Amina Ali

A Biopsy-based 17-gene Genomic Prostate Score Predicts Recurrence After Radical Prostatectomy and Adverse Surgical Pathology in a Racially Diverse Population of Men with Clinically Low- and Intermediate-risk Prostate Cancer

BACKGROUND Biomarkers that are validated in independent cohorts are needed to improve risk assessment for prostate cancer (PCa). OBJECTIVE A racially diverse cohort of men (20% African American [AA]) was used to evaluate the association of the clinically validated 17-gene Genomic Prostate Score (GPS) with recurrence after radical prostatectomy and adverse pathology (AP) at surgery. DESIGN, SETTING, AND PARTICIPANTS Biopsies from 431 men treated for National Comprehensive Cancer Network (NCCN) very low-, low-, or intermediate-risk PCa between 1990 and 2011 at two US military medical centers were tested to validate the association between GPS and biochemical recurrence (BCR) and to confirm the association with AP. Metastatic recurrence (MR) was also evaluated. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Cox proportional hazards models were used for BCR and MR, and logistic regression was used for AP. Central pathology review was performed by one uropathologist. AP was defined as primary Gleason pattern 4 or any pattern 5 and/or pT3 disease. RESULTS AND LIMITATIONS GPS results (scale: 0-100) were obtained in 402 cases (93%); 62 men (15%) experienced BCR, 5 developed metastases, and 163 had AP. Median follow-up was 5.2 yr. GPS predicted time to BCR in univariable analysis (hazard ratio per 20 GPS units [HR/20 units]: 2.9; p<0.001) and after adjusting for NCCN risk group (HR/20 units: 2.7; p<0.001). GPS also predicted time to metastases (HR/20 units: 3.8; p=0.032), although the event rate was low (n=5). GPS was strongly associated with AP (odds ratio per 20 GPS units: 3.3; p<0.001), adjusted for NCCN risk group. In AA and Caucasian men, the median GPS was 30.3 for both, the distributions of GPS results were similar, and GPS was similarly predictive of outcome. CONCLUSIONS The association of GPS with near- and long-term clinical end points establishes the assay as a strong independent measure of PCa aggressiveness. Tumor aggressiveness, as measured by GPS, and outcomes were similar in AA and Caucasian men in this equal-access health care system. PATIENT SUMMARY Predicting outcomes in men with newly diagnosed prostate cancer is challenging. This study demonstrates that a new molecular test, the Genomic Prostate Score, which can be performed on a patients original prostate needle biopsy, can predict the aggressiveness of the cancer and help men make decisions regarding the need for immediate treatment of their cancer.

Evaluation of the ETS-Related Gene mRNA in Urine for the Detection of Prostate Cancer

Purpose: Prevalent gene fusions in prostate cancer involve androgen-regulated promoters (primarily TMPRSS2) and ETS transcription factors (predominantly ETS-regulated gene (ERG)], which result in tumor selective overexpression of ERG in two thirds of patients. Because diverse genomic fusion events lead to ERG overexpression in prostate cancer, we reasoned that it may be more practical to capture such alterations using an assay targeting ERG sequences retained in such gene fusions. This study evaluates the potential of an assay quantitating ERG mRNA in post–digital rectal exam (DRE) urine for improving prostate cancer detection. Experimental Design: Patients scheduled to undergo transrectal ultrasound-guided needle biopsy of the prostate were prospectively enrolled. On the day of biopsy, patients provided a urine sample immediately following a DRE. Urine ERG mRNA was measured and normalized to urine prostate-specific antigen (PSA) mRNA using the DTS 400 system. Demographic traits, clinical characteristics and biopsy results were analyzed for association with urine ERG score. Results: The study was conducted on 237 patients. Prostate cancer was shown on biopsy in 40.9% of study subjects. A higher urine ERG score associated significantly with malignancy on biopsy (P = 0.0145), but not with clinical stage or Gleason score. Urine ERG score performed best in Caucasians and in men with a PSA of ≤4 ng/mL (area under the curve = 0.8). Conclusions: A higher urine ERG score in post-DRE urine is associated with the diagnosis of prostate cancer on biopsy. Urine ERG score performed particularly well in men with a PSA of ≤4.0 ng/mL, a segment of the screening population in which further diagnostic markers are needed to determine in whom biopsy should be done. Clin Cancer Res; 16(5); 1572–6

