The effect of assessing genetic risk of prostate cancer on the use of PSA tests in primary care: A cluster randomized controlled trial.

BACKGROUND: Assessing genetic lifetime risk for prostate cancer has been proposed as a means of risk stratification to identify those for whom prostate-specific antigen (PSA) testing is likely to be most valuable. This project aimed to test the effect of introducing a genetic test for lifetime risk of prostate cancer in general practice on future PSA testing. METHODS AND FINDINGS: We performed a cluster randomized controlled trial with randomization at the level of general practices (73 in each of two arms) in the Central Region (Region Midtjylland) of Denmark. In intervention practices, men were offered a genetic test (based on genotyping of 33 risk-associated single nucleotide polymorphisms) in addition to the standard PSA test that informed them about lifetime genetic risk of prostate cancer and distinguished between "normal" and "high" risk. The primary outcome was the proportion of men having a repeated PSA test within 2 years. A multilevel logistic regression model was used to test the association. After applying the exclusion criteria, 3,558 men were recruited in intervention practices, with 1,235 (34.7%) receiving the genetic test, and 4,242 men were recruited in control practices. Men with high genetic risk had a higher propensity for repeated PSA testing within 2 years than men with normal genetic risk (odds ratio [OR] = 8.94, p < 0.01). The study was conducted in routine practice and had some selection bias, which is evidenced by the relatively large proportion of younger and higher income participants taking the genetic test. CONCLUSIONS: Providing general practitioners (GPs) with access to a genetic test to assess lifetime risk of prostate cancer did not reduce the overall number of future PSA tests. However, among men who had a genetic test, knowledge of genetic risk significantly influenced future PSA testing. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov, number NCT01739062.

Putative cis-regulatory drivers in colorectal cancer.

The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis.

A novel combined miRNA and methylation marker panel (miMe) for prediction of prostate cancer outcome after radical prostatectomy.

Improved prognostic biomarkers are needed to guide personalized prostate cancer (PC) treatment decisions. Due to the prominent molecular heterogeneity of PC, multimarker panels may be more robust. Here, 25 selected top-candidate miRNA and methylation markers for PC were profiled by qPCR in malignant radical prostatectomy (RP) tissue specimens from 198 PC patients (Cohort 1, training). Using GLMnet, we trained a novel multimarker model (miMe) comprising nine miRNAs and three methylation markers that predicted postoperative biochemical recurrence (BCR) independently of the established clinicopathological CAPRA-S nomogram in Cox multivariate regression analysis in Cohort 1 (HR [95% CI]: 1.53 [1.26-1.84], p < 0.001). This result was successfully validated in two independent RP cohorts (Cohort 2, n = 159: HR [95% CI]: 1.35 [1.06-1.73], p = 0.015. TCGA, n = 350: HR [95% CI]: 1.34 [1.01-1.77], p = 0.04). Notably, in CAPRA-S low-risk patients, a high miMe score was associated with >6 times higher risk of BCR, suggesting that miMe may help identify PC patients at high risk of progression despite favorable clinicopathological factors postsurgery. Finally, miMe was a significant predictor of cancer-specific survival (p = 0.019, log-rank test) in a merged analysis of 357 RP patients. In conclusion, we trained, tested and validated a novel 12-marker panel (miMe) that showed significant independent prognostic value in three RP cohorts. In the future, combining miMe score with existing clinical nomograms may improve PC risk stratification and thus help guide treatment decisions.

FGFR3, TERT and OTX1 as a Urinary Biomarker Combination for Surveillance of Patients with Bladder Cancer in a Large Prospective Multicenter Study.

PURPOSE: Patients with nonmuscle invasive bladder cancer are followed with frequent cystoscopies. In this study FGFR3, TERT and OTX1 were investigated as a diagnostic urinary marker combination during followup of patients with primary nonmuscle invasive bladder cancer. MATERIALS AND METHODS: In this international, multicenter, prospective study 977 patients with nonmuscle invasive bladder cancer were included. A total of 2,496 urine samples were collected prior to cystoscopy during regular visits. Sensitivity was estimated to detect concomitant recurrences. Kaplan-Meier curves were used to estimate the development of future recurrences after urinalysis and a negative cystoscopy. RESULTS: Sensitivity of the assay combination for recurrence detection was 57% in patients with primary low grade, nonmuscle invasive bladder cancer. However, sensitivity was 83% for recurrences that were pT1 or muscle invasive bladder cancer. Of the cases 2% progressed to muscle invasive bladder cancer. Sensitivity for recurrence detection in patients with primary high grade disease was 72% and 7% of them had progression to muscle invasive bladder cancer. When no concomitant tumor was found by cystoscopy, positive urine samples were more frequently followed by a recurrence over time compared to a negative urine sample (58% vs 36%, p <0.001). High stage recurrences were identified within 1 year after a positive urine test and a negative cystoscopy. CONCLUSIONS: Recurrences in patients with primary nonmuscle invasive bladder cancer can be detected by a combination of urine assays. This study supports the value of urinalysis as an alternative diagnostic tool in patients presenting with low grade tumors and as a means to identify high stage tumors earlier.

