Effects of butyrate on the colon cancer cell phenotype-chemosensitization and upregulation of proteins implicated in cancer progression

Colon cancer is the second leading cause of cancer mortality in the U.S. Surgery is the only truly effective human colon cancer (HCC) therapy due to marked intrinsic drug resistance. The inefficacy of therapies developed for metastatic HCC suggests that advances in colon cancer chemoprevention and chemotherapy will be needed to reduce HCC mortality. The dietary fiber metabolite butyrate (NaB) is a candidate cancer chemopreventive agent that inhibits growth, promotes differentiation and stimulates apoptosis of HCC cells. Epidemiological and experimental studies suggest that dietary fiber protects against the development of HCC, however, recent large prospective trials have not found significant protection. ^ The first central hypothesis of this dissertation project is that the diversity of phenotypic changes induced by NaB in HCC cells includes molecular alterations that oppose its chemopreventive action and thereby limit its efficacy. We investigated the effect of NaB on the expression/activity of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in HCC HT29 cells. NaB treatment induced a 13-fold increase in EGFR expression in concert with its chemopreventive action in vitro, i.e., induction of growth suppression and G1 arrest, apoptosis and a differentiated phenotype. NaB-induced EGFR was active based on multiple lines of evidence. The EGFR was: (1) heavily phosphorylated at Tyrosine (P-Tyr); (2) associated with the cytoskeleton; (3) localized at the cell surface, and activated in response to EGF; and (4) NaB treatment of the cells induced activation of the EGFR effector Erk1/2. NaB treatment also induced a 7-fold increase in COX-2 expression. The NaB-induced COX-2 was active based on significantly increased PGE2 production. ^ The second central hypothesis is that NaB treatment would render HCC cells more chemosensitive to chemotherapy agents based on the increased apoptotic index induced by NaB. NaB treatment chemosensitized HT29 cells to 5-FU and doxorubicin, despite increases in the expression of P-glycoprotein and a related drug resistance protein (MRP). ^ These results raise the intriguing possibility that the chemopreventive effects of fiber may require concomitant treatment with EGFR and/or COX-2 inhibitors. Similarly, NaB may be a rational drug to combine with existing chemotherapeutic agents for the management of advanced HCC patients. ^

Trastuzumab treatment beyond progression in advanced breast cancer: patterns of care in six Swiss breast cancer centers.

BACKGROUND Trastuzumab is an established treatment for HER2-positive breast cancer (BC). We analyzed Swiss patterns of care in patients with HER2-positive BC after disease progression on trastuzumab-containing therapy for metastatic BC (MBC). PATIENTS AND METHODS A retrospective analysis was performed in six Swiss BC centers. Patients with HER2-positive MBC treated with at least one infusion of trastuzumab for advanced disease between January 2006 and December 2007 were identified. Treatment patterns in first and further lines were analyzed. RESULTS All of the 72 identified patients received trastuzumab as their first palliative anti-HER2 therapy, either as monotherapy (n = 23) or in combination with chemotherapy (typically taxane or vinorelbine; n = 49). Median time to progression was 8.1, 8.0 and 7.9 months in the monotherapy, trastuzumab-taxane and trastuzumab-vinorelbine cohorts, respectively. After progression on first-line anti-HER2 therapy, trastuzumab was continued in 67 of 68 patients who received further therapy. One patient received second-line lapatinib plus capecitabine. The median duration of anti-HER2 therapy was 20 months. Patients received a median of 4 lines of anti-HER2 therapy. CONCLUSIONS Durable responses were achieved with repeated exposure to anti-HER2 therapy. In a selected patient population, trastuzumab monotherapy appears to be a reasonable first-line treatment option.