Prostate Cancer Risk Allele Specific for African Descent Associates with Pathologic Stage at Prostatectomy

Purpose: A region on chromosome 8q24 was recently identified as a novel prostate cancer risk locus. Inherited variation in this region is associated with prostate cancer risk in the general population (21-58%), and specific alleles show a strong association in African-American men. This study was designed to evaluate associations between 8q24 risk alleles and clinical variables, such as pathologic stage, age at diagnosis, and recurrence, in a case series of African-American men. Experimental Design: Peripheral blood DNA samples from 114 African-American men with prostate cancer, including 106 who had undergone radical prostatectomy, were genotyped for six single-nucleotide polymorphisms on three 8q24 regions. The presence of these single-nucleotide polymorphisms was compared with clinicopathologic and follow-up data after radical prostatectomy. Results: The mean age of diagnosis and follow-up time were 57.4 (±8.9) years and 49.1 (±31.6) months, respectively. Patients carrying the Broad11934905 A risk allele, which is specific for African ancestry, were more likely to have a higher pathologic stage (pT3-4) than individuals with the wild type (odds ratio, 4.48; 95% confidence interval, 1.42-14.14; P = 0.011). A trend toward increased frequency of and shorter time to biochemical recurrence was noted in patients with this risk allele on Kaplan-Meier unadjusted survival analysis (P = 0.076). Conclusions: The Broad11934905 polymorphism at 8q24, which is only found in people of African ancestry, is associated with an increase in non-organ-confined prostate cancer at prostatectomy. In addition, for those with this risk allele, there is a trend toward early biochemical recurrence that requires validation in larger studies. Cancer Epidemiol Biomarkers Prev; 19(1); 1–8

Do patients with low volume prostate cancer have prostate specific antigen recurrence following radical prostatectomy

Aims: The objective of this study was to determine the incidence of prostate specific antigen (PSA) relapse in patients with low volume prostate cancer following radical prostatectomy. Methods: Between 1993 and 2001, 50 of 717 patients had total tumour volumes of less than 0.5 cm3 following radical prostatectomy. Biochemical recurrence was defined as two consecutive values of serum PSA levels of 0.2 ng/ml or greater. Results: Median follow-up of the 50 patients was 58 months. In five of the 50 patients (10%), PSA recurrence was observed. All of these five cases had Gleason score of 3+3 (well and/or moderately differentiated), organ confined and surgical margin negative tumours. In three of the five cases, capsular incision resulted in benign glands extending into the surgical margin. Conclusions: Five of 50 cases had PSA failure. In three of the five patients, benign glands located in the margin could explain the “PSA recurrence”. However, in the other two patients, none of the pathological parameters correlated with measurable PSA levels. The explanation for their PSA failure is unclear.

Assessment of circulating tumor cells (CTCs) in prostate cancer patients with low-volume tumors

The objective of this study was to assess the incidence of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors (less than 0.5 cc) after radical prostatectomy (RP). Blood samples were collected from 64 RP patients to assess the incidence of CTCs following RP. The specimens were processed by whole‐mount section. Clinicopathological data (e.g. patient age, race, specimen weight, tumor volume, grade, stage and surgical margin status) and follow‐up PSA data were compared to CTC status. Of the 64 RP patients, nine had ‘low‐volume prostate cancer’. Seven of these patients had detectable levels of CTCs. In two of the seven patients with detectable CTCs, PSA elevation was also observed. Isolation and detection of circulating epithelial cells is possible in low‐volume prostate cancer patients. In the setting of low‐volume prostate cancer, CTCs may be associated with the presence of detectable PSA levels. However, the detection of CTCs did not predict PSA failure.

