High-throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs: functional genomics investigations of biological pathways.

During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.

Analysis of gland volume effect on the performance of PSA density in early detection of non-palpable, isoechoic prostate cancer.

OBJECTIVE: To reassess PSA density (PSAD) in the detection of non-palpable, isoechoic prostate cancer and to analyze the effect of potentially inaccurate non-planimetric transrectal ultrasound volume estimates on the diagnostic performance of this diagnostic tool. METHODS: We prospectively evaluated 343 consecutive men with non-suspicious digital rectal examination and transrectal ultrasound findings and with serum PSA in the intermediate range (4.1-10 ng/mL). All men underwent systematic sextant biopsies of the peripheral zone. We performed a two-fold analysis of PSAD performance first using measured gland volume and then using modified gland volumes ranging from a 25% underestimation to a 25% overestimation, in 5% stepwise increments. RESULTS: With a 0.15 PSAD cut-off, we would have missed 14, 34 and 48% of cancers, if we had respectively used a volume underestimate of 25%, the measured volume and a volume overestimate of 25%. CONCLUSIONS: Potential gland volume under- or overestimation may substantially affect the diagnostic performance of PSAD. Although PSAD may represent a useful adjunct in the early detection of prostate cancer, it may compromise cancer detection in a substantial proportion of cases, if used as the only indicator for biopsy.

Value of repeat ultrasonic prostatic biopsies following negative digitally directed biopsy.

OBJECTIVE: The objective of this study was to determine the benefit of repeat transrectal ultrasound-guided prostatic biopsy (TRUSBx) among men with prior benign histology on digitally guided biopsy despite suspicious digital rectal examination (DRE) findings. PATIENTS AND METHODS: From January 1, 1990 to May 30, 1993, we evaluated 130 consecutive men, referred to us with benign pathology on digitally-guided biopsy and DRE suspicious of cancer. All patients underwent systematic and directed TRUSBx. RESULTS: TRUSBx detected previously undiagnosed malignancy in 67 cases (51%). CONCLUSION: It is mandatory to reevaluate by TRUSBx, all patients with a palpable abnormality on DRE and a prior benign pathology on digitally-guided biopsy, as the likelihood of finding cancer is elevated.

uPM3, a new molecular urine test for the detection of prostate cancer.

OBJECTIVES: To evaluate, in a multicenter study, the diagnostic performance of a new molecular test uPM3 for detecting prostate cancer cells in urine because of the need for better methods to identify patients at risk of prostate cancer. METHODS: The uPM3 test is a nucleic acid amplification assay detecting simultaneously in the urine the relative expression of prostate-specific antigen (PSA) mRNA as a marker of prostate cells and PCA3RNA, which is selectively expressed in most types of prostate cancer. The test is performed using the isothermic nucleic acid-based amplification method, and the two targets are simultaneously detected in real-time fluorescence using specific beacons as probes in a thermostated spectrofluorometer. The test was performed on the first voided urine obtained after careful digital rectal examination of the prostate in men undergoing transrectal ultrasound-guided prostate biopsy. RESULTS: Of 517 patients undergoing biopsy at five centers, 443 (86%) had an assessable sample. Of those, 21%, 55%, and 24% had a total PSA level of less than 4 ng/mL, between 4 and 10 ng/mL, and greater than 10 ng/mL. The corresponding percentage of biopsies positive for cancer in these three groups was 20%, 35%, and 44%. The overall uPM3 sensitivity and specificity was 66% and 89%, respectively. In men with a PSA level less than 4 ng/mL, the sensitivity was 74% and specificity 91%. In those with a PSA level of 4 to 10 ng/mL, the sensitivity was 58% and specificity 91%. In those with a PSA level greater than 10 ng/mL, the sensitivity and specificity was 79% and 80%, respectively. The positive predictive value of uPM3 was 75% compared with 38% for total PSA, and the negative predictive value was 84% compared with 89% and 80% for a PSA cutoff of 2.5 and 4.0 ng/mL, respectively. The overall accuracy was 81% compared with 43% and 47% for total PSA at a cutoff of 2.5 and 4.0 ng/mL, respectively. CONCLUSIONS: These results suggest that the uPM3 molecular urine test may be an important adjunct to current methods for the detection of early prostate cancer.