Targeting myeloid chemotaxis to reverse prostate cancer therapy resistance.

Inflammation is a hallmark of cancer1. In patients with cancer, peripheral blood myeloid expansion, indicated by a high neutrophil-to-lymphocyte ratio, associates with shorter survival and treatment resistance across malignancies and therapeutic modalities2-5. Whether myeloid inflammation drives progression of prostate cancer in humans remain unclear. Here we show that inhibition of myeloid chemotaxis can reduce tumour-elicited myeloid inflammation and reverse therapy resistance in a subset of patients with metastatic castration-resistant prostate cancer (CRPC). We show that a higher blood neutrophil-to-lymphocyte ratio reflects tumour myeloid infiltration and tumour expression of senescence-associated mRNA species, including those that encode myeloid-chemoattracting CXCR2 ligands. To determine whether myeloid cells fuel resistance to androgen receptor signalling inhibitors, and whether inhibiting CXCR2 to block myeloid chemotaxis reverses this, we conducted an investigator-initiated, proof-of-concept clinical trial of a CXCR2 inhibitor (AZD5069) plus enzalutamide in patients with metastatic CRPC that is resistant to androgen receptor signalling inhibitors. This combination was well tolerated without dose-limiting toxicity and it decreased circulating neutrophil levels, reduced intratumour CD11b+HLA-DRloCD15+CD14- myeloid cell infiltration and imparted durable clinical benefit with biochemical and radiological responses in a subset of patients with metastatic CRPC. This study provides clinical evidence that senescence-associated myeloid inflammation can fuel metastatic CRPC progression and resistance to androgen receptor blockade. Targeting myeloid chemotaxis merits broader evaluation in other cancers.

Development and Validation of a New BAG-1L-Specific Antibody to Quantify BAG-1L Protein Expression in Advanced Prostate Cancer.

BCL-2-associated athanogene-1L (BAG-1L) is a critical co-regulator that binds to and enhances the transactivation function of the androgen receptor, leading to prostate cancer development and progression. Studies investigating the clinical importance of BAG-1L protein expression in advanced prostate cancer have been limited by the paucity of antibodies that specifically recognize the long isoform. In this study, we developed and validated a new BAG-1L-specific antibody using multiple orthogonal methods across several cell lines with and without genomic manipulation of BAG-1L and all BAG-1 isoforms. Following this, we performed exploratory immunohistochemistry to determine BAG-1L protein expression in normal human, matched castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC), unmatched primary and metastatic CRPC, and early breast cancer tissues. We demonstrated higher BAG-1L protein expression in CRPC metastases than in unmatched, untreated, castration-sensitive prostatectomies from men who remained recurrence-free for 5 years. In contrast, BAG-1L protein expression did not change between matched, same patient, CSPC and CRPC biopsies, suggesting that BAG-1L protein expression may be associated with more aggressive biology and the development of castration resistance. Finally, in a cohort of patients who universally developed CRPC, there was no association between BAG-1L protein expression at diagnosis and time to CRPC or overall survival, and no association between BAG-1L protein expression at CRPC biopsy and clinical outcome from androgen receptor targeting therapies or docetaxel chemotherapy. The limitations of this study include the requirement to validate the reproducibility of the assay developed, the potential influence of pre-analytical factors, timing of CRPC biopsies, relatively small patient numbers, and heterogenous therapies on BAG-1L protein expression, and the clinical outcome analyses performed. We describe a new BAG-1L-specific antibody that the research community can further develop to elucidate the biological and clinical significance of BAG-1L protein expression in malignant and nonmalignant diseases.

Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.