MP70-10 ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS WITH ERG FUSION STATUS IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Oncogenic activation of ERG resulting from prevalent gene fusions (predominantly as TMPRSS2-ERG) is a key driver event in prostate cancer (CaP) pathogenesis. We and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. However, there is limited data on germline association with ERG fusion status. The goal of the present study is to agnostically identify the inherited risk variants of CaP incidence and progression by ERG-based stratification of CaP. METHODS: Blood derived genomic DNA samples were prepared from 270 AA men and 130 CA men treated by radical prostatectomy. ERG status was determined by IHC for ERG protein expression. SNP genotyping was performed on the Illumina Golden Gate platform using Infinium Oncoarray SNP chip. Data analysis approaches included association analyses based on EMMAX and imputation analysis by IMPUTE2. SNP genotyping was performed using droplet digital polymerase chain reaction (ddPCR) approach. RESULTS: SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. The SNPs most significantly (p<10-5) associated with ERG fusion status of index tumor included rs6698333 (KLF17) and two SNPs, rs1889877 and rs3798999 (ADGRB3). Four SNPsrs10215144 (AGBL3); rs3818136, rs9380660 (TBC1D22B region) and rs1792695 in ncRNA (LOC100505474) were found to be significantly (p<10-5) associated with ERG positive status under any tumor foci positive for fusion. Fine-mapping of the genetic associations found rs34349373 and rs2055272 in TBC1D22B to be significantly associated (<10-7) with ERG fusion status by any tumor foci positive for ERG. Imputed SNP rs2055272 was further experimentally validated by ddPCR approach. Concordance between TaqMan and imputed genotypes was 98.04% (100/102). rs34349373 was found to be significantly associated with CA. KaplaneMeier analysis indicated no association (p 1⁄4 0.9206) between the SNP and biochemical recurrence (BCR). Additional clinicopathological and functional eQTL analysis for the top hits are being performed to understand the biological function of the SNP with ERG fusion status. CONCLUSIONS: This study identified SNPs in TBC1D22B associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance of these SNPs still needs to be determined as it relates to ERG status, independent validation may enhance markers in stratifying patients early (even before CaP is detected) for targeted prevention and/or treatment options.

Abstract A013: Prostate cancer gene expression panel to address racial differences of molecular alterations in prostate cancer

Introduction and Objectives: Prostate cancer (CaP) affects 1 in 7 men in their lifetime. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA) men. Emerging data, including ours, have described significantly lower frequencies of alterations in common CaP driver genes (ERG and PTEN) in AA men as compared to CA men. We have also noted that genes commonly overexpressed in CaP (ERG, AMACR, PCA3), and currently used as diagnostic markers, exhibit much lower frequency and more heterogeneity in AA men. The goal of this study was to define a CaP marker panel that is overexpressed equally well in AA and CA CaPs. Methods: Three platforms (RNA-Seq, NanoString, and qRT-PCR) were used for evaluating CaP-associated gene expression in CA and AA patients (N=144). Candidate genes with robust tumor overexpression (over 4-fold) in CaP in paired normal and tumor specimens from AA and CA patients were selected from NanoString and RNA-Seq data for validation by qRT-PCR (TaqMan) in laser microdissected (LCM) tumor and benign cells of frozen tissue sections (50 CA and 35 AA). An assay protocol (gene specific RT and preamplification followed by TaqMan PCR) was developed for noninvasive early detection of candidate genes in regular urine (non-DRE) using urinary exosomal RNA. Results: Tumor transcriptomes of CA patients consistently revealed elevated expression of PCA3 and AMACR. However, these genes had variable overexpression in the AA cohort. The top genes that were similarly overexpressed in tumors of AA and CA patients were validated by qRT-PCR in LCM tumor and normal epithelial cells (N=85). At least one gene of a six-gene signature (DLX1, HOXC4, NKX2-3, COL10A1, HOXC6, and PSGR) was overexpressed in tumor cells of all AA and CA cases, providing a consistent ethnicity-informed tumor expression signature, which was further validated in silico in TCGA RNA-Seq data. Urinary exosome-based assay was developed and optimized for PSGR, DLX1, HOXC4, NKX2-3, as well as PCA3, PCGEM1, and ERG. All markers have been evaluated in a prospective cohort of 100 patients. In 36 AA patients a sensitivity of 78%, specificity of 68%, and AUC of 0.83 was achieved, surpassing currently used urine CaP markers of ERG and PCA3 in this cohort. Conclusions: A CaP tissue-based gene expression marker panel has been defined with potential diagnostic utility for both CA and AA men in the context of urinary exosomes. Source of Funding: This study is supported by NCI/EDRN ACN12011-001-0 and NCI RO1 CA162383-05 grants to S.S. Citation Format: Indu Kohaar, Sreedatta Banerjee, Lakshmi Ravindranath, Yongmei Chen, Amina Ali, Jacob Kagan, Sudhir Srivastava, Albert Dobi, David McLeod, Inger Rosner, Shiv Srivastava, Gyorgy Petrovics. Prostate cancer gene expression panel to address racial differences of molecular alterations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A013.

