PDF file 32K, Supplementary Table S1 Antibody reagent list used in study

PDF file 54K, Supplementary Table S5. Tumor-specific CDK12 mutations described in publically available sequencing data

PDF file 58K, Supplementary Table S4. DNA repair involvement of PARP inhibitor sensitisation genes found in the screen. PMID numbers refer to pubmed entries

Supplementary Figure 1. Single dose ceralasertib PK. PK from patients in cohorts 1 to 4 was in line with the PK model predictions (shadowed area) built with data from other studies. Supplementary Figure 2. ATM protein expression by IHC in patients with melanoma. Supplementary Figure 3. ctDNA clearance of mutant alleles from a responder to ceralasertib+paclitaxel; example 1. ctDNA was sequenced at baseline, day 61 and day 403. Re-staging scans were performed at day 63, day 117, intermediate days at approximate 8 week intervals (not shown) and day 426 (at the time of PD). The stream plot shows rapid clearance of the mutant alleles in line with RECIST v1.1 response. Supplementary Figure 4. CtDNA clearance of mutant alleles from a responder to ceralasertib+paclitaxel; example 2. ctDNA was sequenced at baseline, day 24, day 52 and day 241. Re-staging scans were performed at day 46, day 98, intermediate days at approximate 8 week intervals (not shown) and day 235. The stream plot shows rapid clearance of the mutant alleles prior to the day of the first re-staging scan which showed a RECIST v1.1 response.

PDF file 120K, Supplementary figure legends and supplementary materials and methods

Supplementary Table 1 - PDF file 57K, Supplementary Table 1. Details of platinum sensitivity and best response to olaparib and post-PARPi chemotherapy of the six patients whose tumor samples were analyzed by massively parallel sequencing. (NE: not evaluable)

PDF file 3292K, Supplementary Figure S1. Genome wide shRNA screen information. Supplementary Figure S2. Validation of ATAD5 as a genetic determinant of olaparib response. Supplementary Figure S3. A working model of PARP1/2 inhibitor-induced DNA repair. Supplementary Figure S4. Inhibition of CDK12 sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S5. CDK12 silencing and the impact on expression of DNA repair proteins. Supplementary Figure S6. Targeting of CCNK sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S7. Animal body weights from in vivo study

PDF file 543K, Supplementary Table S2 shRNA constructs with Olaparib Drug Effect Z scores <-1.96

Supplementary Table 1. Treatment Outcomes for individual trial participants and dose cohort (N=57)

Durvalumab is a programmed death-ligand 1 (PD-L1) inhibitor with clinical activity in advanced urothelial cancer (AUC)1. AUC is characterized by several recurrent targetable genomic alterations2–5. This study ( NCT02546661 , BISCAY) combined durvalumab with relevant targeted therapies in biomarker-selected chemotherapy-refractory AUC populations including: (1) fibroblast growth factor receptor (FGFR) inhibitors in tumors with FGFR DNA alterations (FGFRm); (2) pharmacological inhibitor of the enzyme poly-ADP ribose polymerase (PARP) in tumors with and without DNA homologous recombination repair deficiency (HRRm); and (3) TORC1/2 inhibitors in tumors with DNA alteration to the mTOR/PI3K pathway3–5.This trial adopted a new, biomarker-driven, multiarm adaptive design. Safety, efficacy and relevant biomarkers were evaluated. Overall, 391 patients were screened of whom 135 were allocated to one of six study arms. Response rates (RRs) ranged 9–36% across the study arms, which did not meet efficacy criteria for further development. Overall survival (OS) and progression-free survival (PFS) were similar in the combination arms and durvalumab monotherapy arm. Biomarker analysis showed a correlation between circulating plasma-based DNA (ctDNA) and tissue for FGFRm. Sequential circulating tumor DNA analysis showed that changes to FGFRm correlated with clinical outcome. Our data support the clinical activity of FGFR inhibition and durvalumab monotherapy but do not show increased activity for any of the combinations. These findings question the targeted/immune therapy approach in AUC. The adaptive, biomarker-driven BISCAY trial evaluating durvalumab with targeted agents in patients with metastatic urothelial carcinoma based on tumor genomic alterations finds no added clinical benefit over durvalumab monotherapy.

