Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition.

PURPOSE: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody. METHODS: C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. RESULTS: 177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86. CONCLUSION: In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.

Clinical Pharmacokinetics and Pharmacodynamics of Rucaparib.

Rucaparib is an oral small-molecule poly(ADP-ribose) polymerase inhibitor indicated for patients with recurrent ovarian cancer in the maintenance and treatment settings and for patients with metastatic castration-resistant prostate cancer associated with a deleterious BRCA1 or BRCA2 mutation. Rucaparib has a manageable safety profile; the most common adverse events reported were fatigue and nausea in both indications. Accumulation in plasma exposure occurred after repeated administration of the approved 600-mg twice-daily dosage. Steady state was achieved after continuous twice-daily dosing for a week. Rucaparib has moderate oral bioavailability and can be dosed with or without food. Although a high-fat meal weakly increased maximum concentration and area under the curve, the effect was not clinically significant. A mass balance analysis indicated almost a complete dose recovery of rucaparib over 12 days, with metabolism, renal, and hepatic excretion as the elimination routes. A population pharmacokinetic analysis of rucaparib revealed no effect of age, sex, race, or body weight. No starting dose adjustments were necessary for patients with mild-to-moderate hepatic or renal impairment; the effect of severe organ impairment on rucaparib exposure has not been evaluated. In patients, rucaparib moderately inhibited cytochrome P450 (CYP) 1A2 and weakly inhibited CYP3As, CYP2C9, and CYP2C19. Rucaparib weakly increased systemic exposures of oral contraceptives and oral rosuvastatin and marginally increased the exposure of oral digoxin (a P-glycoprotein substrate). In vitro studies suggested that rucaparib inhibits transporters MATE1, MATE2-K, OCT1, and OCT2. No clinically meaningful drug interactions with rucaparib as a perpetrator were observed. An exposure-response analysis revealed dose-dependent changes in selected clinical efficacy and safety endpoints. Overall, this article provides a comprehensive review of the clinical pharmacokinetics, pharmacodynamics, drug-drug interactions, effects of intrinsic and extrinsic factors, and exposure-response relationships of rucaparib.

The Multi-Kinase Inhibitor Lucitanib Enhances the Antitumor Activity of Coinhibitory and Costimulatory Immune Pathway Modulators in Syngeneic Models.

Lucitanib is a multi-tyrosine kinase inhibitor whose targets are associated with angiogenesis and other key cancer and immune pathways. Its antiangiogenic properties are understood, but lucitanib's immunomodulatory activity is heretofore unknown. Lucitanib exhibited such activity in vivo, increasing CD3 + , CD8 + , and CD4 + T cells and decreasing dendritic cells and monocyte-derived suppressor cells in mouse spleens. Depletion of CD8 + T cells from syngeneic MC38 colon tumor-bearing mice reduced the antitumor efficacy of lucitanib and revealed a CD8 + T-cell-dependent component of lucitanib's activity. The combination of lucitanib and costimulatory immune pathway agonists targeting 4-1BB, glucocorticoid-induced TNFR (GITR), inducible T-cell co-stimulator (ICOS), or OX40 exhibited enhanced antitumor activity compared with each single agent in immunocompetent tumor models. Lucitanib combined with blockade of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein-1 (PD-1) coinhibitory immune pathways also showed enhanced antitumor activity over the single agents in multiple models. In CT26 tumors, lucitanib, alone or combined with anti-PD-1, reduced CD31 + vessels and depleted F4/80 + macrophages. Combination treatment also increased the number of intratumoral T cells. Gene expression in pathways associated with immune activity was upregulated by lucitanib in MC38 tumors and further potentiated by combination with anti-PD-1. Accordingly, lucitanib, alone or combined with anti-PD-1, increased intratumoral CD8 + T-cell abundance. Lucitanib's antitumor and pharmacodynamic activity, alone or combined with anti-PD-1, was not recapitulated by specific vascular endothelial growth factor receptor-2 (VEGFR2) inhibition. These data indicate that lucitanib can modulate vascular and immune components of the tumor microenvironment and cooperate with immunotherapy to enhance antitumor efficacy. They support the clinical development of lucitanib combined with immune pathway modulators to treat cancer.

Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy.

PURPOSE: Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. METHODS: FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent. RESULTS: Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. CONCLUSION: In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.

Population pharmacokinetics of rucaparib in patients with advanced ovarian cancer or other solid tumors.

PURPOSE: To develop a population pharmacokinetics (PPK) model for rucaparib, an oral poly(ADP-ribose) polymerase inhibitor. METHODS: The PPK analysis used PK data from patients in Study 1014 (NCT01009190, n = 35), Study 10 (NCT01482715, n = 123), and ARIEL2 (NCT01891344, n = 300), including intensive intravenous data (12-40 mg), intensive and sparse oral data (12-360 mg single-dose, 40-500 mg once daily, and 240-840 mg twice daily [BID]), and intensive single-dose oral data under fasted conditions and after a high-fat meal (40, 300, and 600 mg). RESULTS: Rucaparib PK was well described by a two-compartment model with sequential zero-order release and first-order absorption and first-order elimination. A high-fat meal slightly increased bioavailability at 600 mg but not at lower doses; this is not considered clinically significant, and rucaparib can be taken with or without food. Covariate effects of baseline creatinine clearance and albumin on rucaparib clearance were identified. Despite numerical increases in exposure with renal impairment, no dose adjustment is recommended for patients with mild or moderate renal impairment. No statistically significant relationships were detected for demographics, hepatic function (normal versus mild impairment), CYP1A2 and CYP2D6 phenotypes, or strong CYP1A2 or CYP2D6 inhibitors. Concomitant proton pump inhibitors showed no clinically significant effect on absorption. External validation of the model with data from ARIEL3 (NCT01968213) and TRITON2 (NCT02952534) studies showed no clinically meaningful PK differences across indications or sex. CONCLUSION: The PPK model adequately described rucaparib PK, and none of the covariates evaluated had a clinically relevant effect. CLINICALTRIALS: GOV: Study 1014 (NCT01009190), Study 10 (NCT01482715), ARIEL2 (NCT01891344), ARIEL3 (NCT01968213), and TRITON2 (NCT02952534).

Characterization of a RAD51C-silenced high-grade serous ovarian cancer model during development of PARP inhibitor resistance.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

BET, SRC, and BCL2 family inhibitors are synergistic drug combinations with PARP inhibitors in ovarian cancer.

BACKGROUND: Homologous recombination deficiencies (HRD) are present in approximately half of epithelial ovarian cancers, for which PARP inhibitors (PARPi) are becoming a preferred treatment option. However, a considerable proportion of these carcinomas acquire resistance or harbour de novo resistance, posing a significant challenge to treatment. METHODS: To identify new combinatorial therapeutics to overcome resistance to PARPi, we employed high-throughput conditional RNAi and drug screening of patient-derived ovarian cancer cells. To prioritise clinically relevant drug combinations, we integrated empirical validation with analysis of The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) datasets to nominate candidate targets and drugs, reaching three main findings. FINDINGS: Firstly, we found that the PARPi rucaparib enhanced the effect of BET inhibitors (CPI-203 & CPI-0610) irrespective of clinical subtype or HRD status. Additional drug combination screens identified that dasatinib, a non-receptor tyrosine kinase inhibitor, augmented the effects of rucaparib and BET inhibitors, proposing a potential broadly applicable triple-drug combination for high-grade serous and clear cell ovarian carcinomas. Secondly, rucaparib synergised with the BCL2 family inhibitor navitoclax, with preferential activity in ovarian carcinomas that harbour alterations in BRCA1/2, BARD1, or MSH2/6. Thirdly, we identified potentially antagonistic drug combinations between the PARPi rucaparib and vinca alkaloids, anthracyclines, and antimetabolites, cautioning their use in the clinic. INTERPRETATION: These findings propose therapeutic strategies to address PARP inhibitor resistance using agents that are already approved or are in clinical development, with the potential for rapid translation to benefit a broad population of ovarian cancer patients.