BACKGROUND: CH5126766 (also known as VS-6766, and previously named RO5126766), a novel MEK-pan-RAF inhibitor, has shown antitumour activity across various solid tumours; however, its initial development was limited by toxicity. We aimed to investigate the safety and toxicity profile of intermittent dosing schedules of CH5126766, and the antitumour activity of this drug in patients with solid tumours and multiple myeloma harbouring RAS-RAF-MEK pathway mutations. METHODS: We did a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study at the Royal Marsden National Health Service Foundation Trust (London, UK). Patients were eligible for the study if they were aged 18 years or older, had cancers that were refractory to conventional treatment or for which no conventional therapy existed, and if they had a WHO performance status score of 0 or 1. For the dose-escalation phase, eligible patients had histologically or cytologically confirmed advanced or metastatic solid tumours. For the basket dose-expansion phase, eligible patients had advanced or metastatic solid tumours or multiple myeloma harbouring RAS-RAF-MEK pathway mutations. During the dose-escalation phase, we evaluated three intermittent oral schedules (28-day cycles) in patients with solid tumours: (1) 4·0 mg or 3·2 mg CH5126766 three times per week; (2) 4·0 mg CH5126766 twice per week; and (3) toxicity-guided dose interruption schedule, in which treatment at the recommended phase 2 dose (4·0 mg CH5126766 twice per week) was de-escalated to 3 weeks on followed by 1 week off if patients had prespecified toxic effects (grade 2 or worse diarrhoea, rash, or creatinine phosphokinase elevation). In the basket dose-expansion phase, we evaluated antitumour activity at the recommended phase 2 dose, determined from the dose-escalation phase, in biomarker-selected patients. The primary endpoints were the recommended phase 2 dose at which no more than one out of six patients had a treatment-related dose-limiting toxicity, and the safety and toxicity profile of each dosing schedule. The key secondary endpoint was investigator-assessed response rate in the dose-expansion phase. Patients who received at least one dose of the study drug were evaluable for safety and patients who received one cycle of the study drug and underwent baseline disease assessment were evaluable for response. This trial is registered with ClinicalTrials.gov, NCT02407509. FINDINGS: Between June 5, 2013, and Jan 10, 2019, 58 eligible patients were enrolled to the study: 29 patients with solid tumours were included in the dose-escalation cohort and 29 patients with solid tumours or multiple myeloma were included in the basket dose-expansion cohort (12 non-small-cell lung cancer, five gynaecological malignancy, four colorectal cancer, one melanoma, and seven multiple myeloma). Median follow-up at the time of data cutoff was 2·3 months (IQR 1·6-3·5). Dose-limiting toxicities included grade 3 bilateral retinal pigment epithelial detachment in one patient who received 4·0 mg CH5126766 three times per week, and grade 3 rash (in two patients) and grade 3 creatinine phosphokinase elevation (in one patient) in those who received 3·2 mg CH5126766 three times per week. 4·0 mg CH5126766 twice per week (on Monday and Thursday or Tuesday and Friday) was established as the recommended phase 2 dose. The most common grade 3-4 treatment-related adverse events were rash (11 [19%] patients), creatinine phosphokinase elevation (six [11%]), hypoalbuminaemia (six [11%]), and fatigue (four [7%]). Five (9%) patients had serious treatment-related adverse events. There were no treatment-related deaths. Eight (14%) of 57 patients died during the trial due to disease progression. Seven (27% [95% CI 11·6-47·8]) of 26 response-evaluable patients in the basket expansion achieved objective responses. INTERPRETATION: To our knowledge, this is the first study to show that highly intermittent schedules of a RAF-MEK inhibitor has antitumour activity across various cancers with RAF-RAS-MEK pathway mutations, and that this inhibitor is tolerable. CH5126766 used as a monotherapy and in combination regimens warrants further evaluation. FUNDING: Chugai Pharmaceutical.

Olaparib in patients with metastatic castration-resistant prostate cancer with DNA repair gene aberrations (TOPARP-B): a multicentre, open-label, randomised, phase 2 trial.

BACKGROUND: Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-B trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer. METHODS: In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-the-winner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to <5 cells per 7·5 mL blood). A confirmed response in a consecutive assessment after at least 4 weeks was required for each component. The primary analysis was done in the evaluable population. If at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. Safety was assessed in all patients who received at least one dose of olaparib. This trial is registered at ClinicalTrials.gov, NCT01682772. Recruitment for the trial has completed and follow-up is ongoing. FINDINGS: 711 patients consented for targeted screening between April 1, 2015, and Aug 30, 2018. 161 patients had DDR gene aberrations, 98 of whom were randomly assigned and treated (49 patients for each olaparib dose), with 92 evaluable for the primary endpoint (46 patients for each olaparib dose). Median follow-up was 24·8 months (IQR 16·7-35·9). Confirmed composite response was achieved in 25 (54·3%; 95% CI 39·0-69·1) of 46 evaluable patients in the 400 mg cohort, and 18 (39·1%; 25·1-54·6) of 46 evaluable patients in the 300 mg cohort. Radiological response was achieved in eight (24·2%; 11·1-42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2-32·0) of 37 in the 300 mg cohort; PSA50 response was achieved in 17 (37·0%; 23·2-52·5) of 46 and 13 (30·2%; 17·2-46·1) of 43; and circulating tumour cell count conversion was achieved in 15 (53·6%; 33·9-72·5) of 28 and 13 (48·1%; 28·7-68·1) of 27. The most common grade 3-4 adverse event in both cohorts was anaemia (15 [31%] of 49 patients in the 300 mg cohort and 18 [37%] of 49 in the 400 mg cohort). 19 serious adverse reactions were reported in 13 patients. One death possibly related to treatment (myocardial infarction) occurred after 11 days of treatment in the 300 mg cohort. INTERPRETATION: Olaparib has antitumour activity against metastatic castration-resistant prostate cancer with DDR gene aberrations, supporting the implementation of genomic stratification of metastatic castration-resistant prostate cancer in clinical practice. FUNDING: Cancer Research UK, AstraZeneca, Prostate Cancer UK, the Prostate Cancer Foundation, the Experimental Cancer Medicine Centres Network, and the National Institute for Health Research Biomedical Research Centres.