AbstractPurpose: Ceralasertib is a potent and selective oral inhibitor of the serine/threonine protein kinase ataxia telangiectasia and Rad3-related (ATR) protein. Patients and Methods: Eligible patients with solid tumors, enriched for melanoma, received ceralasertib in combination with a fixed dose of paclitaxel (80 mg/m2 on D1, D8, D15) in 28-day cycles. The dose of ceralasertib was escalated to reach an MTD in a rolling 6 design. The starting dose of ceralasertib was 40 mg QD. Fifty-seven patients (33 patients with melanoma who failed prior PD1/L1 treatment) were enrolled in 7 dose cohorts ranging from 40 mg QD to 240 mg BD plus weekly paclitaxel. Results: The RP2D was established as ceralasertib 240 mg BD days 1–14 plus paclitaxel 80 mg/m2 on D1, D8, D15 every 28 days. The most common toxicities were neutropenia (n = 39, 68%), anemia (n = 25, 44%), and thrombocytopenia (n = 21, 37%). In the full analysis set of 57 patients, the overall response rate (ORR) was 22.6% (95% CI, 12.5–35.3). In 33 patients with melanoma, resistant to prior anti-PD1 therapy, the ORR was 33.3% (95% CI, 18.0–51.8). In the melanoma subset, the mPFS was 3.6 months (95% CI, 2.0–5.8), the median duration of response was 9.9 months (95% CI, 3.7–23.2), and the mOS was 7.4 months (95% CI, 5.7–11.9). Conclusions: Ceralasertib in combination with paclitaxel was well tolerated in patients with advanced malignancies and showed evidence of antitumor activity. Durable responses were observed in patients with advanced cutaneous, acral, and mucosal melanoma resistant to anti-PD1/L1 treatment. See related commentary by Ashworth, p. 4667

Background: Phase III randomized trial data have confirmed the activity for olaparib in homologous recombination repair (HRR) mutated metastatic castration-resistant prostate cancer (mCRPC) post next-generation hormonal agent (NHA) progression. Preclinical data have suggested the potential for a combined effect between olaparib and NHAs irrespective of whether an HRR gene alteration was present. NCT01972217 was a randomised double-blind Phase II study which evaluated olaparib and abiraterone versus placebo and abiraterone in mCRPC patients who had received prior chemotherapy containing docetaxel. The study showed that radiologic progression was significantly delayed by the combination of olaparib and abiraterone regardless of homologous recombination repair mutation (HRRm) status. The study utilized tumour, blood (germline), and circulating tumour DNA (ctDNA) analysis to profile patient HRRm status, but tumour tissue provision was not mandated, leading to relatively low tissue acquisition and DNA sequencing success rates not representative of real-world testing. Patients and methods: Further analysis of germline and ctDNA samples has been performed for the trial to characterize HRRm status more fully and robustly analyse patient response to treatment. Results: Germline and plasma testing increased the HRRm characterized population from 27% to 68% of 142 randomized patients. Tumour-derived variants were detectable with high confidence in 78% of patients with a baseline plasma sample (71% of randomized patients). There was high concordance across methodologies (plasma vs. tumour; plasma vs. germline). The HR for the exploratory analysis of radiographic progression-free survival was 0.54 (95% CI: 0.32–0.93) in favour of olaparib and abiraterone in the updated HRR wild type (HRRwt) group (n = 73) and 0.62 (95% CI: 0.23–1.65) in the HRRm group (n = 23). Conclusion: Our results confirm the value of plasma testing for HRRm status when there is insufficient high-quality tissue for multi-gene molecular testing. We show that patients with mCRPC benefit from the combination of olaparib and abiraterone treatment regardless of HRRm status. The combination is currently being further investigated in the Phase III PROpel trial.

