Training and validation of a novel 4-miRNA ratio model (MiCaP) for prediction of postoperative outcome in prostate cancer patients.

Background: New molecular biomarkers for prostate cancer (PC) prognosis are urgently needed. Ratio-based models are attractive, as they require no additional normalization. Here, we train and independently validate a novel 4-miRNA prognostic ratio model for PC. Patients and methods: By genome-wide miRNA expression profiling of PC tissue samples from 123 men who underwent radical prostatectomy (RP) (PCA123, training cohort), we identified six top candidate prognostic miRNAs and systematically tested their ability to predict postoperative biochemical recurrence (BCR). The best miRNA-based prognostic ratio model (MiCaP) was validated in two independent cohorts (PCA352 and PCA476) including >800 RP patients in total. Clinical end points were BCR and prostate cancer-specific survival (CSS). The prognostic potential of MiCaP was assessed by univariate and multivariate Cox-regression analyses and Kaplan-Meier analyses. Results: We identified a 4-miRNA ratio model, MiCaP (miR-23a-3p×miR-10b-5p)/(miR-133a×miR-374b-5p), that predicted time to BCR independently of routine clinicopathologic variables in the training cohort (PCA123) and was successfully validated in two independent RP cohorts. In addition, MiCaP was a significant predictor of CSS in univariate analysis [HR 3.35 (95% CI 1.34 - 8.35), P = 0.0096] and in multivariate analysis [HR 2.43 (95% CI 1.45-4.07), P = 0.0210]. As proof-of-principle, we also analyzed MiCaP in plasma samples from 111 RP patients. A high MiCaP score in plasma was significantly associated with BCR (P = 0.0036, Kaplan-Meier analysis). Limitations include low mortality rates (CSS: 5.4%). Conclusions: We identified a novel 4-miRNA ratio model (MiCaP) with significant independent prognostic value in three RP cohorts, indicating promising potential to improve PC risk stratification.

Next-generation sequencing identifies germline MRE11A variants as markers of radiotherapy outcomes in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) can be cured by radical radiotherapy (RT). We previously found tumour MRE11 expression to be predictive of survival following RT in MIBC, and this was independently validated in a separate institute. Here, we investigated germline MRE11A variants as possible predictors of RT outcomes in MIBC, using next-generation sequencing (NGS). PATIENTS AND METHODS: The MRE11A gene was amplified in germline DNA from 186 prospectively recruited MIBC patients treated with RT and sequenced using bar-coded multiplexed NGS. Germline variants were analysed for associations with cancer-specific survival (CSS). For validation as a prognostic or predictive marker, rs1805363 was then genotyped in a cystectomy-treated MIBC cohort of 256 individuals. MRE11A mRNA isoform expression was measured in bladder cancer cell lines and primary tumour samples. RESULTS: Carriage of at least one of six (five novel) rare variants was associated with the worse RT outcome (hazard ratio [HR] 4.04, 95% confidence interval [95% CI] 1.42-11.51, P = 0.009). The single-nucleotide polymorphism (SNP), rs1805363 (minor allele frequency 11%), was also associated with worse CSS (per-allele HR 2.10, 95% CI 1.34-3.28, Ptrend = 0.001) following RT in MIBC, with a gene-dosage effect observed, but no effect seen on CSS in the cystectomy cohort (Ptrend = 0.89). Furthermore, rs1805363 influenced relative MRE11A isoform expression, with increased isoform 2 expression with carriage of the rs1805363 minor A allele. CONCLUSIONS: Germline MRE11A SNP rs1805363 was predictive of RT, but not of cystectomy outcome in MIBC. If successfully validated in an independent RT-treated cohort, this SNP could be a useful clinical tool for selecting patients for bladder-conserving treatment.

Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer.

BACKGROUND: Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC. METHODS: Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays representing 267 radical prostatectomy (RP) and 111 conservatively treated (CT) PC patients. Clinical end points were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. The ANPEP promoter methylation levels were determined by bisulphite sequencing or MethyLight analysis in 278 nonmalignant and PC tissue samples, and in cell lines. RESULTS: The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2'-deoxycytidine induced ANPEP expression in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis marker (vascular endothelial growth factor or microvessel density) improved risk prediction significantly in both cohorts. CONCLUSION: Our results suggest negative APN immunoreactivity as a new independent adverse prognostic factor for patients with clinically localised PC and, furthermore, that epigenetic mechanisms are involved in silencing of ANPEP in PC.

