Inger Rosner

The clinical implications of the genetics of renal cell carcinoma.

Over the last several decades, the advances in molecular genetics have elucidated kidney cancer gene pathways. Kidney cancer is a heterogeneous disorder. Each specific type of kidney cancer has its own histologic features, gene, and clinical course. Insight into the genetic basis of kidney cancer has been learned largely from the study of the familial or hereditary forms of kidney cancer. Extirpative surgery is currently the treatment of choice for kidney cancer that is confined to the kidney. Treatment for advanced or metastatic kidney cancer is a formidable challenge with the traditional therapies currently available. However, investigation of the Mendelian single-gene syndromes, like von Hippel Lindau (VHL: VHL gene), hereditary papillary renal carcinoma (HPRC: c-Met gene), Birt-Hogg-Dubé (BHD: BHD gene), and hereditary leiomyomatosis renal cell cancer (HLRCC: fumarate hydratase gene) provides an opportunity to develop pathway specific therapies. Advances in molecular therapeutics offer novel treatment options for patients with advanced disease.

Elevated alkaline phosphatase velocity strongly predicts overall survival and the risk of bone metastases in castrate-resistant prostate cancer.

OBJECTIVES In patients with a rising prostate-specific antigen (PSA) level during treatment with androgen deprivation therapy, identification of men who progress to bone metastasis and death remains problematic. Accurate risk stratification models are needed to better predict risk for bone metastasis and death among patients with castration-resistant prostate cancer (CRPC). This study evaluates whether alkaline phosphatase (AP) kinetics predicts bone metastasis and death in patients with CRPC. METHODS AND MATERIALS A retrospective cohort study of 9,547 patients who underwent treatment for prostate cancer was conducted using the Center for Prostate Disease Research Multi-center National Database. From the entire cohort, 347 were found to have CRPC and, of those, 165 had 2 or more AP measurements during follow-up. To determine the AP velocity (APV), the slope of the linear regression line of all AP values was plotted over time. Rapid APV was defined as the uppermost quartile of APV values, which was found to be ≥6.3 IU/l/y. CRPC was defined as 2 consecutive rising PSA values after achieving a PSA nadir<4 ng/ml and documented testosterone values less than 50 ng/dl. The primary study outcomes included bone metastasis-free survival (BMFS) and overall survival (OS). RESULTS Rapid APV and PSA doubling time (PSADT) less than 10 months were strong predictors of both BMFS and OS in a multivariable analysis. Faster PSADT was a stronger predictor for BMFS (odds ratio [OR] = 12.1, P<0.0001 vs. OR = 2.7, P = 0.011), whereas rapid APV was a stronger predictor of poorer OS (OR = 5.11, P = 0.0001 vs. OR = 3.98, P = 0.0034). In those with both a rapid APV and a faster PSADT, the odds of developing bone metastasis and death exceeded 50%. CONCLUSION APV is an independent predictor of OS and BMFS in patients with CRPC. APV, in conjunction with PSA-based clinical parameters, may be used to better identify patients with CRPC who are at the highest risk of metastasis and death. These findings need validation in prospective studies.

Familial Renal Cancer: Molecular Genetics and Surgical Management

Familial renal cancer (FRC) is a heterogeneous disorder comprised of a variety of subtypes. Each subtype is known to have unique histologic features, genetic alterations, and response to therapy. Through the study of families affected by hereditary forms of kidney cancer, insights into the genetic basis of this disease have been identified. This has resulted in the elucidation of a number of kidney cancer gene pathways. Study of these pathways has led to the development of novel targeted molecular treatments for patients affected by systemic disease. As a result, the treatments for families affected by von Hippel-Lindau (VHL), hereditary papillary renal carcinoma (HPRC), hereditary leiomyomatosis renal cell carcinoma (HLRCC), and Birt-Hogg-Dubé (BHD) are rapidly changing. We review the genetics and contemporary surgical management of familial forms of kidney cancer.