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer.

BACKGROUND: Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS: We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS: Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconi's anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS: Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

PTEN loss in circulating tumour cells correlates with PTEN loss in fresh tumour tissue from castration-resistant prostate cancer patients.

BACKGROUND: PTEN gene loss occurs frequently in castration-resistant prostate cancer (CRPC) and may drive progression through activation of the PI3K/AKT pathway. Here, we developed a novel CTC-based assay to determine PTEN status and examined the correlation between PTEN status in CTCs and matched tumour tissue samples. METHODS: PTEN gene status in CTCs was evaluated on an enrichment-free platform (Epic Sciences) by fluorescence in situ hybridisation (FISH). PTEN status in archival and fresh tumour tissue was evaluated by FISH and immunohistochemistry. RESULTS: Peripheral blood was collected from 76 patients. Matched archival and fresh cancer tissue was available for 48 patients. PTEN gene status detected in CTCs was concordant with PTEN status in matched fresh tissues and archival tissue in 32 of 38 patients (84%) and 24 of 39 patients (62%), respectively. CTC counts were prognostic (continuous, P=0.001). PTEN loss in CTCs associated with worse survival in univariate analysis (HR 2.05; 95% CI 1.17-3.62; P=0.01) and with high lactate dehydrogenase (LDH) in metastatic CRPC patients. CONCLUSIONS: Our results illustrate the potential use of CTCs as a non-invasive, real-time liquid biopsy to determine PTEN gene status. The prognostic and predictive value of PTEN in CTCs warrants investigation in CRPC clinical trials of PI3K/AKT-targeted therapies.

The poly(ADP-ribose) polymerase inhibitor niraparib (MK4827) in BRCA mutation carriers and patients with sporadic cancer: a phase 1 dose-escalation trial.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is implicated in DNA repair and transcription regulation. Niraparib (MK4827) is an oral potent, selective PARP-1 and PARP-2 inhibitor that induces synthetic lethality in preclinical tumour models with loss of BRCA and PTEN function. We investigated the safety, tolerability, maximum tolerated dose, pharmacokinetic and pharmacodynamic profiles, and preliminary antitumour activity of niraparib. METHODS: In a phase 1 dose-escalation study, we enrolled patients with advanced solid tumours at one site in the UK and two sites in the USA. Eligible patients were aged at least 18 years; had a life expectancy of at least 12 weeks; had an Eastern Cooperative Oncology Group performance status of 2 or less; had assessable disease; were not suitable to receive any established treatments; had adequate organ function; and had discontinued any previous anticancer treatments at least 4 weeks previously. In part A, cohorts of three to six patients, enriched for BRCA1 and BRCA2 mutation carriers, received niraparib daily at ten escalating doses from 30 mg to 400 mg in a 21-day cycle to establish the maximum tolerated dose. Dose expansion at the maximum tolerated dose was pursued in 15 patients to confirm tolerability. In part B, we further investigated the maximum tolerated dose in patients with sporadic platinum-resistant high-grade serous ovarian cancer and sporadic prostate cancer. We obtained blood, circulating tumour cells, and optional paired tumour biopsies for pharmacokinetic and pharmacodynamic assessments. Toxic effects were assessed by common toxicity criteria and tumour responses ascribed by Response Evaluation Criteria in Solid Tumors (RECIST). Circulating tumour cells and archival tumour tissue in prostate patients were analysed for exploratory putative predictive biomarkers, such as loss of PTEN expression and ETS rearrangements. This trial is registered with ClinicalTrials.gov, NCT00749502. FINDINGS: Between Sept 15, 2008, and Jan 14, 2011, we enrolled 100 patients: 60 in part A and 40 in part B. 300 mg/day was established as the maximum tolerated dose. Dose-limiting toxic effects reported in the first cycle were grade 3 fatigue (one patient given 30 mg/day), grade 3 pneumonitis (one given 60 mg/day), and grade 4 thrombocytopenia (two given 400 mg/day). Common treatment-related toxic effects were anaemia (48 patients [48%]), nausea (42 [42%]), fatigue (42 [42%]), thrombocytopenia (35 [35%]), anorexia (26 [26%]), neutropenia (24 [24%]), constipation (23 [23%]), and vomiting (20 [20%]), and were predominantly grade 1 or 2. Pharmacokinetics were dose proportional and the mean terminal elimination half-life was 36·4 h (range 32·8-46·0). Pharmacodynamic analyses confirmed PARP inhibition exceeded 50% at doses greater than 80 mg/day and antitumour activity was documented beyond doses of 60 mg/day. Eight (40% [95% CI 19-64]) of 20 BRCA1 or BRCA2 mutation carriers with ovarian cancer had RECIST partial responses, as did two (50% [7-93]) of four mutation carriers with breast cancer. Antitumour activity was also reported in sporadic high-grade serous ovarian cancer, non-small-cell lung cancer, and prostate cancer. We recorded no correlation between loss of PTEN expression or ETS rearrangements and measures of antitumour activity in patients with prostate cancer. INTERPRETATION: A recommended phase 2 dose of 300 mg/day niraparib is well tolerated. Niraparib should be further assessed in inherited and sporadic cancers with homologous recombination DNA repair defects and to target PARP-mediated transcription in cancer. FUNDING: Merck Sharp and Dohme.

