biotechnology.

Background: Circulating tumour DNA (ctDNA) testing may provide a more current assessment of the genetic profile of advanced breast cancer (BC) compared with analysis of the primary tumour, with repeat advanced disease biopsy conducted infrequently in routine clinical practice. The plasmaMATCH trial was designed to assess the clinical utility of using ctDNA testing to select patients for targeted therapies. Methods: The plasmaMATCH trial was an open-label, multi-centre, multi-cohort platform trial, consisting of ctDNA testing in ~1000 patients with advanced BC, with patients recruited into four parallel treatment cohorts with therapies matched to mutations identified in ctDNA (A: ESR1 mutation - extended-dose fulvestrant 500mg every 2 weeks, B: HER2 mutation - neratinib +/- fulvestrant (standard dosing), C: AKT1 in ER positive BC -capivasertib + fulvestrant (standard dosing), D: AKT1 in ER negative BC or PTEN inactivating mutation - capivasertib). A fifth cohort (E) recruited patients with triple negative BC with no actionable mutation to receive olaparib + AZD6738, and will be reported separately. Each cohort had a specific phase II single arm design. ctDNA testing was conducted with two technologies: digital droplet PCR (ddPCR) at a central laboratory prospectively in all patients, and error corrected sequencing with Guardant360 prospectively from part-way through recruitment and retrospectively for the remaining patients. Tumour sequencing from an advanced disease biopsy was conducted retrospectively, not influencing cohort entry. The primary endpoint for Cohorts A-D is confirmed objective response rate by RECIST v1.1. Secondary endpoints include clinical benefit rate, progression-free survival, safety and frequency of mutations identified in ctDNA screening. Results: Entry into ctDNA testing for Cohorts A-D was closed on 26/Apr/2019 with 1044 patients registered. ctDNA screening results were received for 1033 patients (99%), with 142 patients entered into Cohorts A-D (A 84, B 21, C 18, D 19). Agreement between ctDNA digital PCR and sequencing results was high (individual gene level agreement 95.5%-99.4%, kappa 0.89-0.93). Predefined efficacy criteria were met in Cohorts B (neratinib for HER2 mutations) and C (capivasertib for AKT mutations), with exploratory analysis of Cohort D identifying activity of capivasertib in AKT1 mutations (Table 1). Efficacy criteria were not met in Cohort A (extended-dose fulvestrant for ESR1 mutations). Adverse events were consistent with prior reports, with extended-dose fulvestrant well tolerated. Table 1: Efficacy results from plasmaMATCHMutationCohortConfirmed response rate, % (95%CI; n/N)Median PFS (IQR), monthsAll patientsFirst 16 evaluable patients*ESR1A8.1% (3.0-16.8; 6/74)-2.2 (1.7-5.3)HER2B25.0% (8.7-49.1; 5/20)25.0% (7.3-52.4; 4/16)5.4 (3.4-9.1)AKT1C22.2% (6.4-47.6; 4/18)18.8% (4.0-45.6; 3/16)10.2 (3.2-18.2)AKT basketD10.5% (1.3-33.1; 2/19)12.5% (1.6-38.3; 2/16)3.4 (1.8-5.5)AKT133.3% (4.3-77.7; 2/6)--PTEN0 % (0/13)--*Predefined cohort efficacy thresholds for response were set: 13/78 (A); 3/16 (B, C, D) Conclusion: Circulating tumour DNA testing offers accurate tumour genotyping, sufficient for routine clinical practice. This approach can be used to identify patients with rare HER2 and AKT1 mutations, who have clinically relevant response rates with matched targeted therapies. Citation Format: Nicholas Turner, Belinda Kingston, Lucy Kilburn, Sarah Kernaghan, Andrew M Wardley, Iain Macpherson, Richard D Baird, Rebecca Roylance, Peter Stephens, Olga Oikonomidou, Jeremy P Braybrooke, Mark Tuthill, Jacinta Abraham, Matthew C Winter, Hannah Bye, Michael Hubank, Claire Snowdon, Daniel Rea, David Cameron, Abeer Shaaban, Katrina Randle, Katie Wilkinson, Laura Moretti, Judith M Bliss, Alistair Ring, on behalf of the plasmaMATCH Trial Management Group. Results from the plasmaMATCH trial: A multiple parallel cohort, multi-centre clinical trial of circulating tumour DNA testing to direct targeted therapies in patients with advanced breast cancer (CRUK/15/010) [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 GS3-06.