Testosterone Recovery after Polytrauma and Scrotal Injury in Patients from Operation Enduring Freedom and Operation Iraqi Freedom

PURPOSE We examined the long-term natural history of testosterone recovery in patients with complex battle injuries. MATERIALS AND METHODS We retrospectively reviewed the charts of patients who participated in Operation Enduring Freedom and Operation Iraqi Freedom, and underwent urological surgical consultation at Walter Reed Army Medical Center, Washington, D.C. or the National Naval Medical Center, Bethesda, Maryland, from 2001 to August 2011. Of the 192 patient charts reviewed 138 (72%) had testosterone values available. The study inclusion criterion of at least 2 testosterone measurements, including 1 made within 40 days of injury, was met by 84 patients (61%) with testosterone data available. Those treated with bilateral orchiectomy were not required to meet this criterion. RESULTS Initial patient testosterone after injury in the testosterone recovery group was inversely proportional to the degree of scrotal injury. In patients in whom testosterone recovered to at least 250 ng/dl the recovery occurred a mean of 4.5 months after injury. Patients who required testosterone replacement had lower initial testosterone (p = 0.0063) and lower testosterone velocity (p <0.0001). CONCLUSIONS Monitoring the velocity of testosterone recovery is a viable approach in male patients who receive significant genitourinary trauma. In patients in whom testosterone recovered the recovery occurred within a mean of 5 months after injury. It is reasonable to observe patients with scrotal injuries since testosterone may recover in many of them without intervention.

MP70-13 DEPLETION OF PMEPA1 GENE CONFERS INCREASED CELL PROLIFERATION IN MOUSE PROSTATE EPITHELIUM

expression profiles of PC3CR by microarray to investigate the mechanisms of CBZ resistance. RESULTS: We incubated PC3 cells with gradually increasing concentrations of CBZ for 1.5 years, and established a CBZ-resistant cell line, PC3CR. PC3CR cells underwent cell division with 3 nM CBZ. We compared the cytotoxic effect of CBZ on PC3 and PC3CR cells using a cell viability assay. The half maximal inhibitory concentration (IC50) of CBZ in PC3 and PC3CR cells were 11.0 nM and 3.7 nM, respectively. Mice PC3CR xenograft tumors also had CBZ resistance (Fig. A) Functional annotation clustering (FAC) analysis using microarray data demonstrated cell division (GO: 0051301) and mitotic nuclear division (GO: 0007067) were the most enhanced clusters in PC3CR compared to PC3. These results suggested that enhancement of cell cycle progression signaling was related with CBZ resistance in CRPC. We perform in silico screening for compounds overcoming CBZ resistance by Connectivity Map (CMAP) analysis. Etoposide (VP16) was one of the candidate drugs which reverted gene expression pattern of CBZ-resistant cells into CBZ-sensitive cells. Topoisomerase IIa (TOP2A) is a main target of VP16, so we examined TOP2A expression in PC3CR. Quantitative PCR showed up-regulation of TOP2A in PC3CR. In cell viability assay, 1mM etoposide significantly inhibited PC3CR proliferation (80.3 3.2%). VP16 is ordinary used with platinum-based agents for neuroendocrine tumor. We tested cytotoxic effect of CDDP for CBZ-resistant CPRC. The relative cell viabilities of PC3CR treated with 3mM CDDP was 86.4 1.6%. CDDP was also effective for CBZ-resistant CRPC cells. Next, we tested anti-tumor effect of VP16 and CDDP using PC3CR xenograft tumor model. Both singleagent treatments with VP16 and CDDP significantly inhibited PC3CR xenograft tumors (Fig. B). Moreover, VP16 and CDDP in combination use had a synergic effect for the xenograft tumors. CONCLUSIONS: Etoposide based chemotherapy may be an optimal treatment for CPRC in the post-cabazitaxel setting.

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 A005: Patterns in distant metastasis at diagnosis in a racially diverse, longitudinal cohort of prostate cancer patients: 1989-2013