Variation in precursor lesions of pancreatic cancer among high-risk groups

PURPOSE Pancreatic ductal adenocarcinoma (PDAC) surveillance programs are currently offered to high-risk individuals aiming to detect precursor lesions or PDAC at an early stage. We assessed differences in frequency and behavior of precursor lesions and PDAC between two high-risk groups. EXPERIMENTAL DESIGN Individuals with a p16-Leiden germline mutation (N = 116; median age 54 years) and individuals from familial pancreatic cancer (FPC) families (N = 125; median age 47 years) were offered annual surveillance by MRI and magnetic resonance cholangiopancreatography (MRCP) with or without endoscopic ultrasound (EUS) for a median surveillance period of 34 months (0-127 months) or 36 months (0-110 months), respectively. Detailed information was collected on pancreatic cystic lesions detected on MRCP and precursor lesions in surgical specimens of patients who underwent pancreatic surgery. RESULTS Cystic lesions were more common in the FPC cohort (42% vs. 16% in p16-Leiden cohort), whereas PDAC was more common in the p16-Leiden cohort (7% vs. 0.8% in FPC cohort). Intraductal papillary mucinous neoplasm (IPMN) was a common finding in surgical specimens of FPC-individuals, and was only found in two patients of the p16-Leiden cohort. In the p16-Leiden cohort, a substantial proportion of cystic lesions showed growth or malignant transformation during follow-up, whereas in FPC individuals most cystic lesions remain stable. CONCLUSION In p16-Leiden mutation carriers, cystic lesions have a higher malignant potential than in FPC-individuals. On the basis of these findings, a more intensive surveillance program may be considered in this high-risk group.

Discrete Improvement in Racial Disparity in Survival among Patients with Stage IV Colorectal Cancer: a 21-Year Population-Based Analysis

Purpose Recently, multiple clinical trials have demonstrated improved outcomes in patients with metastatic colorectal cancer. This study investigated if the improved survival is race dependent. Patients and Methods Overall and cancer-specific survival of 77,490 White and Black patients with metastatic colorectal cancer from the 1988–2008 Surveillance Epidemiology and End Results registry were compared using unadjusted and multivariable adjusted Cox proportional hazard regression as well as competing risk analyses. Results Median age was 69 years, 47.4 % were female and 86.0 % White. Median survival was 11 months overall, with an overall increase from 8 to 14 months between 1988 and 2008. Overall survival increased from 8 to 14 months for White, and from 6 to 13 months for Black patients. After multivariable adjustment, the following parameters were associated with better survival: White, female, younger, better educated and married patients, patients with higher income and living in urban areas, patients with rectosigmoid junction and rectal cancer, undergoing cancer-directed surgery, having well/moderately differentiated, and N0 tumors (p<0.05 for all covariates). Discrepancies in overall survival based on race did not change significantly over time; however, there was a significant decrease of cancer-specific survival discrepancies over time between White and Black patients with a hazard ratio of 0.995 (95 % confidence interval 0.991–1.000) per year (p=0.03). Conclusion A clinically relevant overall survival increase was found from 1988 to 2008 in this population-based analysis for both White and Black patients with metastatic colorectal cancer. Although both White and Black patients benefitted from this improvement, a slight discrepancy between the two groups remained.

Prognostic role of FGFR1 amplification in early-stage non-small cell lung cancer.

Background:Recently, fibroblast growth factor receptor 1 (FGFR1) was discovered in squamous cell carcinomas (SCC) of the lung with FGFR1 amplification described as a promising predictive marker for anti-FGFR inhibitor treatment. Only few data are available regarding prevalence, prognostic significance and clinico-pathological characteristics of FGFR1-amplified and early-stage non-small cell lung carcinomas (NSCLC). We therefore investigated the FGFR1 gene status in a large number of well-characterised early-stage NSCLC.Methods:FGFR1 gene status was evaluated using a commercially available fluorescent in situ hybridisation (FISH) probe on a tissue microarray (TMA). This TMA harbours 329 resected, formalin-fixed and paraffin-embedded, nodal-negative NSCLC with a UICC stage I-II. The FISH results were correlated with clinico-pathological features and overall survival (OS).Results:The prevalence of an FGFR1 amplification was 12.5% (41/329) and was significantly (P<0.0001) higher in squamous cell carcinoma (SCC) (20.7%) than in adenocarcinoma (2.2%) and large cell carcinoma (13%). Multivariate analysis revealed significantly (P=0.0367) worse 5-year OS in patients with an FGFR1-amplified NSCLC.Conclusions:FGFR1 amplification is common in early-stage SCC of the lung and is an independent and adverse prognostic marker. Its potential role as a predictive marker for targeted therapies or adjuvant treatment needs further investigation.

The adherence to initial processes of care in elderly patients with acute venous thromboembolism.

