An automated micro solid phase extraction gas chromatography-mass spectrometry (µSPE-GC-MS) detection method for geosmin and 2-methylisoborneol in drinking water.

Geosmin and 2-methylisoborneol (2-MIB) are amongst the most common earthy and musty taste and odour (T&O) compounds found in drinking water. With low odour threshold detection limits below 10 ng L-1, and the complexity of raw water matrices, these two compounds provide a significant challenge for water companies globally. In this research, for the first time, a novel and fully automated micro-solid phase-extraction (µSPE) method coupled with gas chromatography (GC)-mass spectrometry (MS) has been developed for the detection of geosmin and 2-MIB for drinking water analysis. The new automated method described herein is environmentally friendly requiring low raw water sample volumes, of 25 mL, and only 50 µL of elution solvent. Our µSPE-GC-MS method exhibits excellent linearity for both compounds (R2 > 0.999) and low limits of detection of 2.0 ng L-1 and 4.3 ng L-1 for geosmin and 2-MIB, respectively. The method showed excellent recovery rates (95.1-100.1%) and good precision (RSD < 7%) in raw sample matrices. Our approach is fully automated onto a robotic workstation which can be readily integrated into a laboratory workflow for routine water analysis. Furthermore, the method has excellent potential to be incorporated within a portable system for onsite analysis.

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer.

BACKGROUND: miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. METHODS: RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). RESULTS: miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. CONCLUSIONS: A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity.

BACKGROUND: Autophagy is a survival mechanism that allows recycling of cellular breakdown products, particularly in stressed cells. Here we evaluate the hypotheses that up-regulation of autophagy is a common mechanism of resistance to chemotherapy, and that drug resistance can be reversed by inhibiting autophagy with a proton pump inhibitor. METHODS: We exposed human PC3, LNCaP and MCF7 cells to seven clinically-used chemotherapy drugs ± pantoprazole, examined the up-regulation of autophagy and the effect on cellular proliferation by Western Blots, MTS assay and colony-forming assay. The distribution of drug effects and of autophagy was quantified in LNCaP tumor sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3 and p62. RESULTS: All anticancer drugs led to up-regulation of autophagy in cultured tumor cells. Pantoprazole inhibited the induction of autophagy in a time- and dose-dependent manner, and sensitized cancer cells to the seven anti-cancer drugs. Treatment of LNCaP xenografts with paclitaxel induced both DNA damage and autophagy; autophagy was inhibited and markers of toxicity were increased by pantoprazole. CONCLUSIONS: Induction of autophagy is a general mechanism associated with resistance to anticancer drugs and that its inhibition is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes.

Perioperative fluid status and surgical outcomes in patients undergoing cytoreductive surgery for advanced epithelial ovarian cancer.

OBJECTIVE: The objective of this study is to investigate the impact of fluid status on perioperative outcomes of patients undergoing cytoreductive surgery (CRS) for advanced epithelial ovarian cancer (EOC). METHODS: Patients undergoing CRS for stage III or IV EOC at a comprehensive cancer center from 12/2010 to 05/2015 were identified. Those who underwent upper abdominal procedures or colon resections were included. Demographic, perioperative, and 30-day complication data were collected. Perioperative weight change was utilized as a surrogate for fluid status. The time to diuresis (tD) was defined as the postoperative day the patient's weight began to downtrend. RESULTS: One hundred ten patients were included. Median age was 62years and median BMI 25.8kg/m2. The majority (74.5%) were stage IIIC. At least 1 bowel resection was performed in 60 cases (54.5%). A median of 5381mL of crystalloid (range 1000-17,550mL) and 500mL of colloids (range 0-2783mL) was given intraoperatively. The median perioperative weight change was +7.3kg (range-0.9kg to +35.7kg). The median tD was 3days (range 1-17days). On univariate analysis, net positive fluid status was associated with unscheduled reoperation, anastomotic leak, surgical site infections (SSI), and length of stay >5days. On multivariate analysis, fluid status was independently associated with SSI (p=0.01). CONCLUSIONS: Perioperative fluid excess is common in patients undergoing CRS for EOC and is independently associated with SSI.

Hypoxia Marker GLUT-1 (Glucose Transporter 1) is an Independent Prognostic Factor for Survival in Bladder Cancer Patients Treated with Radical Cystectomy.