1024 Background: The plasmaMATCH trial was an open label platform trial, consisting of circulating tumour DNA (ctDNA) testing in ̃1000 patients with advanced breast cancer (ABC) linked to parallel treatment cohorts with therapies matched to mutations identified in ctDNA. Cohorts A-D have already reported (Turner N et al, Lancet Oncol 2020). Cohort E recruited patients with triple negative breast cancer (TNBC) without a targetable mutation identified at ctDNA screening, treating with olaparib (PARP inhibitor) plus ceralasertib (ATR inhibitor). Methods: Patients with TNBC who had received 1 or 2 lines of chemotherapy for advanced disease or relapsed within 12 months of (neo)adjuvant chemotherapy were eligible. Treatment was olaparib 300mg b.i.d continuously and ceralasertib 160mg qd on days 1–7 on a 28 day cycle, until disease progression. The primary endpoint was confirmed objective response rate by RECIST v1.1. Secondary endpoints included clinical benefit rate, progression-free survival (PFS) and safety. Biomarker analysis included response according to BRCA and somatic DNA repair gene status and ATM loss. Using a two-stage design with a target response rate of 25%, unacceptable response rate of 10%, alpha=2% and power=90%, ≥13 responses out of 69 evaluable stage 2 patients were required to infer efficacy (5/37 stage 1). Results: Between 17/09/18 and 5/10/20 75 patients enrolled in Cohort E of whom 70 were evaluable for response. The median age was 55.6 years. 42 (56%) patients had 1 and 13 (17.3%) had 2 prior line(s) of chemotherapy for metastatic disease. Efficacy is shown in Table. The most common grade ≥3 adverse events were: hypertension 12 (17%) and anaemia 9 (13%). Dose reductions and interruptions occurred in 19 (26.4%) and 34 (47.2%) patients respectively. Conclusions: The response rate to olaparib and ceralasertib did not meet pre-specified criteria for efficacy in the overall evaluable population. Responses were observed in patients without germline or somatic BRCA1/2 mutations. Translational analyses are underway to identify potential biomarkers of response in this population and will be presented at the meeting. Clinical trial information: ISRCTN16945804. [Table: see text]

Background: Circulating tumor DNA (ctDNA) testing may provide a contemporary assessment of the genomic profile of advanced cancer, without need to repeat tumor biopsy. The plasmaMATCH trial was designed to assess the clinical utility of using ctDNA testing to select advanced breast cancer patients for targeted treatment. Methods: plasmaMATCH is an open label, multi-center, multi-cohort platform trial of ctDNA testing in ~1000 patients with advanced breast cancer, with patients recruited into four parallel treatment cohorts matched to mutations identified in ctDNA: A:ESR1 mutation, B:HER2 mutation, C:AKT1 mutation (ER-positive cancer), D:AKT activation basket (AKT1 mutation ER-negative cancer or PTEN mutation). ctDNA testing was conducted with digital PCR and error-corrected targeted sequencing. Tumour sequencing from an advanced disease biopsy was conducted retrospectively, not influencing cohort entry. Each cohort had a phase II single-arm design with primary endpoint of confirmed objective response rate using RECISTv1.1 in evaluable patients. Findings: 1051 patients registered, with ctDNA testing results available for 1034 patients. Agreement between ctDNA digital PCR and targeted sequencing was 95.5%-99.4% (n=800, kappa 0.89-0.93). ctDNA testing had 93-98% sensitivity for mutations identified in tissue sequencing. In patients with HER2 mutations, response rate with neratinib, plus fulvestrant in ER-positive cancer, was 25.0% (5/20, 95%CI 8.7-49.1%). In patients with AKT1 mutations and ER-positive cancer, response rate with capivasertib plus fulvestrant was 22.2% (4/18, 95%CI 6.4-47.6%). In patients with AKT1 mutations and ER-negative cancer, 33.3% (2/6, 95%CI,4.3-77.7) responded to capivasertib, although 0/13 patients with PTEN mutation responded. Response rate of ESR1 mutations with extended-dose fulvestrant was 8.1% (6/74, 95%CI 3.0-16.8%). Adverse events were consistent with prior reports. Interpretation: ctDNA testing offers accurate, rapid genotyping that enables the selection of patients for mutation-directed therapies. Our results demonstrate clinically relevant