Validation of a DNA Methylation-Mutation Urine Assay to Select Patients with Hematuria for Cystoscopy.

PURPOSE: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay. MATERIALS AND METHODS: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer. RESULTS: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92-0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy. CONCLUSIONS: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.

Analysis of circulating tumour DNA to monitor disease burden following colorectal cancer surgery.

OBJECTIVE: To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence. DESIGN: We have established efficient procedures for identification of SSVs by next-generation sequencing and subsequent quantification of 3-6 SSVs in plasma. The consequence of intratumour heterogeneity on our approach was assessed. The level of circulating tumour DNA (ctDNA) was quantified in 151 serial plasma samples from six relapsing and five non-relapsing colorectal cancer (CRC) patients by droplet digital PCR, and correlated to clinical findings. RESULTS: Up to six personalised assays were designed for each patient. Our approach enabled efficient temporal assessment of disease status, response to surgical and oncological intervention, and early detection of incipient recurrence. Our approach provided 2-15 (mean 10) months' lead time on detection of metastatic recurrence compared to conventional follow-up. The sensitivity and specificity of the SSVs in terms of detecting postsurgery relapse were 100%. CONCLUSIONS: We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

BS-SNPer: SNP calling in bisulfite-seq data.

UNLABELLED: Sodium bisulfite conversion followed by sequencing (BS-Seq, such as whole genome bisulfite sequencing or reduced representation bisulfite sequencing) has become popular for studying human epigenetic profiles. Identifying single nucleotide polymorphisms (SNPs) is important for quantification of methylation levels and for study of allele-specific epigenetic events such as imprinting. However, SNP calling in such data is complex and time consuming. Here, we present an ultrafast and memory-efficient package named BS-SNPer for the exploration of SNP sites from BS-Seq data. Compared with Bis-SNP, a popular BS-Seq specific SNP caller, BS-SNPer is over 100 times faster and uses less memory. BS-SNPer also offers higher sensitivity and specificity compared with existing methods. AVAILABILITY AND IMPLEMENTATION: BS-SNPer is written in C++ and Perl, and is freely available at https://github.com/hellbelly/BS-Snper.

Prevalence of the HOXB13 G84E mutation in Danish men undergoing radical prostatectomy and its correlations with prostate cancer risk and aggressiveness.

OBJECTIVES: To determine the prevalence of the HOXB13 G84E mutation (rs138213197) in Danish men with or without prostate cancer (PCa) and to investigate possible correlations between HOXB13 mutation status and clinicopathological characteristics associated with tumour aggressiveness. MATERIALS AND METHODS: We conducted a case-control study including 995 men with PCa (cases) who underwent radical prostatectomy (RP) between 1997 and 2011 at the Department of Urology, Aarhus University Hospital, Denmark. As controls, we used 1622 healthy men with a normal prostate specific antigen (PSA) level. RESULTS: The HOXB13 G84E mutation was identified in 0.49% of controls and in 2.51% of PCa cases. The mutation was associated with a 5.12-fold increased relative risk (RR) of PCa (95% confidence interval [CI] 2.26-13.38; P = 13 × 10(-6) ). Furthermore, carriers of the risk allele were significantly more likely to have a higher PSA level at diagnosis (mean PSA 19.9 vs 13.6 ng/mL; P = 0.032), a pathological Gleason score ≥7 (83.3 vs 60.9%; P = 0.032), and positive surgical margins (56.0 vs 28.5%; P = 0.006) than non-carriers. Risk allele carriers were also more likely to have aggressive disease (54.2 vs 28.6%; P = 0.011), as defined by a preoperative PSA ≥20 ng/mL, pathological Gleason score ≥ (4+3) and/or presence of regional/distant disease. At a mean follow-up of 7 months, we found no significant association between HOXB13 mutation status and biochemical recurrence in this cohort of men who underwent RP. CONCLUSIONS: This is the first study to investigate the HOXB13 G84E mutation in Danish men. The mutation was detected in 0.49% of controls and in 2.51% of cases, and was associated with 5.12-fold increased RR of being diagnosed with PCa. In our RP cohort, HOXB13 mutation carriers were more likely to develop aggressive PCa. Further studies are needed to assess the potential of HOXB13 for future targeted screening approaches.