BACKGROUND: Tumour hypoxia, which is frequent in many cancer types, is associated with treatment resistance and poor prognosis. The role of hypoxia in surgically treated bladder cancer (BC) is not well described. We studied the role of hypoxia in two independent series of urothelial bladder cancers treated with radical cystectomy. METHODS: 279 patients from the University Hospital Network (UHN), Toronto, Canada, and Turku University, Finland were studied. Hypoxia biomarkers (HIF1-α, CAIX, GLUT-1) and proliferation marker Ki-67 were analyzed with immunohistochemistry using defined tissue microarrays. Kaplan-Meier methods and Cox proportional hazards regression models were used to investigate prognostic role of the factors. RESULTS: In univariate analyses, strong GLUT-1 positivity and a high Ki-67 index were associated with poor survival. In multivariate model containing clinical prognostic variables, GLUT-1 was an independent prognostic factor associated with worse disease-specific survival (HR 2.9, 95% CI 0.7-12.6, Wald p = 0.15 in the Toronto cohort and HR 3.2, 95% CI 1.3-7.5, Wald p = 0.0085 in the Turku cohort). CONCLUSION: GLUT-1 is frequently upregulated and is an independent prognostic factor in surgically treated bladder cancer. Further studies are needed to evaluate the potential role of hypoxia-based and targeted therapies in hypoxic bladder tumours.

Short-term morbidity in transdiaphragmatic cardiophrenic lymph node resection for advanced stage gynecologic cancer.

Ovarian cancer is commonly diagnosed at an advanced stage, with disease involving the upper abdomen. The finding of enlarged cardiophrenic lymph nodes (CPLNs) on pre-operative imaging often indicates the presence of malignant spread to the mediastinum. Surgical resection of CPLN through a transdiaphragmatic approach can help to achieve cytoreduction to no gross residual. A retrospective chart review was conducted on all patients who underwent transdiaphragmatic cardiophrenic lymph node resection from 8/1/11 through 2/1/15. All relevant pre-, intra-, and post-operative characteristics and findings were recorded. A brief description of the surgical technique is included for reference. Eleven patients were identified who had undergone transdiaphragmatic resection of cardiophrenic lymph nodes. Malignancy was identified in 18/21 (86%) of total lymph nodes submitted. The median number of post-operative days was 7. The overall post-operative morbidity associated with CPLN resection was low, with the most common finding being a small pleural effusion present on chest x-ray between POD# 3-5 (55%). Transdiaphragmatic CPLN resection is a feasible procedure with relatively minor short-term post-operative morbidities that can be used to achieve cytoreduction to no gross residual disease.

Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway.

In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF-EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation.

Excellent translational research in oncology: A journey towards novel and more effective anti-cancer therapies.

Comprehensive Cancer Centres (CCCs) serve as critical drivers for improving cancer survival. In Europe, we have developed an Excellence Designation System (EDS) consisting of criteria to assess "excellence" of CCCs in translational research (bench to bedside and back), with the expectation that many European CCCs will aspire to this status.

Cost-effectiveness analysis of a multivariate index assay compared to modified American College of Obstetricians and Gynecologists criteria and CA-125 in the triage of women with adnexal masses.

OBJECTIVE: To evaluate the cost-effectiveness of the multivariate index assay (MIA) for use in triaging women with an adnexal mass relative to modified American College of Obstetricians and Gynecologists (mACOG) referral guidelines and CA-125 testing alone. METHODS: The MIA triage algorithm was based on qualitative serum testing of five biomarkers: transthyretin, apolipoprotein, A-1, 2-microglobulin, transferrin, and CA-125. An economic analysis was developed to evaluate the clinical and cost implications of adopting MIA in clinical practice versus the mACOG referral guidelines and CA-125 alone, over a lifetime horizon, from the perspective of the public payer. Clinical parameters used to characterize patients' disease status, quality of life, and treatment decisions were estimated using the results of published studies; costs were approximated using reimbursement rates from CMS fee schedules. Model endpoints included overall survival (OS), costs, quality-adjusted life years (QALYs), and incremental cost-effectiveness ratio (ICER). The cost-effectiveness threshold was set to $50,000 per QALY. One-way sensitivity analysis was performed to assess uncertainty of individual parameters included in the analysis. All costs were reported in 2014 US dollars. RESULTS: Use of MIA was cost-effective, resulting in fewer re-operations and pre-treatment CT scans. Overall MIA resulted in an ICER of $35,094/QALY gained. MIA was also cost-saving and QALY-increasing compared to use of CA-125 alone with an ICER of $12,189/QALY gained. One-way sensitivity analysis showed the ICER was most affected by the following parameters: (1) sensitivity of MIA; (2) sensitivity of mACOG; and (3) percentage of patients, not referred to a gynecologic oncologist, who were correctly diagnosed with advanced epithelial ovarian cancer (EOC). CONCLUSION: Use of MIA is a more cost-effective triage strategy than mACOG or CA-125. It is expected to increase the percentage of women with ovarian cancer that are referred to gynecologic oncologists, which is shown to improve clinical outcomes. Limitations include the use of assumptions when published data was unavailable, and the use of multiple sources for survival data.

BRCA1 and BRCA2 mutations sensitize to chemotherapy in patient-derived pancreatic cancer xenografts.