3503 Background: Ataxia Telangiectasia and Rad3 Related (ATR) is an apical kinase with a critical role in the DNA-damage response. During normal DNA replication, ATR is recruited at stalled replication forks which can progress to double strand breaks if left unrepaired. AZD6738 is an oral inhibitor of the serine/threonine protein kinase ATR, a member of the phosphoinositide 3-kinase related kinase (PIKK) family. Methods: Eligible patients (pts) with advanced solid tumours were administered AZD6738 in combination with fixed dose paclitaxel 80 mg/m2 D1, D8, D15 in 28-day cycles. The dose of AZD6738 was escalated to reach a maximum tolerated dose (MTD) in a rolling 6 design. The trial evaluated safety, MTD, pharmacokinetics (PK) and pharmacodynamics (PD). Translational studies on plasma samples included cytokine analysis, panel sequencing of ctDNA, as well as IHC and immunofluorescence of immune cell markers. Results: 58 pts (34 melanoma, 15 gastric cancer (GC), 4 sarcoma, 3 colon cancer, 1 neuroendocrine and 1 hepatocellular cancer) were enrolled in 7 dose cohorts ranging 40mg OD to 240 mg BID. One dose-limiting toxicity (DLT) of neutropenic fever occurred in each cohort of n = 6 evaluable pts at AZD6738 160 mg BD and 240 mg BD days 1-14. Per protocol, the maximum tolerated dose of AZD6738 is 240 mg BID days 1-14. The most common toxicities (all causality, all grade) were: anorexia/nausea (n = 15, 26%), leukopenia (n = 11, 19%) and anemia (n = 11, 19%). 51 pts are evaluable for efficacy; we observed 1 complete response (1.9 %, melanoma), 12 confirmed partial responses (23.5%; 2 gastric, 10 melanoma all of which were post-immunotherapy), 18 stable disease (35.3%) and 20 disease progression (39.2%). The overall confirmed response rate from the dose escalation is 25.5%. Genomic analysis of baseline plasma (27 pts) revealed enrichment of NF1 somatic mutations and activating NRAS mutations amongst melanoma pts (6/18 and 4/18, respectively). Cyclical changes in interleukin-12 levels were observed in three pts with disease control which could reflect an immunological component to the mechanism of response. We will present a comprehensive case report of a patient with dramatic and durable response. Conclusions: We conclude that AZD6738 can be safely combined with weekly paclitaxel and propose a recommended phase II dose and schedule. The combination of AZD6738 and paclitaxel demonstrated promising anti-tumor activity with durable responses, especially in melanoma pts after failing anti-PD1 therapy. Clinical trial information: NCT02630199 .

Supplementary Table from Cediranib in Combination with Olaparib in Patients without a Germline BRCA1/2 Mutation and with Recurrent Platinum-Resistant Ovarian Cancer: Phase IIb CONCERTO Trial

BACKGROUND:Poly (ADP-ribose) polymerase inhibitors combined with immunotherapy have shown antitumour activity in preclinical studies. We aimed to assess the safety and activity of olaparib in combination with the PD-L1-inhibitor, durvalumab, in patients with germline BRCA1-mutated or BRCA2-mutated metastatic breast cancer. METHODS:The MEDIOLA trial is a multicentre, open-label, phase 1/2, basket trial of durvalumab and olaparib in solid tumours. Patients were enrolled into four initial cohorts: germline BRCA-mutated, metastatic breast cancer; germline BRCA-mutated, metastatic ovarian cancer; metastatic gastric cancer; and relapsed small-cell lung cancer. Here, we report on the cohort of patients with breast cancer. Patients who were aged 18 years or older (or aged 19 years or older in South Korea) with germline BRCA1-mutated or BRCA2-mutated or both and histologically confirmed, progressive, HER2-negative, metastatic breast cancer were enrolled from 14 health centres in the UK, the USA, Israel, France, Switzerland, and South Korea. Patients should not have received more than two previous lines of chemotherapy for metastatic breast cancer. Patients received 300 mg olaparib in tablet form orally twice daily for 4 weeks and thereafter a combination of olaparib 300 mg twice daily and durvalumab 1·5 g via intravenous infusion every 4 weeks until disease progression. Primary endpoints were safety and tolerability, and 12-week disease control rate. Safety was analysed in patients who received at least one dose of study treatment, and activity analyses were done in the full-analysis set (patients who received at least one dose of study treatment and were not excluded from the study). Recruitment has completed and the study is ongoing. This trial is registered with ClinicalTrials.gov, NCT02734004. FINDINGS:Between June 14, 2016, and May 2, 2017, 34 patients were enrolled and received both study drugs and were included in the safety analysis. 11 (32%) patients experienced grade 3 or worse adverse events, of which the most common were anaemia (four [12%]), neutropenia (three [9%]), and pancreatitis (two [6%]). Three (9%) patients discontinued due to adverse events and four (12%) patients experienced a total of six serious adverse events. There were no treatment-related deaths. 24 (80%; 90% CI 64·3-90·9) of 30 patients eligible for activity analysis had disease control at 12 weeks. INTERPRETATION:Combination of olaparib and durvalumab showed promising antitumour activity and safety similar to that previously observed in olaparib and durvalumab monotherapy studies. Further research in a randomised setting is needed to determine predictors of therapeutic benefit and whether addition of durvalumab improves long-term clinical outcomes compared with olaparib monotherapy. FUNDING:AstraZeneca.

