Citations

29 Background: Better biomarkers are needed in prostate cancer (PCa) to predict disease recurrence and guide optimal therapy. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor cancer phenotype impact PCa biology and clinical outcomes in localized disease. Methods: Genes associated with self-renewal, drug resistance, and tumorigenicity were analyzed by qRT-PCR on PCa mRNA from radical prostatectomy (RP) specimens (+/- disease recurrence). Wilcoxon rank sum two-sample test, multivariable recursive partitioning, and bootstrap internal validation measured and confirmed associations with recurrence. Further validation was conducted in external cohorts and in-silico, as well as in vitro and in vivo using siRNA knockdown and lentiviral overexpression to determined the effect of gene expression on PCa proliferation, invasion and tumor growth. Results: Four candidate genes were differentially expressed in PCa recurrence, and of these, low AXIN2 expression was internally validated. Validation in external cohorts demonstrated low AXIN2 expression was associated with more aggressive prostate cancer and was independently associated with biochemical recurrence (BCR) and metastasis-free survival (MFS) after RP. In vitro, low AXIN2 expression was associated with a cancer stem-like cell-surface signature, and siRNA knockdown of AXIN2 resulted in significantly greater invasiveness. Conversely, ectopic overexpression of AXIN2 significantly reduced cell proliferation and tumor growth in mice. Conclusions: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts. AXIN2 expression levels in PCa cells significantly impacted invasiveness, proliferation and tumor growth. AXIN2 represents a putative biomarker and potential therapeutic target in early prostate cancer.

255 Background: In a prior study, we found that 8 single nucleotide polymorphisms (SNPs) from 4 candidate hormone-related genes (JAK2, TRMT11, NKX3-1 and HSD17B12) were associated with overall survival (OS) in CRPC stage in a North American cohort of 519 subjects. We attempt to replicate these findings in an independent cohort of Portuguese patients. Methods: A hundred and forty CRPC stage patients in the new cohort were genotyped fort he candidate SNPs. The primary endpoint was overall survival (OS), defined as time from development of CRPC to death. For SNP level results we estimated hazard ratios (HR) and 95% confidence intervals (CI) under the dominant allele model using Cox regression and adjusted for age and Gleason score (GS). Results: The median age of the Portuguese cohort was 69 years (range 53-84). The GS distribution was 48% subjects with GS≥8; 35% with GS=7 and 17% with GS<7. Median time from castration resistance to death for the cohort was 2.2 years (IQ range: 0.8-3.4, 93 deaths). One of the 3 SNPs (rs2295005; C>G; MAF = 0.16) in the TRMT11gene was associated with OS after adjustment for age and GS: (dominant model p=0.05, HR=0.56; 95 CI=0.31-1.00). No other SNP was observed to have statistical significance with OS (Table 1). Conclusions: Variation in the TRMT11 gene is significantly associated with overall survival in patients with CRPC and warrants further validation as a potential prognostic biomarker. [Table: see text]

249 Background: Evaluating tumor specific pathologic and genetic profiles in mCRPC stage is difficult due to the limited availability of mCRPC tissue. We describe findings from a prospective cohort study performed to obtain concomitant histopathology and genetic information in mCRPC. Methods: Patients with mCRPC initiating abiraterone acetate therapy underwent 2 serial (3 months apart) metastatic (met) site needle core biopsies (NCB1/2). Bone lesions were biopsied using 11-13G core needles and 18G core biopsy devices were used for non-osseous masses. Up to 4 cores were obtained at each biopsy with the 1st core (S1) sent for DNA sequencing, the 2nd (S2) for RNA sequencing, and the 3rd/4th (X3, X4) submitted for xenograft implantation. From each, 1-2 mm segments were separated and formalin-fixed for histopathologic examination (HPE). Results: A total of 54 patients, enrolled between June 2013 and July 2014, underwent 94 NCB (54 NCB1, 40 NCB2), rendering a total of 259 samples (94 S1; 75 S2; 59 X3 and 31 X4) of which 85 (32%) were positive (pos) for tumor by HPE. Positivity for tumor in S1, S2, X3, and X4 cores was 42%, 33%, 22%, and 19%, respectively. At least one core pos for met tumor was observed in 62% NCB1 and 52% NCB2 (overall 52/94; 56%). Met sites biopsied include bone (71), lymph nodes (18), liver (3), penile (1) and pelvic (1) soft tissues. HPE revealed met adenocarcinoma in 44/52, and poorly differentiated carcinoma in 8/52. Gleason grade applied to the met ranged from 3+4 to 5+5. 45/85 pos samples (53%) had > 50% tumor cellularity. Pos NCB in bone lesions was observed in 23/71 (32%), compared to 17/23 (73%) of non-bone sites (p=.0004). 85 S1 samples yielded DNA material, of which 66 (77%) had ≥10% tumoral DNA. 24 cases with negative HPE had ≥10% tumoral DNA; 7 cases with pos HPE had <10 tumoral DNA. In all, 76/94 (81%) NCB yielded tumor material either by HPE or DNA analysis. Conclusions: NCB of mCRPC is feasible and provides adequate tissue for pursuing HPE and sequencing studies. HPE of extracted material correlates with DNA sequencing data and provides complementary information on tumor features. Bone lesions yield significantly less tumoral material than non-osseous sites.

