Repurposing FDA approved drugs as radiosensitizers for treating hypoxic prostate cancer.

BACKGROUND: The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. METHODS: Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. RESULTS: Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. CONCLUSION: Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.

Seabirds (Laridae) as a source of Campylobacter spp., Salmonella spp. and antimicrobial resistance in South Africa.

Zoonotic thermophilic Campylobacter and nontyphoidal Salmonella enterica are a major cause of foodborne human gastroenteritis worldwide. There is little information about reservoirs of these zoonotic agents in Africa. Thus, chicks of kelp gulls (Larus dominicanus, n = 129) and greater crested terns (Thalasseus bergii, n = 100) were studied at five colonies on the Western Cape coast (South Africa) during summer 2013/2014. Campylobacter spp. occurrence was 14.0% (CI95% : 9.9-19.3), with C. jejuni the most frequently isolated species, whilst that of Salmonella was 27.5% (CI95% : 21.9-33.9) overall, with a higher prevalence in gulls (43.0%, CI95% : 34.8-52.4) than terns (7.0%, CI95% : 3.1-14.4). Among the 16 different S. enterica serovars found, Anatum, Enteritidis and Hadar were the most frequent. The same or highly similar pulsed-field gel electrophoresis genotype was found in some Salmonella isolates from seabirds and humans presenting with salmonellosis in Cape Town hospitals. Both Campylobacter and Salmonella isolates exhibited antimicrobial resistance to several agents, including critically important antimicrobials (quinolones, tetracyclines and ß-lactams) and multidrug resistance in Salmonella serovars from kelp gulls. Our results highlight the importance of seabirds as reservoirs of Campylobacter and Salmonella resistant strains and their role in the maintenance and transmission of these bacteria in the environment, with implications for public health.

A hypoxia biomarker does not predict benefit from giving chemotherapy with radiotherapy in the BC2001 randomised controlled trial.

BACKGROUND: BC2001 showed combining chemotherapy (5-FU + mitomycin-C) with radiotherapy improves loco-regional disease-free survival in patients with muscle-invasive bladder cancer (MIBC). We previously showed a 24-gene hypoxia-associated signature predicted benefit from hypoxia-modifying radiosensitisation in BCON and hypothesised that only patients with low hypoxia scores (HSs) would benefit from chemotherapy in BC2001. BC2001 allowed conventional (64Gy/32 fractions) or hypofractionated (55Gy/20 fractions) radiotherapy. An exploratory analysis tested an additional hypothesis that hypofractionation reduces reoxygenation and would be detrimental for patients with hypoxic tumours. METHODS: RNA was extracted from pre-treatment biopsies (298 BC2001 patients), transcriptomic data generated (Affymetrix Clariom-S arrays), HSs calculated (median expression of 24-signature genes) and patients stratified as hypoxia-high or -low (cut-off: cohort median). PRIMARY ENDPOINT: invasive loco-regional control (ILRC); secondary overall survival. FINDINGS: Hypoxia affected overall survival (HR = 1.30; 95% CI 0.99-1.70; p = 0.062): more uncertainty for ILRC (HR = 1.29; 95% CI 0.82-2.03; p = 0.264). Benefit from chemotherapy was similar for patients with high or low HSs, with no interaction between HS and treatment arm. High HS associated with poor ILRC following hypofractionated (n = 90, HR 1.69; 95% CI 0.99-2.89 p = 0.057) but not conventional (n = 207, HR 0.70; 95% CI 0.28-1.80, p = 0.461) radiotherapy. The finding was confirmed in an independent cohort (BCON) where hypoxia associated with a poor prognosis for patients receiving hypofractionated (n = 51; HR 14.2; 95% CI 1.7-119; p = 0.015) but not conventional (n = 24, HR 1.04; 95% CI 0.07-15.5, p = 0.978) radiotherapy. INTERPRETATION: Tumour hypoxia status does not affect benefit from BC2001 chemotherapy. Hypoxia appears to affect fractionation sensitivity. Use of HSs to personalise treatment needs testing in a biomarker-stratified trial. FUNDING: Cancer Research UK, NIHR, MRC.

Randomized Phase 3 Trial of the Hypoxia Modifier Nimorazole Added to Radiation Therapy With Benefit Assessed in Hypoxic Head and Neck Cancers Determined Using a Gene Signature (NIMRAD).

PURPOSE: Tumor hypoxia is an adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). We assessed whether patients with hypoxic HNSCC benefited from the addition of nimorazole to definitive intensity modulated radiation therapy (IMRT). METHODS AND MATERIALS: NIMRAD was a phase 3, multicenter, placebo-controlled, double-anonymized trial of patients with HNSCC unsuitable for concurrent platinum chemotherapy or cetuximab with definitive IMRT (NCT01950689). Patients were randomized 1:1 to receive IMRT (65 Gy in 30 fractions over 6 weeks) plus nimorazole (1.2 g/m2 daily, before IMRT) or placebo. The primary endpoint was freedom from locoregional progression (FFLRP) in patients with hypoxic tumors, defined as greater than or equal to the median tumor hypoxia score of the first 50 patients analyzed (≥0.079), using a validated 26-gene signature. The planned sample size was 340 patients, allowing for signature generation in 85% and an assumed hazard ratio (HR) of 0.50 for nimorazole effectiveness in the hypoxic group and requiring 66 locoregional failures to have 80% power in a 2-tail log-rank test at the 5% significance level. RESULTS: Three hundred thirty-eight patients were randomized by 19 centers in the United Kingdom from May 2014 to May 2019, with a median follow-up of 3.1 years (95% CI, 2.9-3.4). Hypoxia scores were available for 286 (85%). The median patient age was 73 years (range, 44-88; IQR, 70-76). There were 36 (25.9%) locoregional failures in the hypoxic group, in which nimorazole + IMRT did not improve FFLRP (adjusted HR, 0.72; 95% CI, 0.36-1.44; P = .35) or overall survival (adjusted HR, 0.96; 95% CI, 0.53-1.72; P = .88) compared with placebo + IMRT. Similarly, nimorazole + IMRT did not improve FFLRP or overall survival in the whole population. In total (N = 338), 73% of patients allocated nimorazole adhered to the drug for ≥50% of IMRT fractions. Nimorazole + IMRT caused more acute nausea compared with placebo + IMRT (Common Terminology Criteria for Adverse Events version 4.0 G1+2: 56.6% vs 42.4%, G3: 10.1% vs 5.3%, respectively; P < .05). CONCLUSIONS: Addition of the hypoxia modifier nimorazole to IMRT for locally advanced HNSCC in older and less fit patients did not improve locoregional control or survival.