Cowden syndrome is an autosomal dominant genetic disease with an estimated incidence of one in 200,000. Affected individuals develop multiple systemic hamartomas and have a cumulative lifetime risk of breast cancer of 85%. Approximately 80% of patients with Cowden syndrome have a germline inactivating mutation in PTEN (10q23.3). PTEN acts as a tumor suppressor gene via numerous mechanisms, one of which is antagonizing the PI3K/AKT/mTOR signaling pathway by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 functions as a secondary messenger in the PI3K pathway that binds and activates proteins that have a pleckstrin homology domain, such as AKT1, and triggers their activation and localization to the plasma membrane, promoting cellular proliferation and survival.

The VIKTORY (targeted agent eValuation In gastric cancer basket KORea) trial was designed to classify patients with metastatic gastric cancer based on clinical sequencing and focused on eight different biomarker groups (RAS aberration, TP53 mutation, PIK3CA mutation/amplification, MET amplification, MET overexpression, all negative, TSC2 deficient, or RICTOR amplification) to assign patients to one of the 10 associated clinical trials in second-line (2L) treatment. Capivasertib (AKT inhibitor), savolitinib (MET inhibitor), selumetinib (MEK inhibitor), adavosertib (WEE1 inhibitor), and vistusertib (TORC inhibitor) were tested with or without chemotherapy. Seven hundred seventy-two patients with gastric cancer were enrolled, and sequencing was successfully achieved in 715 patients (92.6%). When molecular screening was linked to seamless immediate access to parallel matched trials, 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional 2L chemotherapy. Circulating tumor (ctDNA) analysis demonstrated good correlation between high MET copy number by ctDNA and response to savolitinib. SIGNIFICANCE: Prospective clinical sequencing revealed that baseline heterogeneity between tumor samples from different patients affected response to biomarker-selected therapies. VIKTORY is the first and largest platform study in gastric cancer and supports both the feasibility of tumor profiling and its clinical utility.This article is highlighted in the In This Issue feature, p. 1325.

Background Dynamic changes in circulating tumour DNA (ctDNA) levels may predict long-term outcome. We utilised samples from a phase I/II randomised trial (BEECH) to assess ctDNA dynamics as a surrogate for progression-free survival (PFS) and early predictor of drug efficacy. Patients and methods Patients with estrogen receptor-positive advanced metastatic breast cancer (ER+ mBC) in the BEECH study, paclitaxel plus placebo versus paclitaxel plus AKT inhibitor capivasertib, had plasma samples collected for ctDNA analysis at baseline and at multiple time points in the development cohort (safety run-in, part A) and validation cohort (randomised, part B). Baseline sample ctDNA sequencing identified mutations for longitudinal analysis and mutation-specific digital droplet PCR (ddPCR) assays were utilised to assess change in ctDNA abundance (allele fraction) between baseline and 872 on-treatment samples. Primary objective was to assess whether early suppression of ctDNA, based on pre-defined criteria from the development cohort, independently predicted outcome in the validation cohort. Results In the development cohort, suppression of ctDNA was apparent after 8days of treatment (P=0.014), with cycle 2 day 1 (4weeks) identified as the optimal time point to predict PFS from early ctDNA dynamics. In the validation cohort, median PFS was 11.1months in patients with suppressed ctDNA at 4weeks and 6.4months in patients with high ctDNA (hazard ratio=0.20, 95% confidence interval 0.083-0.50, P<0.0001). There was no difference in the level of ctDNA suppression between patients randomised to capivasertib or placebo overall (P=0.904) nor in the PIK3CA mutant subpopulation (P=0.071). Clonal haematopoiesis of indeterminate potential (CHIP) was evident in 30% (18/59) baseline samples, although CHIP had no effect on tolerance of chemotherapy nor on PFS. Conclusion Early on-treatment ctDNA dynamics are a surrogate for PFS. Dynamic ctDNA assessment has the potential to substantially enhance early drug development. Clinical registration number NCT01625286.