238 Background: Cbz/pred extends survival in mCRPC patients (pts) following docetaxel and first-line studies comparing Cbz to docetaxel are ongoing. Mito/pred also has anti-tumor activity in mCRPC and a non-overlapping mechanism of action and toxicity profile with Cbz. A multicenter phase 1 trial was initiated through the DOD PCCTC to establish the maximally tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the combination of Cbz, Mito, and Pred (CAMP). Methods: Chemotherapy (chemo)-naïve pts with mCRPC were enrolled in an accelerated titration design. The primary objective was to determine the MTD and RP2D of CAMP; secondary objectives included PSA response rate and duration of response. Cbz 20 and 25 mg/m2 were each evaluated in combination with escalating doses of Mito (starting dose 4 mg/m2), both administered on day 1 of a 21-day cycle. Pred 5 mg BID and pegfilgrastim were given with each cycle. Results: 20 pts were enrolled. The median age was 66 (range 51-78) and the median baseline PSA was 62.5 (range 3.0-791.2). There were 2 DLTs observed at the dose level of Cbz 25 mg/m2 + Mito 10 mg/m2 (sepsis and febrile neutropenia). No DLTs were observed with Cbz 20 mg/m2 (N = 10), establishing Cbz 20 mg/m2 + Mito 12 mg/m2 as the MTD and RP2D. The most common grade ≥ 3 related adverse events were hematologic (neutropenia, n = 8; thrombocytopenia, n = 3; febrile neutropenia, n = 2). No cardiac adverse events were observed. The median number of treatment cycles was 9 (range 2-16), and 5 pts remain on study. Greater than 50% maximal PSA declines from baseline were observed in 8 of 18 evaluable pts (44%). Objective tumor responses have been observed in 2 of 4 pts (50%) with measurable disease, with evaluation ongoing. The median duration of response has not been reached (range 4.9-10.0+ months). Conclusions: The approved single-agent dose of Mito (12 mg/m2) was safely combined with Cbz 20 mg/m2, a dose with demonstrated activity in mCRPC and potentially less hematologic toxicity than 25 mg/m2. Preliminary efficacy data are encouraging with durable responses observed in a subset of pts. Further study of the treatment combination is warranted. Clinical trial information: NCT01594918.

23 Background: Perineural invasion (PNI) on prostate cancer (PC) biopsies has been associated with disease upgrading among those undergoing radical prostatectomy. However, the clinical significance of PNI in men on active surveillance (AS) has been evaluated by a limited number of studies. Thus, we sought to evaluate the association of PNI with time to clinical and pathological progression in men with PC on AS. Methods: Retrospective analysis of 289 men 48 to 82 years old on AS for low-risk PC (T1c-T2a), Gleason ≤6, ≤3 positive cores, ≤50% of any core involved, prostate-specific antigen (PSA) ≤11ng/ml, life expectancy >5 years and follow-up data in the REduction by Dutasteride of clinical progression Events in Expectant Management study. Progression was divided in pathological (>3 positive cores, >50% core involvement or Gleason >6 in a repeat biopsy) or therapeutic (any treatment for PC) or both. Time to progression was analyzed with Kaplan-Meier plots, log-rank tests and Cox model adjusting for age, PSA density, percent cores involved, maximum core involvement and treatment. Results: A total of 11 (4%) patients had PNI on baseline biopsy. PNI was associated with higher tumor length and maximum core involvement (all P<0.05). PNI was not associated with patient’s age, race, PSA levels or density, percent or number of positive cores. After a median follow-up of 37 months, 125 (43%) patients developed progression. Of these, 95 (76%) patients had pathological and 30 (24%) had therapeutic progression. In univariable analysis, patients with baseline PNI had a shorter time to overall and pathological progression (HR=2.62, 95%CI=1.31-5.23, P=0.006 and HR=2.42, 95%CI=1.03-5.66, P=0.041, respectively). Similar results were obtained in multivariable analysis for overall and pathological progression (HR=2.26, 95%CI=1.10-4.68, P=0.028 and HR=2.13, 95%CI=0.88-5.13, P=0.092, respectively). Conclusions: Among patients with PC on AS, PNI is independently associated with shorter time to progression. Thus, PNI may be used to help select patients for AS and stratify them according to the risk of disease progression.