Introduction: The introduction of PSA screening in the U.S. and its nationwide use as a prostate cancer (PCa) screening modality led to a spike in the incidence of localized PCa in the early 1990s. With this shift toward earlier stage disease at presentation, concerns were raised for overdetection and overtreatment of clinically insignificant PCa. In response to such concerns, professional guidelines on PSA screening practices have changed over time. In 2008 the US Preventive Services Task Force (USPSTF) gave PSA screening for detection of prostate cancer a Grade D recommendation for older men (>=75 years), and in 2012 this grade D was extended to men of all age. More recently, in 2017 a draft of revised guidelines was released, elevating the letter Grade to C, only for men aged 55-69 years. Yet three compelling studies have been published that reveal increases in the diagnosis of metastatic PCa (mPCa) in U.S. men, with provocative associations in time to changes in PSA screening guidelines, prompting concern that a stage shift toward more advanced PCa at diagnosis may be occurring. The primary aim of this study was to examine time trends in mPCa at time of diagnosis, over a 20+-year study period, in a racially diverse longitudinal cohort with equal access to health care. The primary hypotheses were that mPCa at diagnosis declined after the introduction of PSA screening and that such declines would be comparable in both Caucasian (CA) and African American (AA) patients in this cohort. Methodology: The Center for Prostate Disease Research (CPDR) Multi-Center National Database was the source of patients for this study. Men under suspicion for PCa and undergoing TRUS-guided biopsy for PCa detection are eligible for enrolment in this database. This study focused on those with biopsy-confirmed PCa between January 1, 1989 and December 31, 2013. Trends in mPCa at the time of diagnosis were examined for the overall cohort, as well as stratified by race (AA and CA) and patient age at CaP diagnosis ( Results: A total of 462 men presented with mPCa and were eligible for inclusion. The decline in APC for the overall cohort was statistically significant (APC = -8.5%, p Conclusions: In this longitudinal, racially diverse cohort with equal health care access, significant declines in mPCa at diagnosis were observed over a 20+-year study period. This is contrast to other recent studies that have demonstrated increases in mPCa following changes in USPSTF guidelines. There was, however, a difference in the magnitude of decrease in oldest patients (≥75 years) compared to younger men, which may have been influenced by changing PSA screening recommendations in 2008 by the USPSTF. Continued attention to shifts in mPCa at diagnosis is needed to determine the impact of changes in screening recommendation. Citation Format: John McCauley, Huai-Ching Kuo, Inger L. Rosner, Yongmei Chen, Lauren Hurwitz, Sean Stroup, Joseph R. Sterbis, Christopher Porter, Timothy C. Brand, Shiv Srivastava, Jennifer Cullen. Patterns in distant metastasis at diagnosis in a racially diverse, longitudinal cohort of prostate cancer patients: 1989-2013 [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 A005.

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.

MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS

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. ERG status was determined in whole mounted prostate specimens by immunohistochemistry (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 (<10-5) associated with ERG fusion status included rs6698333, an intron variant of Kruppel-like factor 17 (KLF17) and two SNPs (rs1889877, rs3798999) in the intron of adhesion G proteincoupled receptor B3 (ADGRB3). Fine-mapping of SNPs is underway by genotype imputation analysis (IMPUTE2) using the 1000 Genomes reference dataset, followed by independent validation. 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.

PD71-11 P53 FOCAL PROTEIN EXPRESSION IN PRIMARY PROSTATE TUMORS AND LYMPHATIC VESSEL INVASION PREDICT BIOCHEMICAL RECURRENCE AND METASTATIC PROGRESSION

Pathological features of primary M1 tumors are generally indistinguishable from those of high-grade localized (M0) cases, however 5year survival for M0 PC is nearly 100%. Digital image analysis is an evolving 00OMICS00 platform for biomarker development that can be applied to diagnostic histopathology. We hypothesize that novel software analysis tools and machine learning can systematically interrogate digitized prostate needle biopsy (PNBX) slides to extract histomorphometric features that identify discrepant architecture and nuclear texture of M0 and M1 tumors. Herein, algorithms that measure these features were developed in a training set of digital images and then validated in an independent patient cohort. METHODS: We created a biorepository of diagnostic PNBX specimens from 2150 PC patients from the Greater Los Angeles VA Healthcare System between 2000 and 2016. The biorepository was mined to create a matched cohort of M0 (n1⁄444) and M1 (n1⁄461) cases. Slides were digitized at 40X magnification and two pathologists annotated all cancer foci. ~30 image tiles were obtained from each case (n1⁄42857) and 88 features were extracted. Segmentation based fractal texture descriptors (SFTA), Gabor (GF), grey level run length (GLRL), and nuclear texture (CP) features were used to train a classifier to distinguish M0 from M1 tiles. RESULTS: After conversion of M0 and M1 image tiles to digital nuclear masks, training features were used to classify nuclear texture or tissue architecture. The majority vote from nuclear classification was transferred to the tile level and the majority classification of tiles was used to classify each case. For tissue architecture, 45 STFA and 60 Gabor features classified M1 and M0 cases with an accuracy of 71.8% and 80%, respectively. For nuclear features, 44 GLRL and 8 CP classified M0 versus M1 cases with an accuracy of 63% and 75.4%, respectively. A classifier trained with a combined 88 features achieved 86% accuracy in distinguishing M1 from M0 cases. CONCLUSIONS: We applied digital imaging technology and machine learning to extract 88 novel features that accurately differentiate high-grade M0 from M1 PC. The quantification of tissue architecture and nuclear morphology provides an orthogonal approach for biomarker development, which can be applied to prognostication and potential treatment decisions in patients with high-risk localized or metastatic PC.