Performance of the colorectal cancer screening marker Sept9 is influenced by age, diabetes and arthritis: a nested case-control study.

BACKGROUND: Annually, colorectal cancer (CRC) is diagnosed in >1.4 million subjects worldwide and incidence is increasing. Much effort has therefore been focused on screening, which has proven to reduce cancer-related mortality. The Sept9 DNA-methylation assay is among the most well studied blood-based screening markers. However, earlier reported performances may be misleading: the Sept9 test was recently examined in two screening based cohorts and yielded performances lower than expected. We hypothesize that comorbidities and/or demographic characteristics affect the results of the Sept9 test. METHODS: Using a retrospective nested case-control study design, we studied plasma from 150 cancer and 150 controls selected from a well-characterized cohort of 4698 subjects referred for diagnostic colonoscopy due to CRC-related symptoms. The cases and controls were matched on age and gender, and moreover cases were stratified on tumor-site and tumor-stage. The selected cohort included a wide range of comorbidities. Plasma Sept9 levels were assessed using a commercially available PCR based assay (Epi-proColon). RESULTS: Clinical sensitivity for CRC stages I-IV was 37 %, 91 %, 77 %, and 89 %, and the overall sensitivity 73 % (95 % CI, 64-80 %) and specificity 82 % (95 % CI, 75-88 %), respectively. Age >65 was associated with both increased false positive and false negative results (p < 0.05). Arthritis was associated with a higher false negative rate (p = 0.005) whereas Arteriosclerosis was associated with a higher false positive rate (p = 0.007). Diabetes was associated with Sept9 positivity with an OR of 5.2 (95 % CI 1.4-19.1). When the performance of Sept9 was adjusted for these parameters in a final multivariate regression model, the OR for a positive Sept9 test to be associated with CRC increased from 8.25 (95 % CI 4.83-14.09) to 29.46 (95 % CI 12.58-69.02). CONCLUSIONS: The results indicate that the performance of the Sept9 assay is negatively affected by several factors commonly associated with CRC screening populations: early-stage disease, age > 65 years, diabetes, arthritis, and arteriosclerosis. This should be taken into account if the Sept9 assay is used as a single marker for CRC screening, but may also have a wider impact, as it is likely that such factors may affect other blood based DNA markers as well.

A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition.

Disruption of histone acetylation patterns is a common feature of cancer cells, but very little is known about its genetic basis. We have identified truncating mutations in one of the primary human histone deacetylases, HDAC2, in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. The presence of the HDAC2 frameshift mutation causes a loss of HDAC2 protein expression and enzymatic activity and renders these cells more resistant to the usual antiproliferative and proapoptotic effects of histone deacetylase inhibitors. As such drugs may serve as therapeutic agents for cancer, our findings support the use of HDAC2 mutational status in future pharmacogenetic treatment of these individuals.

Multicenter validation of cyclin D1, MCM7, TRIM29, and UBE2C as prognostic protein markers in non-muscle-invasive bladder cancer.

Transcripts from the four genes encoding cyclin D1, MCM7, TRIM29, and UBE2C have previously been included in gene expression signatures for outcome prediction in stage Ta/T1 urothelial carcinomas. We investigated the prognostic value of the protein expressions in Ta/T1 urothelial carcinomas patients. We used four different tissue microarrays (TMAs) with a total of 859 Ta/T1 urothelial carcinomas from Danish, Swedish, Spanish, and Taiwanese patient cohorts with long-term follow-up. Protein expression was measured by IHC, and antibody specificity was validated by Western blotting. We found the expression of cyclin D1, MCM7, TRIM29, and UBE2C to be significantly associated with progression to muscle-invasive bladder cancer (log-rank test; P < 0.001) in the Danish training cohort (n = 283). Multivariate Cox regression analysis identified cyclin D1 (P = 0.003), TRIM29 (P = 0.001), and UBE2C (P < 0.001) as independent prognostic markers. The prognostic value of the four proteins was validated in a joint validation cohort from Sweden, Spain, and Taiwan (n = 576). Computer-assisted image analysis of the prognostic markers produced results comparable to those obtained by manual scoring. Finally, a four-protein maximum-likelihood classifier was trained on the Danish training cohort and applied to the validation cohort. The four protein markers may help optimize treatment of patients with Ta/T1 bladder cancer. Additional prospective studies are needed for further validation of their clinical relevance.