Expression of MAGE-A3, NY-ESO-1, LAGE-1 and PRAME in urothelial carcinoma.

BACKGROUND: The potential for cancer-testis (CT) antigens as targets for immunotherapy in cancer patients has been heavily investigated, and currently cancer vaccine trials based on the CT antigens, MAGE-A3 and NY-ESO-1, are being carried out. METHODS: We used specific q-RT-PCR assays to analyse the expression of the CT genes MAGE-A3, NY-ESO-1 (CTAG1B), LAGE-1 (CTAG2) and PRAME in a panel of bladder tumours from 350 patients with long-term follow-up and detailed treatment information. RESULTS: Overall, 43% of the tumours expressed MAGE-A3, 35% expressed NY-ESO-1, 27% expressed LAGE-1 and 20% expressed PRAME. In all, 56% of the tumours expressed at least one of the CT genes analysed. Univariate Cox regression analysis of CT gene expression in non-muscle-invasive tumours showed that expression of MAGE-A3 (P=0.026), LAGE-1 (P=0.001) and NY-ESO-1 (P=0.040) was significantly associated with a shorter progression-free survival. In addition, we found that patients with tumours expressing PRAME responded poorly to chemotherapy (P=0.02, χ(2)-test). CONCLUSION: Cancer-testis genes are frequently expressed in bladder cancer and especially in tumours of high stage and grade. In addition, the CT gene expression may have both prognostic and predictive value. Development of specific immunotherapy against the CT antigens in bladder cancer may ultimately increase patient survival.

Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; technology transfer from microarrays to PCR.

BACKGROUND: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. METHODS: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. RESULTS: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P<0.001) when adjusting for stage, grade and treatment. Independent validation of the 12-gene panel and the determined cut-off values is needed and ongoing. CONCLUSION: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patient's disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer.

The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer.

BACKGROUND: Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood. METHODS: We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1-T4 tumours using expression microarrays and qRT-PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays. RESULTS: We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3'UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict. CONCLUSION: This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.

Low ANXA10 expression is associated with disease aggressiveness in bladder cancer.

BACKGROUND: Markers for outcome prediction in bladder cancer are urgently needed. We have previously identified a molecular signature for predicting progression in non-muscle-invasive bladder cancer. ANXA10 was one of the markers included in the signature and we now validated the prognostic relevance of ANXA10 at the protein level. METHODS: We investigated ANXA10 expression by immunohistochemistry using a tissue microarray with 249 Ta and T1 urothelial carcinomas. The expression of ANXA10 was also investigated in an additional set of 97 more advanced tumours. The functional role of ANXA10 in cell lines was investigated by siRNA-mediated ANXA10 knockdown using wound-healing assays, proliferation assays, and ingenuity pathway analysis. RESULTS: Low expression of ANXA10 correlated with shorter progression-free survival in patients with stage Ta and T1 tumours (P<0.00001). Furthermore, patients with more advanced tumours and low ANXA10 expression had an unfavourable prognosis (P<0.00001). We found that ANXA10 siRNA transfected cells grew significantly faster compared with control siRNA transfected cells. Furthermore, a wound-healing assay showed that ANXA10 siRNA transfected cells spread along wound edges faster than control transfected cells. CONCLUSION: We conclude that ANXA10 may be a clinical relevant marker for predicting outcome in both early and advanced stages of bladder cancer.

Percutaneous endoscopic gastrostomy in patients with systemic sclerosis.