BACKGROUND We aimed to assess whether elderly patients with acute venous thromboembolism (VTE) receive recommended initial processes of care and to identify predictors of process adherence. METHODS We prospectively studied in- and outpatients aged ≥65 years with acute symptomatic VTE in a multicenter cohort study from nine Swiss university- and non-university hospitals between September 2009 and March 2011. We systematically assessed whether initial processes of care, which are recommended by the 2008 American College of Chest Physicians guidelines, were performed in each patient. We used multivariable logistic models to identify patient factors independently associated with process adherence. RESULTS Our cohort comprised 950 patients (mean age 76 years). Of these, 86% (645/750) received parenteral anticoagulation for ≥5 days, 54% (405/750) had oral anticoagulation started on the first treatment day, and 37% (274/750) had an international normalized ratio (INR) ≥2 for ≥24 hours before parenteral anticoagulation was discontinued. Overall, 35% (53/153) of patients with cancer received low-molecular-weight heparin monotherapy and 72% (304/423) of patients with symptomatic deep vein thrombosis were prescribed compression stockings. In multivariate analyses, symptomatic pulmonary embolism, hospital-acquired VTE, and concomitant antiplatelet therapy were associated with a significantly lower anticoagulation-related process adherence. CONCLUSIONS Adherence to several recommended processes of care was suboptimal in elderly patients with VTE. Quality of care interventions should particularly focus on processes with low adherence, such as the prescription of continued low-molecular-weight heparin therapy in patients with cancer and the achievement of an INR ≥2 for ≥24 hours before parenteral anticoagulants are stopped.

Incidence of second malignancies for prostate cancer

INTRODUCTION There is a need to assess risk of second primary cancers in prostate cancer (PCa) patients, especially since PCa treatment may be associated with increased risk of second primary tumours. METHODS We calculated standardized incidence ratios (SIRs) for second primary tumours comparing men diagnosed with PCa between 1980 and 2010 in the Canton of Zurich, Switzerland (n = 20,559), and the general male population in the Canton. RESULTS A total of 1,718 men developed a second primary tumour after PCa diagnosis, with lung and colon cancer being the most common (15 and 13% respectively). The SIR for overall second primary cancer was 1.11 (95%CI: 1.06-1.17). Site-specific SIRs varied from 1.19 (1.05-1.34) to 2.89 (2.62-4.77) for lung and thyroid cancer, respectively. When stratified by treatment, the highest SIR was observed for thyroid cancer (3.57 (1.30-7.76)) when undergoing surgery, whereas liver cancer was common when treated with radiotherapy (3.21 (1.54-5.90)) and kidney bladder was most prevalent for those on hormonal treatment (3.15 (1.93-4.87)). Stratification by time since PCa diagnosis showed a lower risk of cancer for men with PCa compared to the general population for the first four years, but then a steep increase in risk was observed. CONCLUSION In the Canton of Zurich, there was an increased risk of second primary cancers among men with PCa compared to the general population. Increased diagnostic activity after PCa diagnosis may partly explain increased risks within the first years of diagnosis, but time-stratified analyses indicated that increased risks remained and even increased over time.

Primary prophylaxis for venous thromboembolism in ambulatory cancer patients receiving chemotherapy.