High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer.

BACKGROUND: The standard treatment for non-metastatic muscle-invasive bladder cancer (stages T2-T4a) is radical cystectomy with lymphadenectomy. However, patients undergoing cystectomy show metastatic spread in 25% of cases and these patients will have limited benefit from surgery. Identification of patients with high risk of lymph node metastasis will help select patients that may benefit from neoadjuvant and/or adjuvant chemotherapy. METHODS: RNA was procured by laser micro dissection of primary bladder tumors and corresponding lymph node metastases for Affymetrix U133 Plus 2.0 Gene Chip expression profiling. A publically available dataset was used for identification of the best candidate markers, and these were validated using immunohistochemistry in an independent patient cohort of 368 patients. RESULTS: Gene Set Enrichment Analysis showed significant enrichment for e.g. metastatic signatures in the metastasizing tumors, and a set of 12 genes significantly associated with lymph node metastasis was identified. Tumors did not cluster according to their metastatic ability when analyzing gene expression profiles using hierarchical cluster analysis. However, half (6/12) of the primary tumor clustered together with matching lymph node metastases, indicating a large degree of intra-patient similarity in these patients. Immunohistochemical analysis of 368 tumors from cystectomized patients showed high expression of GEM (P = 0.033; HR = 1.46) and EDNRA (P = 0.046; HR = 1.60) was significantly associated with decreased cancer-specific survival. CONCLUSIONS: GEM and EDNRA were identified as promising prognostic markers for patients with advanced bladder cancer. The clinical relevance of GEM and EDNRA should be evaluated in independent prospective studies.

The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers.

An increasing body of evidence connects alterations in the process of alternative splicing with cancer development and progression. However, a direct role of splicing factors as drivers of cancer development is mostly unknown. We analysed the gene copy number of several splicing factors in colon and lung tumours, and found that the gene encoding for the splicing factor SRSF6 is amplified and over-expressed in these cancers. Moreover, over-expression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them to be tumourigenic in mice. In contrast, knock-down of SRSF6 in lung and colon cancer cell lines inhibited their tumourigenic abilities. SRSF6 up- or down-regulation altered the splicing of several tumour suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumour-suppressive isoforms. Our data suggest that the splicing factor SRSF6 is an oncoprotein that regulates the proliferation and survival of lung and colon cancer cells.

Prognostic DNA methylation markers for prostate cancer.

Prostate cancer (PC) is the most commonly diagnosed neoplasm and the third most common cause of cancer-related death amongst men in the Western world. PC is a clinically highly heterogeneous disease, and distinction between aggressive and indolent disease is a major challenge for the management of PC. Currently, no biomarkers or prognostic tools are able to accurately predict tumor progression at the time of diagnosis. Thus, improved biomarkers for PC prognosis are urgently needed. This review focuses on the prognostic potential of DNA methylation biomarkers for PC. Epigenetic changes are hallmarks of PC and associated with malignant initiation as well as tumor progression. Moreover, DNA methylation is the most frequently studied epigenetic alteration in PC, and the prognostic potential of DNA methylation markers for PC has been demonstrated in multiple studies. The most promising methylation marker candidates identified so far include PITX2, C1orf114 (CCDC181) and the GABRE~miR-452~miR-224 locus, in addition to the three-gene signature AOX1/C1orf114/HAPLN3. Several other biomarker candidates have also been investigated, but with less stringent clinical validation and/or conflicting evidence regarding their possible prognostic value available at this time. Here, we review the current evidence for the prognostic potential of DNA methylation markers in PC.

Non-CpG island promoter hypomethylation and miR-149 regulate the expression of SRPX2 in colorectal cancer.

Gene silencing by DNA hypermethylation of CpG islands is a well-characterized phenomenon in cancer. The effect of hypomethylation in particular of non-CpG island genes is much less well described. By genome-wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ -0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non-CpG island genes and two of them, APOLD1 (Spearman's ρ = -0.82) and SRPX2 (Spearman's ρ = -0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p < 0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR-149 as a potential novel post-transcriptional regulator of SRPX2. In carcinoma tissue, miR-149 was downregulated and inversely correlated to SRPX2 (ρ = -0.77). Furthermore, ectopic expression of miR-149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non-CpG island-associated promoters deregulate gene expression nearly as frequent as do CpG-island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR-149.

MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.

Cathepsin E, maspin, Plk1, and survivin are promising prognostic protein markers for progression in non-muscle invasive bladder cancer.

Bladder cancer is a common cancer with particularly high recurrence after transurethral resection. In this study, we investigated the prognostic value of the protein expression of cathepsin E, maspin, polo-like kinase 1 (Plk1), and survivin in patients with stage Ta and T1 urothelial carcinomas. Transcripts from the four genes encoding these proteins were previously included in gene expression signatures for outcome prediction for Ta/T1 bladder cancer. We used three different tissue microarrays with 693 non-muscle invasive urothelial carcinomas from Danish, Swedish, and Spanish patient cohorts with long-term follow-up. Protein expression was measured by immunohistochemistry, and antibody specificity was validated by Western blotting. In the Danish patient cohort, we found the expression of cathepsin E, maspin, Plk1, and survivin to be significantly associated with progression to stage T2 to T4 bladder cancer (for each marker: log-rank test; P < 0.001). Multivariate Cox regression analysis identified cathepsin E (P < 0.001), Plk1 (P = 0.021), maspin (P = 0.001), and survivin (P = 0.001) as independent prognostic markers. Furthermore, maspin, survivin, and cathepsin E expression significantly subgrouped patients already stratified by European Organization for Research and Treatment of Cancer risk scores. Finally, we successfully validated the results in tumors from 410 patients from both Sweden and Spain. We conclude that all four protein markers may have prognostic value in non-muscle invasive bladder cancer for guiding optimal treatment of patients. Additional prospective studies are needed for further validation of the clinical relevance of this marker panel.

Combinations of urinary biomarkers for surveillance of patients with incident nonmuscle invasive bladder cancer: the European FP7 UROMOL project.

PURPOSE: We determined a combination of markers with optimal sensitivity to detect recurrence in voided urine after resection of an incident low grade, nonmuscle invasive bladder tumor. MATERIALS AND METHODS: A total of 136 patients with G1/G2 nonmuscle invasive bladder tumor were included in the study at transurethral resection of the incident tumor. At least 3 followup urine samples were required for patient selection. DNA was extracted from the incident tumor and cell pellets of subsequently collected urine samples. We performed FGFR3, PIK3CA and RAS mutation analysis, and microsatellite and methylation analysis on tissue and urine DNA samples. RESULTS: We obtained 716 urine samples. The 136 patients experienced a total of 552 recurrences during a median 3-year followup. Sensitivity for detecting a recurrent tumor varied between 66% and 68% for the molecular tests after patient stratification based on tumor DNA analysis. A combination of markers increased sensitivity but decreased the number of patients eligible for a certain test combination. Combining urine cytology with FGFR3 analysis without stratifying for FGFR3 status of the incident tumor increased sensitivity from 56% to 76%. CONCLUSIONS: A combination of markers increased the percentage of patients eligible for urine based followup and the sensitivity of recurrence detection. Adding FGFR3 analysis to urine cytology could be valuable for noninvasive followup of patients with nonmuscle invasive bladder cancer.

Alternative splicing of SLC39A14 in colorectal cancer is regulated by the Wnt pathway.

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ß-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ß-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

Identifying distinct classes of bladder carcinoma using microarrays.

Bladder cancer is a common malignant disease characterized by frequent recurrences. The stage of disease at diagnosis and the presence of surrounding carcinoma in situ are important in determining the disease course of an affected individual. Despite considerable effort, no accepted immunohistological or molecular markers have been identified to define clinically relevant subsets of bladder cancer. Here we report the identification of clinically relevant subclasses of bladder carcinoma using expression microarray analysis of 40 well characterized bladder tumors. Hierarchical cluster analysis identified three major stages, Ta, T1 and T2-4, with the Ta tumors further classified into subgroups. We built a 32-gene molecular classifier using a cross-validation approach that was able to classify benign and muscle-invasive tumors with close correlation to pathological staging in an independent test set of 68 tumors. The classifier provided new predictive information on disease progression in Ta tumors compared with conventional staging (P < 0.005). To delineate non-recurring Ta tumors from frequently recurring Ta tumors, we analyzed expression patterns in 31 tumors by applying a supervised learning classification methodology, which classified 75% of the samples correctly (P < 0.006). Furthermore, gene expression profiles characterizing each stage and subtype identified their biological properties, producing new potential targets for therapy.