A multigene urine test for the detection and stratification of bladder cancer in patients presenting with hematuria.

PURPOSE: We investigated whether the RNA assay uRNA® and its derivative Cxbladder® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, and whether they are useful in risk stratification. MATERIALS AND METHODS: A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cxbladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. RESULTS: uRNA detected 41 of 66 urothelial carcinoma cases (62.1% sensitivity, 95% CI 49.3-73.8) compared with NMP22 ELISA (50.0%, 95% CI 37.4-62.6), BladderChek (37.9%, 95% CI 26.2-50.7) and cytology (56.1%, 95% CI 43.8-68.3). Cxbladder, which was developed on the study data, detected 82%, including 97% of the high grade tumors and 100% of tumors stage 1 or greater. The cutoffs for uRNA and Cxbladder were prespecified to give a specificity of 85%. The specificity of cytology was 94.5% (95% CI 91.9-96.5), NMP22 ELISA 88.0%, (95% CI 84.6-91.0) and BladderChek 96.4% (95% CI 94.2-98.0). Cxbladder distinguished between low grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91% and a specificity of 90%. CONCLUSIONS: uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists.

Predicting clinical outcome through molecular profiling in stage III melanoma.

PURPOSE: Patients with macroscopic stage III melanoma represent a heterogeneous cohort with average 5-year overall survival rates of <30%. With current algorithms, it is not possible to predict which patients will achieve longer-term survival. We hypothesized that molecular profiling could be used to identify prognostic groups within patients with stage III melanoma while also providing a greater understanding of the biological programs underpinning these differences. EXPERIMENTAL DESIGN: Lymph node sections from 29 patients with stage IIIB and IIIC melanoma, with divergent clinical outcome including 16 "poor-prognosis" and 13 "good-prognosis" patients as defined by time to tumor progression, were subjected to molecular profiling using oligonucleotide arrays as an initial training set. Twenty-one differentially expressed genes were validated using quantitative PCR and the 15 genes with strongest cross-platform correlation were used to develop two predictive scores, which were applied to two independent validation sets of 10 and 14 stage III tumor samples. RESULTS: Supervised analysis using differentially expressed genes was able to differentiate the prognostic groups in the training set. The developed predictive scores correlated directly with clinical outcome. When the predictive scores were applied to the two independent validation sets, clinical outcome was accurately predicted in 90% and 85% of patients, respectively. CONCLUSION: We describe a gene expression profile that is capable of distinguishing clinical outcomes in a previously homogeneous group of stage III melanoma patients.

Development of a multiplex RNA urine test for the detection and stratification of transitional cell carcinoma of the bladder.

PURPOSE: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage > or =T1 disease. EXPERIMENTAL DESIGN: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases. RESULTS: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% for Ta tumors > or =1 cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage > or =T1 TCCs with approximately 80% specificity and sensitivity. CONCLUSIONS: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.

Multiple gene expression classifiers from different array platforms predict poor prognosis of colorectal cancer.

PURPOSE: This study aimed to develop gene classifiers to predict colorectal cancer recurrence. We investigated whether gene classifiers derived from two tumor series using different array platforms could be independently validated by application to the alternate series of patients. EXPERIMENTAL DESIGN: Colorectal tumors from New Zealand (n = 149) and Germany (n = 55) patients had a minimum follow-up of 5 years. RNA was profiled using oligonucleotide printed microarrays (New Zealand samples) and Affymetrix arrays (German samples). Classifiers based on clinical data, gene expression data, and a combination of the two were produced and used to predict recurrence. The use of gene expression information was found to improve the predictive ability in both data sets. The New Zealand and German gene classifiers were cross-validated on the German and New Zealand data sets, respectively, to validate their predictive power. Survival analyses were done to evaluate the ability of the classifiers to predict patient survival. RESULTS: The prediction rates for the New Zealand and German gene-based classifiers were 77% and 84%, respectively. Despite significant differences in study design and technologies used, both classifiers retained prognostic power when applied to the alternate series of patients. Survival analyses showed that both classifiers gave a better stratification of patients than the traditional clinical staging. One classifier contained genes associated with cancer progression, whereas the other had a large immune response gene cluster concordant with the role of a host immune response in modulating colorectal cancer outcome. CONCLUSIONS: The successful reciprocal validation of gene-based classifiers on different patient cohorts and technology platforms supports the power of microarray technology for individualized outcome prediction of colorectal cancer patients. Furthermore, many of the genes identified have known biological functions congruent with the predicted outcomes.

Association of CDH1 haplotypes with susceptibility to sporadic diffuse gastric cancer.

Truncating mutations in the gene for the cell to cell adhesion protein E-cadherin are the most consistent genetic alterations observed in sporadic and hereditary diffuse gastric cancer (DGC). In addition to these inactivating mutations, a CDH1 promoter polymorphism at position -160 has been reported to lead to transcriptional downregulation of the gene in vitro. We therefore performed a case-control study to investigate whether this variant is associated with an increased susceptibility to DGC. The frequency of the -160A allele was significantly higher (P<0.005) in 53 diffuse gastric cancer cases compared to 70 matched controls. The odds ratio associated with the A-allele was 2.27 for CA-heterozygotes (95%CI 1.16-4.44) and 7.84 for AA-homozygotes (95%CI 2.89-21.24). Two additional polymorphisms (the 48+6T-->C and the 2076C-->T variant) were genotyped and shown to be equally distributed among cases and controls. Haplotype analysis with the three polymorphisms confirmed an association with disease (P<0.004). However, this analysis suggested the -160C-->A CDH1 promoter polymorphism may be in linkage disequilibrium with a distinct aetiological locus or acts in combination with other functional variants in or near the CDH1 region.

Novel germline CDH1 mutations in hereditary diffuse gastric cancer families.

Hereditary diffuse gastric cancer (HDGC) is a recently defined cancer syndrome caused by inactivating, heterozygous germline mutations in the gene for the cell-to-cell adhesion protein E-cadherin (CDH1). Here, we describe the search for CDH1 mutations in 10 newly identified gastric cancer families. Seven of 10 families met the clinical criteria for HDGC. Germline mutations were identified in four of these seven families and one family that was borderline for the clinical criteria. Of the mutations identified in the five new families, four were previously unreported and consisted of two frameshift and two donor splice site mutations. One splice site mutation occurred at the 100% conserved +1 position. The second splice site mutation occurred at the +5 position and was shown to lead to abnormal splicing. Additional CDH1 variants detected include the heterozygous -160 C-->A promoter polymorphism, which has previously been reported to be associated with decreased CDH1 transcription. We, however, found this polymorphism to be common in a control population, suggesting that a major role for this polymorphism in gastric cancer susceptibility is unlikely.