activity of targeted therapies against rare HER2 and AKT1 mutations. Trial Registration: ISRCTN16945804 ClinicalTrials.gov Identifier: NCT03182634 Funding: Cancer Research UK (CRUK/15/010,C30746/A19505), AstraZeneca, and Puma Biotechnology. Declaration of Interests: NCT, AR, JMB, LSK, CS, LM, SK, KW, SM, HB, MH, BK and RC report grants from Cancer Research UK, grants and non-financial support in the form of study drug provision from AstraZeneca and Puma Biotechnology and non-financial support in the form of ctDNA sequencing from Guardant Health and provision of reagents from BioRad during the conduct of the study. NCT also reports grants and personal fees from AstraZeneca, Pfizer, and Roche/Genentech, personal fees from Bristol-Myers Squibb, Lilly, MSD, Novartis, Bicycle Theraputics, Taiiho, Zeno Pharmaceuticals and Repare Therapeutics and grants from BioRad, Clovis, Merck Sharpe and Dohme, and Guardant Health outside the submitted work. BK also reports personal fees from Guardant Health outside the submitted work. AMW reports personal fees from Roche, personal fees and other support from Novartis, Pfizer, Lilly, Daiichi-Sankyo, MSD, AstraZeneca, Athenex and other support from Seattle Genetics, Andrew Wardley Ltd, Manchester Cancer Academy and Outreach Research and Innovation Group Limited outside the submitted work. IRM reports personal fees and non-financial support from Roche Products UK Ltd, Eli Lilly and Eisai and personal fees from Novartis, Pfizer, Daichi Sankyo, Genomic Health, Pierre Fabre and MSD outside the submitted work. RDB reports grants from AstraZeneca and Roche/Genentech outside the submitted work. RR reports personal fees from Novartis, Eli-Lilly and Pfizer, personal fees and non-financial support from Daiichi Sankyo and G1Therapeutics and non-financial support from Roche and AstraZeneca outside the submitted work. PS reports personal fees from Novartis, Eisai and Daiichi Sankyo outside the submitted work.OO reports grants and personal fees from Pfizer and Eisai, personal fees from Roche/GNE and Tesaro, non-financial support from AstraZeneca, personal fees and non-financial support from Eli Lilly, grants from Novartis outside the submitted work. MT reports personal fees from Pfizer, Novartis, Roche, Vaccitech, Oxford Vacmedix, Lilly, Astellas, Genomic Health and Esai, personal fees and non-financial support from Janssen, BMS, Ipsen and non-financial support from EUSA Pharma outside the submitted work. JA reports grant and personal fees from Eisai and personal fees from Merck outside the submitted work. MCW reports personal fees and non-financial support from Easai, Lilly and Roche and personal fees from Pfizer, Genomic Health and Novartis outside the submitted work. HB also reports personal fees from AstraZeneca outside of the submitted work. MH also reports personal fees from Bristol Myers Squibb, Boehringer Ingelheim, Roche Diagnostics and Eli Lilly during the conduct of the study. AS reports grants from Ventana Roche and Genomic Heath, personal fees from Daiichi Sankyo, Hologic, Genomic Health and Ventana Roche outside the submitted work. JMB also reports grants and non-financial support from AstraZeneca, Novartis, Janssen-Cilag, Merck Sharpe & Dohme, Pfizer, Roche, and Clovis Oncology and grants from Medivation outside the submitted work. AR also reports personal fees from Roche Products Limited, Pfizer, Novartis, Lilly and MSD outside the submitted work. JPB, HG, DR, DC and KR have nothing to disclose. Ethics Approval Statement: The study was co-sponsored by The Institute of Cancer Research and the Royal Marsden NHS Foundation Trust and approved by a Research Ethics Committee (16/SC/0271). All participants gave written informed consent prior to registration for ctDNA testing, and again prior to treatment cohort entry. Safety and efficacy data were reviewed regularly by an Independent Data Monitoring Committee (IDMC). Trial oversight was provided by an independent Trial Steering Committee.

An efficient Pd-catalyzed cyclization of o-picolylbromoarenes into pyridoindoles has been developed. This novel transformation, which proceeds through a not common for Pd catalysis L-to X-type pyridine ligand swap, provides an efficient route to aryl-, as well as to cyanopyrido[1,2-a]indoles, not easily accessible by existing cyclization methods.