BACKGROUND: Germline mutations of the BRCA tumour suppressors have been associated with increased risk of pancreatic cancer. Clinical evidence suggests that these patients may be more sensitive to treatment with cisplatin. As the frequency of germline BRCA mutations is low, definitive experimental data to support the clinical observations are still missing. METHODS: We tested gemcitabine and cisplatin sensitivity of four BRCA1 and BRCA2 mutant and three BRCA1 and BRCA2 wild-type (WT) patient-derived pancreatic cancer xenografts. RESULTS: We observed treatment sensitivity to gemcitabine and cisplatin in the BRCA WT and mutant models. The BRCA1 and BRCA2 mutant xenografts were significantly more sensitive to cisplatin although these models also showed sensitivity to gemcitabine. The BRCA1 and BRCA2 WT models showed sensitivity to gemcitabine but not cisplatin. Treatment sensitivity in the xenograft models closely resembled treatment response in the corresponding patients. DISCUSSION: We have characterised a panel of xenografts derived from pancreatic cancer patients carrying germline BRCA mutations, and shown that their genetic features resemble the patient donor. Our results support further clinical testing of treatment regimens combining gemcitabine and platinum drugs in this patient population, as well as preclinical research aiming to identify mechanisms of cisplatin resistance in BRCA mutant pancreatic cancers.

Costs of treatment for elderly women with advanced ovarian cancer in a Medicare population.

OBJECTIVE: To analyze the cost of treating women with advanced stage epithelial ovarian cancer (EOC) undergoing primary debulking surgery (PDS) or neo-adjuvant chemotherapy (NACT). METHODS: The Surveillance, Epidemiology, and End Results (SEER) - Medicare database (1992 to 2009) was used to evaluate the 7-month cost of care following PDS and NACT for advanced EOC. Multivariate analyses were used to evaluate differences between women treated by PDS and NACT on cost and survival. RESULTS: Of the 4506 women eligible for analysis, 82.4% underwent PDS and 17.6% received NACT. Eighty-five percent with stage IIIC and 78.5% with stage IV EOC underwent PDS (p<0.0001). No significant difference in the median cost of care between PDS and NACT existed in women with stage IIIC EOC ($59,801 vs. $59,905). There was a 12% increase in adjusted cost of care for stage IV patients ($63,131 vs. $55,302) who received PDS (p<0.0001). Increasing Charlson score was associated with an increase in 7-month cost of care in both stages. NACT was associated with a decreased 5-year overall survival in women with stage IIIC EOC (HR=1.27, 95% CI: 1.10-1.47) and stage IV EOC (HR=1.19, 95% CI: 1.03-1.37) compared to PDS. CONCLUSION: NACT and PDS are comparable in cost for women with stage IIIC EOC, and PDS is minimally more expensive for women with stage IV EOC. PDS was associated with an increase 5-year overall survival. Future investigations should include cost-effectiveness analyses where additional measures such as quality adjusted life years and propensity scored survival are included.

Cooperation of Blm and Mus81 in development, fertility, genomic integrity and cancer suppression.

BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81(-/-) and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.

New paradigms in the surgical and adjuvant treatment of ovarian cancer.

Advanced stage epithelial ovarian cancer is difficult to treat. Despite advances in surgical resection and adjuvant chemotherapy the majority of patients suffer from disease recurrence. In an effort to improve oncologic outcomes, including progression free and overall survival, novel surgical paradigms and chemotherapeutic techniques have emerged over the past decade. An emphasis has been placed on achieving maximal surgical cytoreduction (defined as no visible residual disease) at completion of surgery, in combination with intra-peritoneal (IP) chemotherapy, as well as hyperthermic IP chemotherapy (HIPEC). This review article will discuss the evolution of surgical cytoreduction in the treatment of advanced stage epithelial ovarian cancer, as well as the development of adjuvant treatments that increasingly utilize the biologic advantage provided by microscopic residual disease.

An arranged marriage for precision medicine: hypoxia and genomic assays in localized prostate cancer radiotherapy.

Prostate cancer (CaP) is the most commonly diagnosed malignancy in males in the Western world with one in six males diagnosed in their lifetime. Current clinical prognostication groupings use pathologic Gleason score, pre-treatment prostatic-specific antigen and Union for International Cancer Control-TNM staging to place patients with localized CaP into low-, intermediate- and high-risk categories. These categories represent an increasing risk of biochemical failure and CaP-specific mortality rates, they also reflect the need for increasing treatment intensity and justification for increased side effects. In this article, we point out that 30-50% of patients will still fail image-guided radiotherapy or surgery despite the judicious use of clinical risk categories owing to interpatient heterogeneity in treatment response. To improve treatment individualization, better predictors of prognosis and radiotherapy treatment response are needed to triage patients to bespoke and intensified CaP treatment protocols. These should include the use of pre-treatment genomic tests based on DNA or RNA indices and/or assays that reflect cancer metabolism, such as hypoxia assays, to define patient-specific CaP progression and aggression. More importantly, it is argued that these novel prognostic assays could be even more useful if combined together to drive forward precision cancer medicine for localized CaP.