Capivasertib in combination with enzalutamide for metastatic castration resistant prostate cancer after docetaxel and abiraterone: Results from the randomized phase II RE-AKT trial.

BACKGROUND: PTEN loss and aberrations in PI3K/AKT signaling kinases associate with poorer response to abiraterone acetate (AA) in metastatic castration-resistant prostate cancer (mCRPC). In this study, we assessed antitumor activity of the AKT inhibitor capivasertib combined with enzalutamide in mCRPC with prior progression on AA and docetaxel. METHODS: This double-blind, placebo-controlled, randomized phase 2 trial, recruited men ≥ 18 years with progressing mCRPC and performance status 0-2 from 15 UK centers. Randomized participants (1:1) received enzalutamide (160 mg orally, once daily) with capivasertib (400 mg)/ placebo orally, twice daily on an intermittent (4 days on, 3 days off) schedule. Primary endpoint was composite response rate (RR): RECIST 1.1 objective response, ≥ 50 % PSA decrease from baseline, or circulating tumor cell count conversion (from ≥ 5 at baseline to < 5 cells/7.5 mL). Subgroup analyses by PTENIHC status were pre-planned. RESULTS: Overall, 100 participants were randomized (50:50); 95 were evaluable for primary endpoint (47:48); median follow-up was 43 months. RR were 9/47 (19.1 %) enzalutamide/capivasertib and 9/48 (18.8 %) enzalutamide/placebo (absolute difference 0.4 % 90 %CI -12.8 to 13.6, p = 0.58), with similar results in the PTENIHC loss subgroup. Irrespective of treatment, OS was significantly worse for PTENIHC loss (10.1 months [95 %CI: 4.6-13.9] vs 14.8 months [95 %CI: 10.8-18]; p = 0.02). Most common treatment-emergent grade ≥ 3 adverse events for the combination were diarrhea (13 % vs 2 %) and fatigue (10 % vs 6 %). CONCLUSIONS: Combined capivasertib/enzalutamide was well tolerated but didn't significantly improve outcomes from abiraterone pre-treated mCRPC.

RNASEH2B loss and PARP inhibition in advanced prostate cancer.

BACKGROUND: Clinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and preclinically sensitizes to PARP inhibition. The current study reports on RNASEH2B protein loss in advanced prostate cancer and its association with RB1 protein loss, clinical outcome and clonal dynamics during treatment with PARP inhibition in a prospective clinical trial. METHODS: Whole tumor biopsies from multiple cohorts of patients with advanced prostate cancer were interrogated using whole-exome sequencing (WES), RNA sequencing (bulk and single nucleus) and immunohistochemistry (IHC) for RNASEH2B and RB1. Biopsies from patients treated with olaparib in the TOPARP-A and TOPARP-B clinical trials were used to evaluate RNASEH2B clonal selection during olaparib treatment. RESULTS: Shallow co-deletion of RNASEH2B and adjacent RB1, co-located at chromosome 13q14, was common, deep co-deletion infrequent, and gene loss associated with lower mRNA expression. In castration-resistant PC (CRPC) biopsies, RNASEH2B and RB1 mRNA expression correlated, but single nucleus RNA sequencing indicated discordant loss of expression. IHC studies showed that loss of the two proteins often occurred independently, arguably due to stochastic second allele loss. Pre- and post-treatment metastatic CRPC (mCRPC) biopsy studies from BRCA1/2 wildtype tumors, treated on the TOPARP phase II trial, indicated that olaparib eradicates RNASEH2B-loss tumor subclones. CONCLUSION: PARP inhibition may benefit men suffering from mCRPC by eradicating tumor subclones with RNASEH2B loss. CLINICALTRIALS: gov NCT01682772FUNDING. AstraZeneca; Cancer Research UK; Medical Research Council; Cancer Research UK; Prostate Cancer UK; Movember Foundation; Prostate Cancer Foundation.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Systematic study of tissue factor expression in solid tumors.

