Summary 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...

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

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Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.