Gastrointestinal (GI) problems are common in patients with systemic sclerosis (SSc) and some develop pronounced underweight despite dietary intervention. Two patients with SSc were referred to our department because of severe GI symptoms and critical underweight. They had lost 24 and 23 kg, respectively, and their body mass index (BMI) was only 15 and 17 kg/m(2), respectively. After careful GI evaluation, both were treated with percutaneous endoscopic gastrostomy (PEG). Eight months later one patient had gained 13 kg (BMI 19 kg/m(2)) and after 5 months follow-up the other had gained 9 kg (BMI 20 kg/m(2)). Weight was no longer critically low and quality of life improved in both. In conclusion, PEG is an option in selected patients with severe malnutrition due to SSc. However, we advocate that thorough GI evaluation is performed in advance.

dc-Sheet resistance as sensitive monitoring tool of protein immobilization on thin metal films.

The suitability of high resolution, in situ dc-sheet resistance monitoring (SRM) as a simplified and reliable sensing technique towards detection and tracking of protein immobilization has been explored. Non-specific adsorption of bovine serum albumin (BSA) onto a very thin gold film, acting as the sensing resistor, has been employed as a model system. For comparison, the novel sensing method was combined with surface plasmon resonance (SPR) spectroscopy, using the same flow cell and sensing surface. Two different, well known adsorption states, involving a composite layer of irreversibly and reversibly bound BSA, were clearly resolved by both methods. Clearly structured, pronounced and fully reproducible film resistance modulations have been resolved in the associated SRM data. The transition from reversibly bound BSA to the diluted protein phase is associated with an unusually large decrease in the dc-sheet resistance. The observed resistance modulation magnitude for an adsorbed BSA monolayer corresponds to approximately 1%, and up to 100 mOmega at a 10 Omega sensing resistor. The sheet resistance of irreversibly bound BSA was determined to 0.24 kOmega/cm2, and the associated specific resistivity estimated to 1-2x10(4) Omega cm.

Association between immunohistochemical expression of vascular endothelial growth factor (VEGF), VEGF-expressing neuroendocrine-differentiated tumor cells, and outcome in prostate cancer patients subjected to watchful waiting.

Tumor growth is dependent on angiogenesis, which is thought to be controlled by angiogenic factors. Therefore, the immunoreactivity of the angiogenic cytokine vascular endothelial growth factor (VEGF) was semiquantitatively scored in archival prostate tumors obtained at diagnosis in 221 patients followed expectantly. At diagnosis, 125 patients suffered from clinically localized disease. Median length of follow-up was 15 years, and 57% of the patients eventually died of prostate cancer. All of the tumors exhibited cytoplasmic staining for VEGF. The staining intensity was weak in 47 tumors and moderate and strong in 107 and 67, respectively. VEGF expression was significantly correlated with microvessel density (MVD; median, 43; range, 16-151; P = 0.014), increasing T-classification (P = 0.001), dedifferentiation (P < 0.001), and disease-specific survival (P = 0.013). Strongly VEGF-immunoreactive, neuroendocrine-differentiated (NE) tumor cells were observed in 125 tumors. NE expression was significantly correlated with increasing MVD, increasing T-classification, dedifferentiation, and survival (all, P < 0.001). MVD and NE tumor cell expressions were significant variables in a multivariate analysis that included patients with clinically localized prostate cancer only. VEGF and NE expression were significantly correlated with MVD, clinical characteristics, and disease-specific survival. NE expression was a significant prognostic marker in localized prostate cancer patients, whereas the applied semiquantitatively scoring of VEGF expression was inadequate to make this growth factor provide any additional prognostic information. Moreover, the significant VEGF expression of NE tumor cells suggests an additional important character of these cells in the involvement in disease progression.

Quantification of angiogenesis as a prognostic marker in human carcinomas: a critical evaluation of histopathological methods for estimation of vascular density.

Chalkley counts have been suggested as the primary method for immunohistochemical evaluation of angiogenesis, however, most studies have used microvessel density (MVD). We present paired Chalkley and MVD estimates in carcinomas of the prostate, breast, bladder and lung. The clinical data has previously been reported. In prostate carcinomas, high MVD indicated poor prognosis, whereas high Chalkley counts in breast carcinoma were associated with a poor prognosis. In bladder carcinoma, high estimates using both methods showed good prognosis and were associated with a high degree of inflammation. Neither of the counts revealed prognostic value in lung carcinomas, where the vascular pattern indicated that this cancer was non-angiogenic. We highlight methodological problems with both counting methods. Since angiogenic processes in lung and bladder cancers may be different from those occuring in prostate cancer, we suggest that future analyses also focus on measuring angiogenic factors to obtain more information on the biology of angiogenesis.