BACKGROUND: Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. AIMS: To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. METHODS AND RESULTS: TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. CONCLUSION: This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.

Germline ATM Mutations Detected by Somatic DNA Sequencing in Lethal Prostate Cancer.

Background: Germline mutations in the ataxia telangiectasia mutated (ATM) gene occur in 0.5-1% of the overall population and are associated with tumour predisposition. The clinical and pathological features of ATM-mutated prostate cancer (PC) are poorly defined but have been associated with lethal PC. Objective: To report on the clinical characteristics including family history and clinical outcomes of a cohort of patients with advanced metastatic castration-resistant PC (CRPC) who were found to have germline ATM mutations after mutation detection by initial tumour DNA sequencing. Design setting and participants: We acquired germline ATM mutation data by saliva next-generation sequencing from patients with ATM mutations in PC biopsies sequenced between January 2014 and January 2022. Demographics, family history, and clinical data were collected retrospectively. Outcome measurements and statistical analysis: Outcome endpoints were based on overall survival (OS) and time from diagnosis to CRPC. Data were analysed using R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria). Results and limitations: Overall, seven patients (n = 7/1217; 0.6%) had germline ATM mutations detected, with five of them having a family history of malignancies, including breast, prostate, pancreas, and gastric cancer; leukaemia; and lymphoma. Two patients had concomitant somatic mutations in tumour biopsies in genes other than ATM, while two patients were found to carry more than one ATM pathogenic mutation. Five tumours in germline ATM variant carriers had loss of ATM by immunohistochemistry. The median OS from diagnosis was 7.1 yr (range 2.9-14 yr) and the median OS from CRPC was 5.3 yr (range 2.2-7.3 yr). When comparing these data with PC patients sequenced by The Cancer Genome Atlas, we found that the spatial localisation of mutations was similar, with distribution of alterations occurring on similar positions in the ATM gene. Interestingly, these include a mutation within the FRAP-ATM-TRRAP (FAT) domain, suggesting that this represents a mutational hotspot for ATM. Conclusions: Germline ATM mutations are rare in patients with lethal PC but occur at mutational hotspots; further research is warranted to better characterise the family histories of these men and PC clinical course. Patient summary: In this report, we studied the clinical and pathological features of advanced prostate cancers associated with germline mutations in the ATM gene. We found that most patients had a strong family history of cancer and that this mutation might predict the course of these prostate cancers, as well as response to specific treatments.

Efficacy and Safety of Weekly Paclitaxel Plus Vistusertib vs Paclitaxel Alone in Patients With Platinum-Resistant Ovarian High-Grade Serous Carcinoma: The OCTOPUS Multicenter, Phase 2, Randomized Clinical Trial.

Importance: Patients with platinum-resistant or refractory ovarian high-grade serous carcinoma (PR-HGSC) have a poor prognosis and few therapeutic options. Preclinical studies support targeting PI3K/AKT/mTOR signaling in this setting, and a phase 1 study of the dual mTORC1/mTORC2 inhibitor vistusertib with weekly paclitaxel showed activity. Objective: To evaluate whether the addition of vistusertib to weekly paclitaxel improves clinical outcomes in patients with PR-HGSC. Design, Setting, and Participants: This phase 2, double-blind, placebo-controlled multicenter randomized clinical trial recruited patients from UK cancer centers between January 2016 and March 2018. Patients with PR-HGSC of ovarian, fallopian tube, or primary peritoneal origin and with measurable or evaluable disease (Response Evaluation Criteria in Solid Tumors version 1.1 and/or Gynecological Cancer Intergroup cancer antigen 125 criteria) were eligible. There were no restrictions on number of lines of prior therapy. Data analysis was performed from May 2019 to January 2022. Interventions: Patients were randomized (1:1) to weekly paclitaxel (80 mg/m2 days 1, 8, and 15 of a 28-day cycle) plus oral vistusertib (50 mg twice daily) or placebo. Main Outcomes and Measures: The primary end point was progression-free survival in the intention-to-treat population. Secondary end points included response rate, overall survival, and quality of life. Results: A total of 140 patients (median [range] age, 63 [36-86] years; 17.9% with platinum-refractory disease; 53.6% with ≥3 prior therapies) were randomized. In the paclitaxel plus vistusertib vs paclitaxel plus placebo groups, there was no difference in progression-free survival (median, 4.5 vs 4.1 months; hazard ratio [HR], 0.84; 80% CI, 0.67-1.07; 1-sided P = .18), overall survival (median, 9.7 vs 11.1 months; HR, 1.21; 80% CI, 0.91-1.60) or response rate (odds ratio, 0.86; 80% CI, 0.55-1.36). Grade 3 to 4 adverse events were 41.2% (weekly paclitaxel plus vistusertib) vs 36.7% (weekly paclitaxel plus placebo), and there was no difference in quality of life. Conclusions and Relevance: In this randomized clinical trial of weekly paclitaxel and dual mTORC1/2 inhibition in patients with PR-HGSC, vistusertib did not improve clinical activity of weekly paclitaxel. Trial Registration: isrctn.org Identifier: ISRCTN16426935.