MP87-18 ASSOCIATION OF GERMLINE GENETIC VARIANTS WITH TMPRSS2-ERG FUSION STATUS IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Despite the role of interferon-g (IFNg) in tumor immune surveillance; studies have implicated the dark side of IFNg based on its pro-tumorigenic activity. IFNg can induce transcriptional activation of IFN-stimulated genes (ISGs) via JAK-STAT signaling pathway. The most highly induced ISGs are interferon-induced tetratricopeptide repeat (IFIT) family members. By studying a differential regulation on a unique tumor suppressor miR-363 from its polycistronic miR-106a-363 cluster, we unveiled a new microRNA (miRNA) turnover machinery composed of IFIT5, which is first described as a viral RNA binding protein. Up to date, the impact of IFIT5 on cancer metastasis is unclear. METHODS: Luciferase reporter gene assay was for examining the IFNg-induced IFIT5 gene activation. Transwell migration assay was for demonstrating the function of IFIT5 with cancer metastasis. Sitedirected mutagenesis, in vitro transcription, RNA pull down and in vitro RNA degradation assay were for determining the specificity of miRNA species regulated by IFIT5-mediated turnover machinery. RESULTS: IFIT5 gene promoter activity and protein level were significantly elevated by IFNg. IFIT5 complex represents unique posttranscriptional machinery for turnover of a specific population of tumor suppressor miRNAs. We examined several IFIT5-regulated miRNA candidates, and found IFNg can suppress miR-101, miR-335, miR-203, and miR-128, and phenocopied the miRNA expression profile of IFIT5overexpressing cells. Seed regions of miR-101 and miR-128 have sequence-matched target sites at ZEB1 mRNA 30UTR, and indeed can suppress ZEB1. Meanwhile, IFIT5 is elevated in higher grade prostatic tumors, and positively correlated with ZEB1, ZEB2 and Slug in prostate cancer. Knockdown of IFIT5 lead to suppression of ZEB1 and Slug, along with decreased migration mobility in cancer cells. On the contrary, IFNg treatment enhanced cell migration, and this effect is diminished by the loss of IFIT5. We also modified the 50end structure of precursor miR101 and miR-128, and examined its regulation by IFIT5-mediated miRNA turnover machinery. Both pre-miR-101 and pre-miR-128 with mutated blunt end are resistant to degradation in an IFIT5-expressing PC3 cell and show greater impact on suppressing cell migration, compared to the mutant precursor with single stranded overhang. CONCLUSIONS: We demonstrated that IFNg potentiate prostate cancer metastasis via IFIT5-mediated miRNA turnover machinery, which regulates specific tumor suppressor miRNAs that target critical EMT factors including ZEB1 and Slug.

Abstract 1290: Association of common genetic variants withTMPRSS2 ERGfusion status in prostate cancer