BACKGROUND Venous thromboembolism (VTE) often complicates the clinical course of cancer. The risk is further increased by chemotherapy, but the safety and efficacy of primary thromboprophylaxis in cancer patients treated with chemotherapy is uncertain. This is an update of a review first published in February 2012. OBJECTIVES To assess the efficacy and safety of primary thromboprophylaxis for VTE in ambulatory cancer patients receiving chemotherapy compared with placebo or no thromboprophylaxis. SEARCH METHODS For this update, the Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched May 2013), CENTRAL (2013, Issue 5), and clinical trials registries (up to June 2013). SELECTION CRITERIA Randomised controlled trials (RCTs) comparing any oral or parenteral anticoagulant or mechanical intervention to no intervention or placebo, or comparing two different anticoagulants. DATA COLLECTION AND ANALYSIS Data were extracted on methodological quality, patients, interventions, and outcomes including symptomatic VTE and major bleeding as the primary effectiveness and safety outcomes, respectively. MAIN RESULTS We identified 12 additional RCTs (6323 patients) in the updated search so that this update considered 21 trials with a total of 9861 patients, all evaluating pharmacological interventions and performed mainly in patients with advanced cancer. Overall, the risk of bias varied from low to high. One large trial of 3212 patients found a 64% (risk ratio (RR) 0.36, 95% confidence interval (CI) 0.22 to 0.60) reduction of symptomatic VTE with the ultra-low molecular weight heparin (uLMWH) semuloparin relative to placebo, with no apparent difference in major bleeding (RR 1.05, 95% CI 0.55 to 2.00). LMWH, when compared with inactive control, significantly reduced the incidence of symptomatic VTE (RR 0.53, 95% CI 0.38 to 0.75; no heterogeneity, Tau(2) = 0%) with similar rates of major bleeding events (RR 1.30, 95% CI 0.75 to 2.23). In patients with multiple myeloma, LMWH was associated with a significant reduction in symptomatic VTE when compared with the vitamin K antagonist warfarin (RR 0.33, 95% CI 0.14 to 0.83), while the difference between LMWH and aspirin was not statistically significant (RR 0.51, 95% CI 0.22 to 1.17). No major bleeding was observed in the patients treated with LMWH or warfarin and in less than 1% of those treated with aspirin. Only one study evaluated unfractionated heparin against inactive control and found an incidence of major bleeding of 1% in both study groups while not reporting on VTE. When compared with placebo, warfarin was associated with a statistically insignificant reduction of symptomatic VTE (RR 0.15, 95% CI 0.02 to 1.20). Antithrombin, evaluated in one study involving paediatric patients, had no significant effect on VTE nor major bleeding when compared with inactive control. The new oral factor Xa inhibitor apixaban was evaluated in a phase-II dose finding study that suggested a promising low rate of major bleeding (2.1% versus 3.3%) and symptomatic VTE (1.1% versus 10%) in comparison with placebo. AUTHORS' CONCLUSIONS In this update, we confirmed that primary thromboprophylaxis with LMWH significantly reduced the incidence of symptomatic VTE in ambulatory cancer patients treated with chemotherapy. In addition, the uLMWH semuloparin significantly reduced the incidence of symptomatic VTE. However, the broad confidence intervals around the estimates for major bleeding suggest caution in the use of anticoagulation and mandate additional studies to determine the risk to benefit ratio of anticoagulants in this setting. Despite the encouraging results of this review, routine prophylaxis in ambulatory cancer patients cannot be recommended before safety issues are adequately addressed.

Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study.

BACKGROUND Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. METHODS We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. FINDINGS Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. INTERPRETATION This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. FUNDING Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

Early development of human lymphomas in a prostate cancer xenograft program using triple knock-out immunocompromised mice

BACKGROUND There is an urgent need for preclinical models of prostate cancer; however, clinically relevant patient-derived prostate cancer xenografts (PDXs) are demanding to establish. METHODS Sixty-seven patients who were undergoing palliative transurethral surgery or radical prostatectomy for histologically confirmed, clinically relevant prostate cancer were included in the study. Fresh prostate cancer tissue was identified by frozen analysis in 48 patients. The cancer tissue was transplanted subcutaneously and under the renal capsule of NSG and NOG mice supplemented with human testosterone. All growing PDXs were evaluated by histology and immunohistochemistry. RESULTS Early assessment of the animals at least three months after transplantation included 27/48 (56.3%) eligible PDX cohorts. PDX growth was detected in 10/27 (37%) mouse cohorts. Eight of the ten PDXs were identified as human donor derived lymphomas, including seven Epstein Barr virus (EBV)-positive diffuse large B-cell lymphomas and one EBV-negative peripheral T-cell lymphoma. One sample consisted of benign prostatic tissue, and one sample comprised a benign epithelial cyst. Prostate cancer was not detected in any of the samples. CONCLUSIONS Tumors that arise within the first three months after prostate cancer xenografting may represent patient-derived EBV-positive lymphomas in up to 80% of the early growing PDXs when using triple knockout NSG immunocompromised mice. Therefore, lymphoma should be excluded in prostate cancer xenografts that do not resemble typical prostatic adenocarcinoma. Prostate 9999: XX-XX, 2014. © 2015 Wiley Periodicals, Inc.