Olaparib and Ceralasertib (AZD6738) in Patients with Triple-Negative Advanced Breast Cancer: Results from Cohort E of the plasmaMATCH Trial (CRUK/15/010).

PURPOSE: Approximately 10% to 15% of triple-negative breast cancers (TNBC) have deleterious mutations in BRCA1 and BRCA2 and may benefit from PARP inhibitor treatment. PARP inhibitors may also increase exogenous replication stress and thereby increase sensitivity to inhibitors of ataxia telangiectasia and Rad3-related (ATR) protein. This phase II study examined the activity of the combination of PARP inhibitor, olaparib, and ATR inhibitor, ceralasertib (AZD6738), in patients with advanced TNBC. PATIENTS AND METHODS: Patients with TNBC on most recent biopsy who had received 1 or 2 lines of chemotherapy for advanced disease or had relapsed within 12 months of (neo)adjuvant chemotherapy were eligible. Treatment was olaparib 300 mg twice a day continuously and celarasertib 160 mg on days 1-7 on a 28-day cycle until disease progression. The primary endpoint was confirmed objective response rate (ORR). Tissue and plasma biomarker analyses were preplanned to identify predictors of response. RESULTS: 70 evaluable patients were enrolled. Germline BRCA1/2 mutations were present in 10 (14%) patients and 3 (4%) patients had somatic BRCA mutations. The confirmed ORR was 12/70; 17.1% (95% confidence interval, 10.4-25.5). Responses were observed in patients without germline or somatic BRCA1/2 mutations, including patients with mutations in other homologous recombination repair genes and tumors with functional homologous recombination deficiency by RAD51 foci. CONCLUSIONS: The response rate to olaparib and ceralasertib did not meet prespecified criteria for activity in the overall evaluable population, but responses were observed in patients who would not be expected to respond to olaparib monotherapy.

B7-H3 as a Therapeutic Target in Advanced Prostate Cancer.

BACKGROUND: B7-H3 is a cell surface immunomodulatory glycoprotein overexpressed in prostate cancers (PCs). Understanding its longitudinal expression at emergence of castration resistance and association with tumour genomics are critical to the development of and patient selection for B7-H3 targeted therapies. OBJECTIVE: To characterise B7-H3 expression in same-patient hormone-sensitive (HSPC) and castration-resistant (CRPC) PC biopsies, associating this with PC genomics, and to evaluate the antitumour activity of an anti-B7-H3 antibody-drug conjugate (ADC) in human CRPC in vitro and in vivo. DESIGN, SETTING, AND PARTICIPANTS: We performed immunohistochemistry and next-generation sequencing on a cohort of 98 clinically annotated CRPC biopsies, including 72 patients who also had HSPC biopsies for analyses. We analysed two CRPC transcriptome and exome datasets, and PC scRNASeq datasets. PC organoids (patient-derived xenograft [PDX]-derived organoids [PDX-Os]) were derived from PDXs generated from human CRPC biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We evaluated B7-H3 mRNA expression in relation to a panel of 770 immune-related genes, compared B7-H3 protein expression between same-patient HSPC and CRPC biopsies, determined associations with PC genomic alterations, and evaluated the antitumour activity of DS-7300a, a topoisomerase-1 inhibitor payload anti-B7-H3 ADC, in human PC cell lines, organoids (PDX-Os), and xenografts (PDXs) of different histologies, B7-H3 expressions, and genomics. RESULTS AND LIMITATIONS: B7-H3 was among the most highly expressed immunomodulatory genes in CRPCs. Most CRPCs (93%) expressed B7-H3, and in patients who developed CRPC, B7-H3 expression was frequently expressed at the time of HSPC diagnosis (97%). Conversion from B7-H3 positive to negative, or vice versa, during progression from HSPC to CRPC was uncommon. CRPC with neuroendocrine features were more likely to be B7-H3 negative (28%) than adenocarcinomas. B7-H3 is overexpressed in tumours with defective DNA repair gene (ATM and BRCA2) alterations and is associated with ERG expression, androgen receptor (AR) expression, and AR activity signature. DS7300a had antitumour activity against B7-H3 expressing human PC models including cell lines, PDX-Os, and PDXs of adenocarcinoma and neuroendocrine histology. CONCLUSIONS: The frequent overexpression of B7-H3 in CRPC compared with normal tissue and other B7 family members implicates it as a highly relevant therapeutic target in these diseases. Mechanisms driving differences in B7-H3 expression across genomic subsets warrant investigation for understanding the role of B7-H3 in cancer growth and for the clinical development of B7-H3 targeted therapies. PATIENT SUMMARY: B7-H3, a protein expressed on the surface of the most lethal prostate cancers, in particular those with specific mutations, can be targeted using drugs that bind B7-H3. These findings are relevant for the development of such drugs and for deciding which patients to treat with these new drugs.