Introduction and Objectives: Oncogenic activation of ERG resulting from prevalent gene fusions is present in two thirds of prostate cancer (CaP) patients of European Ancestry including Caucasian Americans (CA). Our laboratory and others have recently reported that major cancer driver genes, including ERG, show significant racial/ethnic differences in CaP with lower frequencies in African Americans (AA), Africans and Asians. Racial differences of CaP associated SNPs have also been extensively described. However, there is limited data on germline association with ERG fusion status. The goal of this study is to identify germline molecular determinants associating with ERG status of CaP. Methods: Blood derived genomic DNA samples were prepared from 270 AA men and 129 CA men treated by radical prostatectomy at Walter Reed National Military Medical Center (WRNMMC). ERG status was determined in whole mounted prostate specimens by immuno-histochemistry (IHC) for ERG protein expression as a surrogate for the TMPRSS2-ERG fusion. Blinded blood samples were genotyped for SNPs on the Illumina Golden Gate platform using Infinium Oncoarray, a 500K genome wide BeadChip kit from Illumina. Data analysis approaches included association analyses based on logistic regression, Principal Component Analysis (PCA) and Efficient Mixed-Model Association eXpedited (EMMAX) analysis. Genotype imputation analysis is being performed by IMPUTE2 program. Results: After applying rigorous sample and SNP QC steps on the datasets, SNP genotyping analysis was performed in 321 patients with 478,299 SNPs. Logistic regression, principal component analysis by EIGENSTRAT and a variance component approach, EMMAX analysis were performed to account for population structure. By EMMAX we identified SNPs associated with ERG status. The SNPs most significantly ( Conclusions: This study identified SNPs differentially associated with ERG status of CaP, a major driver oncogene in CaP. Although the biological significance as it relates to ERG status of CaP still needs to be determined, these SNPs, with independent validation, may help as markers in stratifying patients early (even before CaP is detected) for targeted prevention and treatment options. Citation Format: Indu Kohaar, Lakshmi Ravindranath, Denise Young, Amina Ali, Qiyuan Li, Albert Dobi, David McLeod, Inger L. Rosner, Isabell Sesterhenn, Matthew Freedman, Shiv Srivastava, Gyorgy Petrovics. Association of common genetic variants with TMPRSS2 ERG fusion status in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2017-1290

Abstract 1859: Potential impact on the biology and biomarker utility of ERG-typing in the context of ethnic differences of prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: In comparison to other ethnicities African American men have the highest incidence and mortality rates of prostate cancer (CaP). Recent reports on ethnic differences of ERG oncogenic activation, the most common CaP genomic alteration, provide new insights into the ERG based stratification of CaP in the context of the increasingly diverse ethnic patient populations in the United States. This rapidly advancing knowledge has potential to stratify CaP by ERG typing in a given patient population that may eventually lead to new targeted treatments. Methods: Whole-mounted prostate sections of 91 Caucasian American (CA) and 91 African American (AA) men matched for age, Gleason score and pathologic stage were analyzed for ERG oncoprotein expression. High grade tumors with a primary Gleason pattern of 4 or 5 or cumulative Gleason score of 8-10 from a total of 63 AA patients and 63 CA patients were also assessed for ERG. Hormone naive AA CaP patients (n=306) with a minimum of two years of follow up after radical prostatectomy were evaluated for ERG frequencies in whole-mounted prostate specimens. Performance of PCA3, ERG and PSA biomarkers were evaluated in 306 post-DRE urine samples from CA and AA patients by the DTS® 400 system and by whole transcriptome analyses in selected CaP samples. The associations of biomarker scores with clinical parameters were assessed in matching biopsies and were stratified by ethnicity. Results: Remarkably higher frequencies of ERG expression was noted in the index tumors of CA (63.3%) compared to AA (28.6%) men. The observed difference was even more pronounced in higher grade disease AA (16%) and CA (49%), respectively. ERG positive tumors were more likely to be present in index tumors of younger AA men. Furthermore, ERG negative tumors were significantly associated with higher pathologic T stage. In urine assays the combination of ERG and PCA3 scores increased the AUC performance as compared to serum PSA alone (0.68 vs. 0.53, p = 0.0062) for predicting CaP in CA men. In contrast, the biomarker panel showed strikingly poor performance in AA men (AUC = 0.49). Conclusions: ERG-based stratification defines molecular subtypes and ethnic differences in the biology of CaP. Association of ERG negative status with high grade tumors and poor prognostic features in AA patients warrants the development of informative marker panels that includes ERG to aid in diagnostic, prognostic and targeted therapy settings. Citation Format: Albert Dobi, Yongmei Chen, Amina Ali, Denise Young, Philip Rosen, James Farrell, Michael Degon, Sudhir Srivastava, Jacob Kagan, Jocelyn Lee, Jennifer Cullen, Gyorgy Petrovics, David G. McLeod, Isabell A. Sesterhenn, Shiv Srivastava. Potential impact on the biology and biomarker utility of ERG-typing in the context of ethnic differences of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1859. doi:10.1158/1538-7445.AM2014-1859