Comparison of multiple gene expression platforms for measuring a bladder cancer hypoxia signature.

Tumour hypoxia status provides prognostic information and predicts response to hypoxia­modifying treatments. A previous study by our group derived a 24­gene signature to assess hypoxia in bladder cancer. The objectives of the present study were to compare platforms for generating signature scores, identify cut­off values for prospective studies, assess intra­tumour heterogeneity and confirm hypoxia relevance. Briefly, RNA was extracted from prospectively collected diagnostic biopsies of muscle invasive bladder cancer (51 patients), and gene expression was measured using customised Taqman Low Density Array (TLDA) cards, NanoString and Clariom S arrays. Cross­platform transferability of the gene signature was assessed using regression and concordance analysis. The cut­off values were the cohort median expression values. Intra­ and inter­tumour variability were determined in a retrospective patient cohort (n=51) with multiple blocks (2­18) from the same tumour. To demonstrate relevance, bladder cancer cell lines were exposed to hypoxia (0.1% oxygen, 24 h), and extracted RNA was run on custom TLDA cards. Hypoxia scores (HS) values showed good agreement between platforms: Clariom S vs. TLDA (r=0.72, P<0.0001; concordance 73%); Clariom S vs. NanoString (r=0.84, P<0.0001; 78%); TLDA vs. NanoString (r=0.80, P<0.0001; 78%). Cut­off values were 0.047 (TLDA), 7.328 (NanoString) and 6.667 (Clariom S). Intra­tumour heterogeneity in gene expression and HS (coefficient of variation 3.9%) was less than inter­tumour (7.9%) variability. HS values were higher in bladder cancer cells exposed to hypoxia compared with normoxia (P<0.02). In conclusion, the present study revealed that application of the 24­gene bladder cancer hypoxia signature was platform agnostic, cut­off values determined prospectively can be used in a clinical trial, intra­tumour heterogeneity was low and the signature was sensitive to changes in oxygen levels in vitro.

Developing Tumor Radiosensitivity Signatures Using LncRNAs.

Long non-coding RNAs (lncRNAs) are involved in diverse biological processes, including DNA damage repair, and are of interest as potential biomarkers of radiosensitivity. We investigated whether lncRNA radiosensitivity signatures could be derived for use in cancer patients treated with radiotherapy. Signature development involved radiosensitivity measurements for cell lines and primary tumor samples, and patient outcome after radiotherapy. A 10-lncRNA signature trained on radiosensitivity measurements in bladder cell lines showed a trend towards independent validation. In multivariable analyses, patients with tumors classified as radioresistant by the lncRNA signature had poorer local relapse-free survival (P = 0.065) in 151 patients with muscle-invasive bladder cancer who underwent radiotherapy. An mRNA-based radiosensitivity index signature performed similarly to the lncRNA bladder signature for local relapse-free survival (P = 0.055). Pathway analysis showed the lncRNA signature associated with molecular processes involved in radiation responses. Knockdown of one of the lncRNAs in the signature showed a modest increase in radiosensitivity in one cell line. An alternative approach involved training on primary cervical tumor radiosensitivity or local control after radiotherapy. Both approaches failed to generate a cervix lncRNA radiosensitivity signature, which was attributed to the age of samples in our cohorts. Our work highlights challenges in validating lncRNA signatures as biomarkers in archival tissue from radiotherapy cohorts, but supports continued investigation of lncRNAs for a role in radiosensitivity.

Do humans spread zoonotic enteric bacteria in Antarctica?

Reports of enteric bacteria in Antarctic wildlife have suggested its spread from people to seabirds and seals, but evidence is scarce and fragmentary. We investigated the occurrence of zoonotic enteric bacteria in seabirds across the Antarctic and subantarctic region; for comparison purposes, in addition to seabirds, poultry in a subantarctic island was also sampled. Three findings suggest reverse zoonosis from humans to seabirds: the detection of a zoonotic Salmonella serovar (ser. Enteritidis) and Campylobacter species (e.g. C. jejuni), typical of human infections; the resistance of C. lari isolates to ciprofloxacin and enrofloxacin, antibiotics commonly used in human and veterinary medicine; and most importantly, the presence of C. jejuni genotypes mostly found in humans and domestic animals but rarely or never found in wild birds so far. We also show further spread of zoonotic agents among Antarctic wildlife is facilitated by substantial connectivity among populations of opportunistic seabirds, notably skuas (Stercorarius). Our results highlight the need for even stricter biosecurity measures to limit human impacts in Antarctica.