Treatment and outcomes of urethral recurrence of urinary bladder cancer in women after radical cystectomy and orthotopic neobladder: a series of 12 cases

INTRODUCTION The incidence, treatment, and outcome of urethral recurrence (UR) after radical cystectomy (RC) for muscle-invasive bladder cancer with orthotopic neobladder in women have rarely been addressed in the literature. PATIENTS AND METHODS A total of 12 patients (median age at recurrence: 60 years) who experienced UR after RC with an orthotopic neobladder were selected for this study from a cohort of 456 women from participating institutions. The primary clinical and pathological characteristics at RC, including the manifestation of the UR and its treatment and outcome, were reviewed. RESULTS The primary bladder tumors in the 12 patients were urothelial carcinoma in 8 patients, squamous cell carcinoma and adenocarcinoma in 1 patient each, and mixed histology in 2 patients. Three patients (25%) had lymph node-positive disease at RC. The median time from RC to the detection of UR was 8 months (range 4-55). Eight recurrences manifested with clinical symptoms and 4 were detected during follow-up or during a diagnostic work-up for clinical symptoms caused by distant metastases. Treatment modalities were surgery, chemotherapy, radiotherapy, and bacillus Calmette-Guérin urethral instillations. Nine patients died of cancer. The median survival after the diagnosis of UR was 6 months. CONCLUSIONS UR after RC with an orthotopic neobladder in females is rare. Solitary, noninvasive recurrences have a favorable prognosis when detected early. Invasive recurrences are often associated with local and distant metastases and have a poor prognosis. © 2014 S. Karger AG, Basel.

Natural history of surgically treated high-risk prostate cancer

BACKGROUND No data exist on the patterns of biochemical recurrence (BCR) and their effect on survival in patients with high-risk prostate cancer (PCa) treated with surgery. The aim of our investigation was to evaluate the natural history of PCa in patients treated with radical prostatectomy (RP) alone. MATERIALS AND METHODS Overall, 2,065 patients with high-risk PCa treated with RP at 7 tertiary referral centers between 1991 and 2011 were identified. First, we calculated the probability of experiencing BCR after surgery. Particularly, we relied on conditional survival estimates for BCR after RP. Competing-risks regression analyses were then used to evaluate the effect of time to BCR on the risk of cancer-specific mortality (CSM). RESULTS Median follow-up was 70 months. Overall, the 5-year BCR-free survival rate was 55.2%. Given the BCR-free survivorship at 1, 2, 3, 4, and 5 years, the BCR-free survival rates improved by+7.6%,+4.1%,+4.8%,+3.2%, and+3.7%, respectively. Overall, the 10-year CSM rate was 14.8%. When patients were stratified according to time to BCR, patients experiencing BCR within 36 months from surgery had higher 10-year CSM rates compared with those experiencing late BCR (19.1% vs. 4.4%; P<0.001). At multivariate analyses, time to BCR represented an independent predictor of CSM (P<0.001). CONCLUSIONS Increasing time from surgery is associated with a reduction of the risk of subsequent BCR. Additionally, time to BCR represents a predictor of CSM in these patients. These results might help provide clinicians with better follow-up strategies and more aggressive treatments for early BCR.

Impact of synchronous metastasis distribution on cancer specific survival in renal cell carcinoma after radical nephrectomy with tumor thrombectomy

PURPOSE Metastatic renal cell carcinoma can be clinically diverse in terms of the pattern of metastatic disease and response to treatment. We studied the impact of metastasis and location on cancer specific survival. MATERIALS AND METHODS The records of 2,017 patients with renal cell cancer and tumor thrombus who underwent radical nephrectomy and tumor thrombectomy from 1971 to 2012 at 22 centers in the United States and Europe were analyzed. Number and location of synchronous metastases were compared with respect to patient cancer specific survival. Multivariable Cox regression models were used to quantify the impact of covariates. RESULTS Lymph node metastasis (155) or distant metastasis (725) was present in 880 (44%) patients. Of the patients with distant disease 385 (53%) had an isolated metastasis. The 5-year cancer specific survival was 51.3% (95% CI 48.6-53.9) for the entire group. On univariable analysis patients with isolated lymph node metastasis had a significantly worse cancer specific survival than those with a solitary distant metastasis. The location of distant metastasis did not have any significant effect on cancer specific survival. On multivariable analysis the presence of lymph node metastasis, isolated distant metastasis and multiple distant metastases were independently associated with cancer specific survival. Moreover higher tumor thrombus level, papillary histology and the use of postoperative systemic therapy were independently associated with worse cancer specific survival. CONCLUSIONS In our multi-institutional series of patients with renal cell cancer who underwent radical nephrectomy and tumor thrombectomy, almost half of the patients had synchronous lymph node or distant organ metastasis. Survival was superior in patients with solitary distant metastasis compared to isolated lymph node disease.

Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer

PURPOSE To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa). METHODS A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR. RESULTS A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89). CONCLUSIONS Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma

BACKGROUND Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. METHODS Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). RESULTS RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease. CONCLUSIONS A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.