Novel, gross chromosomal alterations involving PTEN cooperate with allelic loss in prostate cancer.

There is increasing evidence that multiple chromosomal rearrangements occur in prostate cancer. PTEN loss is considered to be a key event in prostate carcinogenesis but the mechanisms of loss remain to be fully elucidated. We hypothesised that gross rearrangements may exist that cause disruption of the PTEN gene in the absence of genomic deletion. We therefore designed a novel fluorescence in situ hybridisation (FISH) assay with probes overlying regions 3' and 5' of PTEN and a third probe overlying the gene. We aimed to identify both genomic deletions and gross rearrangements of PTEN that would be overlooked by previously reported single-probe FISH assays. We proceeded to evaluate a tissue microarray with radical prostatectomy and trans-urethral resection of the prostate specimens from 187 patients. We identified PTEN genomic loss in 45/150 (30%) radical prostatectomy patients and 16/37 (43%) trans-urethral resection of the prostate patients. Importantly, our assay detected novel chromosomal alterations in the PTEN gene (characterised by splitting of FISH signals) in 13 tumours (6.9% of all prostate cancers; 21% of PTEN-lost cancers). All PTEN-rearranged tumours had genomic loss at the other allele and had no expression of PTEN by immunohistochemistry. PTEN-rearranged tumours were significantly more likely to have an underlying ERG rearrangement. Our assay differentiated loss of the probe overlying PTEN in isolation or in combination with either one of or both the probes overlying the 3' and 5' regions. This gave an indication of the size of genomic loss and we observed considerable inter-tumoural heterogeneity in the extent of genomic loss in PTEN-lost tumours. In summary, gross rearrangements of the PTEN locus occur in prostate cancer and can be detected by a 'break-apart' FISH assay. This observation could explain the absence of PTEN protein expression in a subgroup of tumours previously classified as having heterozygous genomic loss using single-probe traditional FISH assays.

Prostate-Specific Membrane Antigen Expression and Response to DNA Damaging Agents in Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) targeting therapies such as Lutetium-177 (177Lu)-PSMA-617 are affecting outcomes from metastatic castration-resistant prostate cancer (mCRPC). However, a significant subset of patients have prostate cancer cells lacking PSMA expression, raising concerns about treatment resistance attributable at least in part to heterogeneous PSMA expression. We have previously demonstrated an association between high PSMA expression and DNA damage repair defects in mCRPC biopsies and therefore hypothesized that DNA damage upregulates PSMA expression. EXPERIMENTAL DESIGN: To test this relationship between PSMA and DNA damage we conducted a screen of 147 anticancer agents (NCI/NIH FDA-approved anticancer "Oncology Set") and treated tumor cells with repeated ionizing irradiation. RESULTS: The topoisomerase-2 inhibitors, daunorubicin and mitoxantrone, were identified from the screen to upregulate PSMA protein expression in castration-resistant LNCaP95 cells; this result was validated in vitro in LNCaP, LNCaP95, and 22Rv1 cell lines and in vivo using an mCRPC patient-derived xenograft model CP286 identified to have heterogeneous PSMA expression. As double-strand DNA break induction by topoisomerase-2 inhibitors upregulated PSMA, we next studied the impact of ionizing radiation on PSMA expression; this also upregulated PSMA protein expression in a dose-dependent fashion. CONCLUSIONS: The results presented herein are the first, to our knowledge, to demonstrate that PSMA is upregulated in response to double-strand DNA damage by anticancer treatment. These data support the study of rational combinations that maximize the antitumor activity of PSMA-targeted therapeutic strategies by upregulating PSMA.

