Presentation Authors: Indu Kohaar*, Sreedatta Banerjee, Yongmei Chen, Amina Ali, Rockville, MD, Jacob Kagan, Sudhir Srivastava, Bethesda, MD, Jennifer Cullen, Inger Rosner, Shiv Srivastava, Gyorgy Petrovics, Rockville, MD
Introduction: Prostate cancer (CaP) affects 1 in 7 men in their life time. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA) men. We and others have also noted that the genes commonly overexpressed in CaP (e.g., ERG) and currently used as diagnostic markers, exhibit much lower frequency and more heterogeneity in AA men. The goals of this study are to develop CaP diagnostic/prognostic marker panels based on ethnicity associated differences in molecular alterations.
Methods: A quantitative expression assay protocol have been developed for noninvasive detection of candidate genes in exosomal mRNAs from regular urine. The selected genes were reverse transcribed in a single reaction using gene specific primer pool (GSP) followed by pre-amplification and PCR based assay of target genes.
Results: We have developed a multi-gene RT-PCR expression assay for use with non-DRE urine specimens from patients undergoing diagnostic biopsy procedure. We used Droplet Digital PCR (ddPCR) based approach to measure the analytical sensitivity, specificity and accuracy of the gene assays (GAPDH, SPDEF, HOXC6, DLX1, PCA3, ERG, PSGR, PCGEM1, ARv7, PCAT1, SPARC, SChLaP1 and TGFÎ² by serially diluting RNA sample by 10-fold over a linear range of 5 concentrations from VCaP and LNCaP cell lines. ERG expression was found to be robust in VCaP. The results demonstrate the performance of gene assays to quantify cDNA over a wide range of input concentrations that can be used in non-DRE urine specimens from patients coming for diagnostic biopsy procedure. After establishing the assay robustness, we are further evaluating and validating the mRNA expression for urine markers by ddPCR approach in 250 patients. Preliminary analysis on 135 patients for selected markers (SPDEF, GAPDH, PCA3, ERG, PSGR, PCGEM1, HOXC6, DLX1 showed that a five gene panel (PCA3, ERG, PSGR, PCGEM1, HOXC6) yielded a sensitivity of 88.0% and specificity of 39.0% in AA men while a panel comprising of PCA3, ERG, PSGR, HOXC6 provided a sensitivity of 83.0% and specificity of 46.0% in CA men.Taken together, these findings are promising towards the development of ethnicity informed diagnostic and prognostic CaP biomarkers using urinary exosomes by ddPCR approach.
Conclusions: Given the prostate cancer genomic differences between racial/ethnic groups, this study highlights the potential for developing broadly applicable CaP diagnostic/prognostic biomarker panels.
Source of Funding: This work was supported by the National Cancer Institute, Early Detection Network, IAA ACN1700500100000 to S.S., the CPDR Program HU00011020002 to I.L.R. and S.S. the NIH Grants NCI RO1 CA16238305 grants to S.S.