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein.

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.

Multiple genes code for high-molecular-mass rhoptry proteins of Plasmodium yoelii.

We have examined the number of genes coding for a group of high-molecular-mass rhoptry protein(s) in the malaria parasite Plasmodium yoelii, and studied variation in the gene family within the parasite's genome. A region of the genes was amplified using oligonucleotides based on conserved DNA sequences and the products cloned. The sequences could be divided into 7 groups by restriction-fragment-length polymorphism. Further variation was detected by sequence analysis; 11 different sequences were detected in the 16 clones analyzed. The genes in the family were distributed on 6 chromosomes probably at 9 or more loci.

Primary structure and localization of a conserved immunogenic Plasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle.

A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.

Immunohistochemical determination of tumor angiogenesis measured by the maximal microvessel density in human prostate cancer.

Tumor growth beyond a certain size requires angiogenesis. Experimental evidence shows that once tumors leave the pre-angiogenic phenotype to become angiogenic, metastases often start to evolve. The aim of this study was to develop a reproducible immunohistochemical technique and method to characterize the neovascularization in archival prostate cancer tissue by quantifying the microvessel density (MVD). Archival tumor specimens from 64 consecutively diagnosed prostate cancer patients were immunostained for von Willebrand Factor (vWF), endothelial antigen and for CD31 combined with the use of different digestive enzymes (trypsin and pronase) and heating in a microwave oven. Both the mean and the maximal MVD, and the reproducibility of the method were estimated. Finally, the mean MVD, the maximal MVD, and clinical characteristics were correlated with the crude survival of the patient population. The immunohistochemical staining for vWF to measure the maximal MVD was found to be a reproducible method of characterizing the individual tumor. Both a univariate and a multivariate analysis demonstrated that the maximal MVD, in contrast to the mean MVD, was significantly associated with survival in prostate cancer patients. We conclude that evaluation of angiogenesis by immunostaining the endothelial cells for vWF measured by the MVD in the most vascularized areas of the tumor is a reproducible method of characterizing the individual prostate tumor. Maximal MVD proved to be an independent prognostic parameter useful in conjunction with other known prognostic markers in human prostate cancer.

Association between in vivo iododeoxyuridine labeling, MIB-1 expression, malignancy grade and clinical stage in human prostate cancer.

Large variability in the biological behavior of prostate cancer makes prognostic markers important. The extent of tumor cell proliferation has been suggested as an important predictor of clinical outcome. Fifty-five patients suspected of having or with previously diagnosed prostate cancer were labeled in vivo with IdUrd (a thymidine analogue incorporated into DNA in S-phase cells) by intravenous infusion before transurethral resection. IdUrd-labeled cells and MIB-1-positive cells were detected by immunohistochemistry. We found statistically significant associations between the tumor cell proliferation rates measured by in vivo IdUrd labeling and MIB-1 expression in formalin-fixed paraffin-embedded tumors. Good correlations were also found between S-phase fraction, MIB-1 expression, clinical stage and malignancy grade. These results make larger retrospective studies on archival tissue meaningful.

Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay.

A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P. falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP.

Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein: evidence for protection of individuals living in a holoendemic area of Liberia.

A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155/RESA) (EENV)6 were examined in 423 individuals (age range 30 days-78 years) living in a malaria holoendemic area of Liberia. In the 5-9-year-old age group, subjects with anti-GLURP489-1271 antibody concentrations greater than the mean value of the group had lower parasite densities than those with low antibody concentrations (P = 0.0151). High levels of anti-GLURP899-916 antibodies did not correlate with low parasite densities. However, high levels of anti-(EENV)6 antibodies were associated with significantly lower parasite densities in the 2-4-year-old age group (P = 0.0189). There was no correlation between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA.

Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results.

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.

Development of a PCR-based technique for detection of Helicobacter pylori.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.