Immune Biomarkers in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease in which molecular stratification is needed to improve clinical outcomes. The identification of predictive biomarkers can have a major impact on the care of these patients, but the availability of metastatic tissue samples for research in this setting is limited. OBJECTIVE: To study the prevalence of immune biomarkers of potential clinical utility to immunotherapy in mCRPC and to determine their association with overall survival (OS). DESIGN, SETTING, AND PARTICIPANTS: From 100 patients, mCRPC biopsies were assayed by whole exome sequencing, targeted next-generation sequencing, RNA sequencing, tumor mutational burden, T-cell-inflamed gene expression profile (TcellinfGEP) score (Nanostring), and immunohistochemistry for programmed cell death 1 ligand 1 (PD-L1), ataxia-telangiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), SRY homology box 2 (SOX2), and the presence of neuroendocrine features. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The phi coefficient determined correlations between biomarkers of interest. OS was assessed using Kaplan-Meier curves and adjusted hazard ratios (aHRs) from Cox regression. RESULTS AND LIMITATIONS: PD-L1 and SOX2 protein expression was detected by immunohistochemistry (combined positive score ≥1 and >5% cells, respectively) in 24 (33%) and 27 (27%) mCRPC biopsies, respectively; 23 (26%) mCRPC biopsies had high TcellinfGEP scores (>-0.318). PD-L1 protein expression and TcellinfGEP scores were positively correlated (phi 0.63 [0.45; 0.76]). PD-L1 protein expression (aHR: 1.90 [1.05; 3.45]), high TcellinfGEP score (aHR: 1.86 [1.04; 3.31]), and SOX2 expression (aHR: 2.09 [1.20; 3.64]) were associated with worse OS. CONCLUSIONS: PD-L1, TcellinfGEP score, and SOX2 are prognostic of outcome from the mCRPC setting. If validated, predictive biomarker studies incorporating survival endpoints need to take these findings into consideration. PATIENT SUMMARY: This study presents an analysis of immune biomarkers in biopsies from patients with metastatic prostate cancer. We describe tumor alterations that predict prognosis that can impact future studies.

SMG8/SMG9 Heterodimer Loss Modulates SMG1 Kinase to Drive ATR Inhibitor Resistance.

Gastric cancer represents the third leading cause of global cancer mortality and an area of unmet clinical need. Drugs that target the DNA damage response, including ATR inhibitors (ATRi), have been proposed as novel targeted agents in gastric cancer. Here, we sought to evaluate the efficacy of ATRi in preclinical models of gastric cancer and to understand how ATRi resistance might emerge as a means to identify predictors of ATRi response. A positive selection genome-wide CRISPR-Cas9 screen identified candidate regulators of ATRi resistance in gastric cancer. Loss-of-function mutations in either SMG8 or SMG9 caused ATRi resistance by an SMG1-mediated mechanism. Although ATRi still impaired ATR/CHK1 signaling in SMG8/9-defective cells, other characteristic responses to ATRi exposure were not seen, such as changes in ATM/CHK2, γH2AX, phospho-RPA, or 53BP1 status or changes in the proportions of cells in S- or G2-M-phases of the cell cycle. Transcription/replication conflicts (TRC) elicited by ATRi exposure are a likely cause of ATRi sensitivity, and SMG8/9-defective cells exhibited a reduced level of ATRi-induced TRCs, which could contribute to ATRi resistance. These observations suggest ATRi elicits antitumor efficacy in gastric cancer but that drug resistance could emerge via alterations in the SMG8/9/1 pathway. SIGNIFICANCE: These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors.

A Phase I Trial of CT900, a Novel α-Folate Receptor-Mediated Thymidylate Synthase Inhibitor, in Patients with Solid Tumors with Expansion Cohorts in Patients with High-Grade Serous Ovarian Cancer.

PURPOSE: CT900 is a novel small molecule thymidylate synthase inhibitor that binds to α-folate receptor (α-FR) and thus is selectively taken up by α-FR-overexpressing tumors. PATIENTS AND METHODS: A 3+3 dose escalation design was used. During dose escalation, CT900 doses of 1-6 mg/m2 weekly and 2-12 mg/m2 every 2 weeks (q2Wk) intravenously were evaluated. Patients with high-grade serous ovarian cancer were enrolled in the expansion cohorts. RESULTS: 109 patients were enrolled: 42 patients in the dose escalation and 67 patients in the expansion cohorts. At the dose/schedule of 12 mg/m2/q2Wk (with and without dexamethasone, n = 40), the most common treatment-related adverse events were fatigue, nausea, diarrhea, cough, anemia, and pneumonitis, which were predominantly grade 1 and grade 2. Levels of CT900 more than 600 nmol/L needed for growth inhibition in preclinical models were achieved for >65 hours at a dose of 12 mg/m2. In the expansion cohorts, the overall response rate (ORR), was 14/64 (21.9%). Thirty-eight response-evaluable patients in the expansion cohorts receiving 12 mg/m2/q2Wk had tumor evaluable for quantification of α-FR. Patients with high or medium expression had an objective response rate of 9/25 (36%) compared with 1/13 (7.7%) in patients with negative/very low or low expression of α-FR. CONCLUSIONS: The dose of 12 mg/m2/q2Wk was declared the recommended phase II dose/schedule. At this dose/schedule, CT900 exhibited an acceptable side effect profile with clinical benefit in patients with high/medium α-FR expression and warrants further investigation.