Evaluation of in vitro absorption, distribution, metabolism, and excretion and assessment of drug-drug interaction of rucaparib, an orally potent poly(ADP-ribose) polymerase inhibitor.

1. The absorption, distribution, metabolism, elimination, and drug-drug interaction (DDI) potential of the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib was characterised in vitro.2. Rucaparib showed moderate cellular permeability, moderate human plasma protein binding (70.2%), and slow metabolism in human liver microsomes (HLMs). In HLMs, cytochrome P450 (CYP) 1A2 and CYP3A contributed to the metabolism of rucaparib to its major metabolite M324 with estimated fractions of metabolism catalysed by CYP (fm,CYP) of 0.27 and 0.64, respectively. Rucaparib reversibly inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3As (IC50, 3.55, 12.9, 5.42, 41.6, and 17.2-22.9 µM [2 substrates], respectively), but not CYP2B6 or CYP2C8 (>190 µM). No time-dependent inhibition of any CYP was observed. In cultured human hepatocytes, rucaparib showed concentration-dependent induction of CYP1A2 mRNA and downregulation of CYP3A4 and CYP2B6 mRNA. In transfected cells expressing drug transporters, rucaparib was a substrate for P-gp and BCRP, but not for OATP1B1, OATP1B3, OAT1, OAT3, or OCT2. Rucaparib inhibited P-gp and BCRP (IC50, 169 and 55 µM, respectively) and slightly inhibited OATP1B1, OATP1B3, OAT1, and OAT3 (66%, 58%, 58%, and 42% inhibition, respectively) at 300 µM. Rucaparib inhibited OCT1, OCT2, MATE1, and MATE2-K (IC50, 4.3, 31, 0.63, and 0.19 µM, respectively).3. DDI risk assessment using static models suggested potential CYP-related DDIs, with rucaparib as a perpetrator. Caution is advised when co-administering rucaparib with sensitive substrates of MATEs, OCT1, and OCT2.

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance.

BRCA Reversion Mutations in Circulating Tumor DNA Predict Primary and Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma.

A key resistance mechanism to platinum-based chemotherapies and PARP inhibitors in BRCA-mutant cancers is the acquisition of BRCA reversion mutations that restore protein function. To estimate the prevalence of BRCA reversion mutations in high-grade ovarian carcinoma (HGOC), we performed targeted next-generation sequencing of circulating cell-free DNA (cfDNA) extracted from pretreatment and postprogression plasma in patients with deleterious germline or somatic BRCA mutations treated with the PARP inhibitor rucaparib. BRCA reversion mutations were identified in pretreatment cfDNA from 18% (2/11) of platinum-refractory and 13% (5/38) of platinum-resistant cancers, compared with 2% (1/48) of platinum-sensitive cancers (P = 0.049). Patients without BRCA reversion mutations detected in pretreatment cfDNA had significantly longer rucaparib progression-free survival than those with reversion mutations (median, 9.0 vs. 1.8 months; HR, 0.12; P < 0.0001). To study acquired resistance, we sequenced 78 postprogression cfDNA, identifying eight additional patients with BRCA reversion mutations not found in pretreatment cfDNA. SIGNIFICANCE: BRCA reversion mutations are detected in cfDNA from platinum-resistant or platinum-refractory HGOC and are associated with decreased clinical benefit from rucaparib treatment. Sequencing of cfDNA can detect multiple BRCA reversion mutations, highlighting the ability to capture multiclonal heterogeneity.This article is highlighted in the In This Issue feature, p. 151.