4553 Background: BISCAY is a biomarker-directed Ph1b multi-arm platform study exploring the combination of targeted therapies with anti-PD-L1, Durvalumab, in advanced urothelial cancer. Methods: Next generation sequencing (NGS) of tumour tissue samples from > 380 patients(pts) was performed using the FoundationOne assay alongside IHC for PD-L1. ct DNA from pts enrolled in trial modules at treatment initiation was profiled using the Guardant Health OMNI platform assessing a panel of 500 genes. For a subset of pts, serial plasma samples were also analysed to monitor early signs of response vs. resistance and changes in ct DNA dynamics using a bespoke NGS panel of 10 genes. Results: To date 149 pts have been actively enrolled across 7 different biomarker selected and unselected treatment modules. Across all screened pts the most prevalent genomic alterations in tumour tissue were TERT promoter (65%), TP53 (59%), KMT2D 21%, KDM6A 21%, with the most common CNV CDKN2A/ B loss (32 %). All enrolled pts tested had detectable ctDNA in plasma. Similar genomic alterations, both frequency and type, were detected in both plasma ctDNA and tumour tissue with high concordance for module specific biomarkers used for patient allocation (80% (8/10) for ATM, BRCA1 and 2). Alterations in putative biomarkers predictive of response to anti-PD-L1, such as HRR/MMR alterations and high bTMB levels ( > 20mut/Mb) were observed in22% and 40% patient plasma samples, respectively. Correlations between biomarkers across modules treatment efficacy have been explored. Conclusions: All pts with advanced bladder cancer enrolled on BISCAY who were plasma profiled had detectable ctDNA; frequencies of genomic alterations (in both tumour tissue and plasma) were comparable to prior published data sets. ctDNA may be an attractive alternative to tissue-based NGS, providing comprehensive dynamic snapshots of genomic landscapes at the start and during therapy, and warrants further prospective investigation in trials.

4061 Background: This trial was a phase II study with selumetinib (AZD6244)/docetaxel as second-line treatment for metastatic GC patients with MEK signature or RAS gene alterations as part of the GC umbrella trial, guided through the molecular screening, the VIKTORY trial (NCT#02299648). Methods: Selumetinib was administered orally 75mg twice a day continuously. Docetaxel was administered as an IV infusion over 1 hour at 60 mg/m2 every 3 week of a 21 days schedule. The primary objective was to investigate response rate (RR) and secondary objectives were to perform pre-planned analysis using ctDNA and tumors to identify an optimal biomarker for selumetinib. Results: Twenty seven patients who were identified through the VIKTORY screening to harbor KRAS mutation, KRAS amplification or MEK signature were enrolled onto the study. Of 27 patients, 12 patients had GC harboring KRAS mutation and two KRAS amplification. The remaining 13 patients were KRAS wild type with either low MEK signature (N = 7) or high MEK signature (N = 6). Only one of 12 KRAS mutant GC had high MEK signature. Of 25 patients who received the study drug, there were 7 partial responses (PRs), 8 stable diseases (SDs), 6 progressive diseases (PDs) and 4 non-evaluable diseases. The overall RR for the study combination was 28.0% (95 CI, 0.12 – 0.49). The most commonly observed any grade adverse events (AEs) were skin rash (7.4%) but no grade 3 or 4 skin rash. The second common AE was diarrhea (5.5%) which was all grade 1/2. In biomarker analysis, the two patients with had typical KRAS mutation (G12) or KRAS amplification with high MEKsignature achieved a PR from selumetinib/docetaxel . However, three with atypical KRAS mutation (i.e. A146P, Q61R, Q61H) with low/intermediate MEK signature showed PD to the combination therapy. Conclusions: Selumetinib plus docetaxel as 2nd line therapy revealed the useful efficacy and tolerable safety in GC patients with MEK signature or RAS gene alterations. Especially, the specific subset of patients with had typical KRAS mutation or KRAS amplification with high MEK signature were likely to benefit from selumetinib/docetaxel. Clinical trial information: NCT02448290.