Cell-Free DNA Next-Generation Sequencing Prediction of Response and Resistance to Third-Generation EGFR Inhibitor.

INTRODUCTION: The genomic alterations driving resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs) are not well established, and collecting tissue biopsy samples poses potential complications from invasive procedures. Cell-free circulating DNA (cfDNA) testing provides a noninvasive approach to identify potentially targetable mechanisms of resistance. Here we utilized a 70-gene cfDNA next-generation sequencing test to interrogate pretreatment and progression samples from 77 EGFR-mutated non-small cell lung cancer (NSCLC) patients treated with a third-generation EGFR TKI. PATIENTS AND METHODS: Rociletinib was evaluated in advanced or metastatic (second line or higher) disease with EGFR T790M-positive NSCLC in the TIGER-X (NCT01526928) and TIGER-2 (NCT02147990) studies. Plasma samples were collected at baseline and at the time of systemic progression while receiving rociletinib. The critical exons in 70 genes were sequenced in cfDNA isolated from plasma samples to elucidate a comprehensive genomic profile of alterations for each patient. RESULTS: Plasma-based cfDNA analysis identified 93% of the initial EGFR activating and 85% of the EGFR T790M resistance mutations in pretreatment samples with detectable tumor DNA. Profiling of progression samples revealed significant heterogeneity, with different variant types (eg, mutations, amplifications, and fusions) detected in multiple genes (EGFR, MET, RB1) that may be driving resistance in patients. Novel alterations not previously described in association with resistance to third-generation TKIs were also detected, such as an NTRK1 fusion. CONCLUSION: cfDNA next-generation sequencing identified initial EGFR activating and secondary T790M resistance mutations in NSCLC patients with high sensitivity, predicted treatment response equivalent to tissue analysis, and identified multiple novel and established resistance alterations.

Methylation of all BRCA1 copies predicts response to the PARP inhibitor rucaparib in ovarian carcinoma.

Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.

A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance.

Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage.

Genomic loss of heterozygosity and survival in the REAL3 trial.

BACKGROUND: Homologous recombination deficiency (HRD) measured using a genomic signature for loss of heterozygosity (LOH) predicts benefit from rucaparib in ovarian cancer. We hypothesized that some oesophagogastric cancers will have high-LOH which would be prognostic in patients treated with platinum chemotherapy. METHODS: Diagnostic biopsy DNA from patients treated in the REAL3 trial was sequenced using the Foundation Medicine T5 next-generation sequencing (NGS) assay. An algorithm quantified the percentage of interrogable genome with LOH. Multidimensional optimization was performed to identify a cut-off dichotomizing the population into LOH-high and low groups associated with differential survival outcomes. RESULTS: Of 158 available samples, 117 were successfully sequenced; LOH was derived for 74 of these. A cut-off of 21% genomic LOH defined an LOH-high subgroup (n=10, 14% of population) who had median overall survival (OS) of 18.3 months (m) versus 11m for the LOH-low group (HR 0.55 95% CI 0.19-0.97, p= 0.10). Progression free survival (PFS) for LOH-high and LOH-low groups was 10.7m and 7.3m (HR 0.61 (95% CI 0.21 - 1.09, p=0.09). Sensitivity analysis censoring operated patients (n=4), demonstrated OS of 18.3m vs. 10.2m (HR 0.43, 95% CI (0.20-0.92), p=0.02; PFS was 10.5m vs. 7.2m (HR 0.55, (95% CI 0.26-1.17), p=0.09 for LOH-high and LOH-low. CONCLUSION: HRD assessment using an algorithmically derived LOH signature on a standard NGS panel identifies oesophagogastric cancer patients with high LOH who have prolonged survival when treated with platinum chemotherapy. Validation work will determine the signature's predictive value in patients treated with a PARP inhibitor and with platinum chemotherapy.

Rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy (ARIEL3): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Rucaparib, a poly(ADP-ribose) polymerase inhibitor, has anticancer activity in recurrent ovarian carcinoma harbouring a BRCA mutation or high percentage of genome-wide loss of heterozygosity. In this trial we assessed rucaparib versus placebo after response to second-line or later platinum-based chemotherapy in patients with high-grade, recurrent, platinum-sensitive ovarian carcinoma. METHODS: In this randomised, double-blind, placebo-controlled, phase 3 trial, we recruited patients from 87 hospitals and cancer centres across 11 countries. Eligible patients were aged 18 years or older, had a platinum-sensitive, high-grade serous or endometrioid ovarian, primary peritoneal, or fallopian tube carcinoma, had received at least two previous platinum-based chemotherapy regimens, had achieved complete or partial response to their last platinum-based regimen, had a cancer antigen 125 concentration of less than the upper limit of normal, had a performance status of 0-1, and had adequate organ function. Patients were ineligible if they had symptomatic or untreated central nervous system metastases, had received anticancer therapy 14 days or fewer before starting the study, or had received previous treatment with a poly(ADP-ribose) polymerase inhibitor. We randomly allocated patients 2:1 to receive oral rucaparib 600 mg twice daily or placebo in 28 day cycles using a computer-generated sequence (block size of six, stratified by homologous recombination repair gene mutation status, progression-free interval after the penultimate platinum-based regimen, and best response to the most recent platinum-based regimen). Patients, investigators, site staff, assessors, and the funder were masked to assignments. The primary outcome was investigator-assessed progression-free survival evaluated with use of an ordered step-down procedure for three nested cohorts: patients with BRCA mutations (carcinoma associated with deleterious germline or somatic BRCA mutations), patients with homologous recombination deficiencies (BRCA mutant or BRCA wild-type and high loss of heterozygosity), and the intention-to-treat population, assessed at screening and every 12 weeks thereafter. This trial is registered with ClinicalTrials.gov, number NCT01968213; enrolment is complete. FINDINGS: Between April 7, 2014, and July 19, 2016, we randomly allocated 564 patients: 375 (66%) to rucaparib and 189 (34%) to placebo. Median progression-free survival in patients with a BRCA-mutant carcinoma was 16·6 months (95% CI 13·4-22·9; 130 [35%] patients) in the rucaparib group versus 5·4 months (3·4-6·7; 66 [35%] patients) in the placebo group (hazard ratio 0·23 [95% CI 0·16-0·34]; p<0·0001). In patients with a homologous recombination deficient carcinoma (236 [63%] vs 118 [62%]), it was 13·6 months (10·9-16·2) versus 5·4 months (5·1-5·6; 0·32 [0·24-0·42]; p<0·0001). In the intention-to-treat population, it was 10·8 months (8·3-11·4) versus 5·4 months (5·3-5·5; 0·36 [0·30-0·45]; p<0·0001). Treatment-emergent adverse events of grade 3 or higher in the safety population (372 [99%] patients in the rucaparib group vs 189 [100%] in the placebo group) were reported in 209 (56%) patients in the rucaparib group versus 28 (15%) in the placebo group, the most common of which were anaemia or decreased haemoglobin concentration (70 [19%] vs one [1%]) and increased alanine or aspartate aminotransferase concentration (39 [10%] vs none). INTERPRETATION: Across all primary analysis groups, rucaparib significantly improved progression-free survival in patients with platinum-sensitive ovarian cancer who had achieved a response to platinum-based chemotherapy. ARIEL3 provides further evidence that use of a poly(ADP-ribose) polymerase inhibitor in the maintenance treatment setting versus placebo could be considered a new standard of care for women with platinum-sensitive ovarian cancer following a complete or partial response to second-line or later platinum-based chemotherapy. FUNDING: Clovis Oncology.

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma.

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51CIn vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations.Significance: Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. Cancer Discov; 7(9); 984-98. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Quigley et al., p. 999See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.

Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 Part 1): an international, multicentre, open-label, phase 2 trial.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have activity in ovarian carcinomas with homologous recombination deficiency. Along with BRCA1 and BRCA2 (BRCA) mutations genomic loss of heterozygosity (LOH) might also represent homologous recombination deficiency. In ARIEL2, we assessed the ability of tumour genomic LOH, quantified with a next-generation sequencing assay, to predict response to rucaparib, an oral PARP inhibitor. METHODS: ARIEL2 is an international, multicentre, two-part, phase 2, open-label study done at 49 hospitals and cancer centres in Australia, Canada, France, Spain, the UK, and the USA. In ARIEL2 Part 1, patients with recurrent, platinum-sensitive, high-grade ovarian carcinoma were classified into one of three predefined homologous recombination deficiency subgroups on the basis of tumour mutational analysis: BRCA mutant (deleterious germline or somatic), BRCA wild-type and LOH high (LOH high group), or BRCA wild-type and LOH low (LOH low group). We prespecified a cutoff of 14% or more genomic LOH for LOH high. Patients began treatment with oral rucaparib at 600 mg twice per day for continuous 28 day cycles until disease progression or any other reason for discontinuation. The primary endpoint was progression-free survival. All patients treated with at least one dose of rucaparib were included in the safety analyses and all treated patients who were classified were included in the primary endpoint analysis. This trial is registered with ClinicalTrials.gov, number NCT01891344. Enrolment into ARIEL2 Part 1 is complete, although an extension (Part 2) is ongoing. FINDINGS: 256 patients were screened and 206 were enrolled between Oct 30, 2013, and Dec 19, 2014. At the data cutoff date (Jan 18, 2016), 204 patients had received rucaparib, with 28 patients remaining in the study. 192 patients could be classified into one of the three predefined homologous recombination deficiency subgroups: BRCA mutant (n=40), LOH high (n=82), or LOH low (n=70). Tumours from 12 patients were established as BRCA wild-type, but could not be classified for LOH, because of insufficient neoplastic nuclei in the sample. The median duration of treatment for the 204 patients was 5·7 months (IQR 2·8-10·1). 24 patients in the BRCA mutant subgroup, 56 patients in the LOH high subgroup, and 59 patients in the LOH low subgroup had disease progression or died. Median progression-free survival after rucaparib treatment was 12·8 months (95% CI 9·0-14·7) in the BRCA mutant subgroup, 5·7 months (5·3-7·6) in the LOH high subgroup, and 5·2 months (3·6-5·5) in the LOH low subgroup. Progression-free survival was significantly longer in the BRCA mutant (hazard ratio 0·27, 95% CI 0·16-0·44, p<0·0001) and LOH high (0·62, 0·42-0·90, p=0·011) subgroups compared with the LOH low subgroup. The most common grade 3 or worse treatment-emergent adverse events were anaemia or decreased haemoglobin (45 [22%] patients), and elevations in alanine aminotransferase or aspartate aminotransferase (25 [12%]). Common serious adverse events included small intestinal obstruction (10 [5%] of 204 patients), malignant neoplasm progression (10 [5%]), and anaemia (nine [4%]). Three patients died during the study (two because of disease progression and one because of sepsis and disease progression). No treatment-related deaths occurred. INTERPRETATION: In patients with BRCA mutant or BRCA wild-type and LOH high platinum-sensitive ovarian carcinomas treated with rucaparib, progression-free survival was longer than in patients with BRCA wild-type LOH low carcinomas. Our results suggest that assessment of tumour LOH can be used to identify patients with BRCA wild-type platinum-sensitive ovarian cancers who might benefit from rucaparib. These results extend the potential usefulness of PARP inhibitors in the treatment setting beyond BRCA mutant tumours. FUNDING: Clovis Oncology, US Department of Defense Ovarian Cancer Research Program, Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Research Grant, and V Foundation Translational Award.

Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.