209 Background: Predictive markers of response to AA are not known. In a prior study, we found that 8 single nucleotide polymorphisms (SNPs) from 4 candidate hormone-related genes (JAK2, TRMT11, NKX3-1 and HSD17B12) were associated with response to AA in HSPC in a North American cohort of 519 subjects. We attempt to replicate these findings in a cohort of Portuguese patients. Methods: The Portuguese cohort consisted of 308 HSPC patients, and the primary endpoint was time to AA failure, defined as time from initiating continuous AA to two serial PSA increases over the nadir, initiation of chemotherapy for progressive disease, or clinical or imaging based progression, whichever came first. For each SNP, we calculated hazard ratios (HRs) and 95% confidence intervals (CI) for time to AA failure using a recessive model with adjustment for Gleason score (GS). Results: The median age of the Portuguese cohort was 69 years (range 43, 85). The GS distribution was 35% subjects with GS≥8; 35% with GS=7 and 30% with GS<7. Median time to AA failure for the cohort was 1.8 years (IQ range: 0.3, 3.6). Association of each SNP with time to AA failure is shown in the Table. None of the SNPs replicated. However, there was a suggestive inverse association for rs10503733 (NKX3.1) with time to AA failure (P=0.07). Conclusions: While we were not able to replicate our initial findings, there was an unexpected association with rs10503733 (NKX3.1 gene) that may warrant additional clinical validation. [Table: see text]

190 Background: It is not known if biopsy of CRPC stage metastases alters CTC results. We evaluated pre & post biopsy CTC counts in a prospective cohort undergoing pre-chemotherapy abiraterone acetate/prednisone (AA/P). Methods: CTC enumeration was performed on 7.5mL blood using the FDA cleared immunomagnetic/immunofluorescence assay (CellSearch). Time of blood draw for CTC counts pre/post biopsy was recorded for each biopsy. Biopsies were performed using 11/13 gauge core biopsy device for bone lesions and 18 gauge core needle biopsy device for soft tissue/nodal metastases. All patients (pts) underwent two serial biopsies 3 months apart. Change in CTC counts pre/post biopsy for visits 1 and 2 were evaluated using Wilcoxon signed rank test (significance at p<0.05). Differences in CTC count change (visit1) pre/post biopsy based on metastatic site (skeletal vs. non-skeletal) was compared using Wilcoxon rank-sum test. Results: Median age of the cohort was 74 years (IQR: 68-78); median PSA at V1 was 16.4 ng/ml (IQR: 6.4-87.6); at V2 was 11.5 ng/ml (IQR: 1.8-23.7). Of 59 pts undergoing biopsies 42 were in bone and 17 in nodal/soft tissue masses. At V1, pre/post biopsy CTC counts were measured on 50 and 49 pts, respectively; at V2, pre/post CTC counts were measured on 43 and 36 pts. High volume metastatic disease at V1 was observed in 67.8 % of all pts. Table lists CTC counts and time of collection pre/post biopsy for both visits. Mean change in pre/post biopsy CTC counts for skeletal sites was 0.7 (range: -12 to 64) compared to 12.1 (range: -21 to 13) for non-skeletal sites (p=0.23). Conclusions: An increase in mean CTC counts after biopsy of CRPC metastases was observed. No significant differences in change of CTC counts based on site biopsied (skeletal versus non-skeletal) was noted. Correlation with clinical outcomes is on-going. Clinical trial information: NCT# 01953640. [Table: see text]

174 Background: AA/P is an FDA approved treatment for mCRPC. Since markers of early resistance to AA/P are unknown, we report initial findings of androgen receptor (AR) based associations with short term (12 week) progression on AA/P. Methods: mCRPC stage patients (pts) initiating pre-chemotherapy AA/P underwent metastatic site biopsies at baseline (pre AA/P) and after 12 weeks. Baseline somatic whole exome DNA, tumor gene expression for AR full length (ARFL), AR splice variant 7 (ARV7) and ARV7/ARFL ratios were compared in progessors versus non-progressors. Progression at or within 12 weeks of AA/P therapy was defined as death or disease progression by PCWG2 “composite progression (CP)” and/or “radiographic progression” endpoints. Wilcoxon rank-sum tests were used to test for differences in the two groups for comparing ARFL, ARV7 expressions and ARV7/ARFL ratios and chi square tests were used for differences in copy number variation. Results: Between 1/2013 and 6/2014, 59 pts were enrolled of which 44 have disease assessment data at the12-week time point. CP was observed in 17/44 patients. DNA seq and clinical data was available for 42/44 pts. Using radiographic progression at 12 weeks, AR Amplification/Gain was observed in 20/26 non-progressors (13 with Amplification) and in 9/16 progressors (P-value = 0.19; OR 2.5). ARFL and ARV7 gene expressions in both groups is provided in table. Conclusions: A trend towards higher ARV7/ARFL gene expression ratio in metastases was observed with early progression on AA/P. AR gain/amplification is observed less often in pts with early progression. Validation of these findings is on-going in this prospective trial. Clinical trial information: NCT# 01953640. [Table: see text]

168 Background: The optimal timing of postoperative radiotherapy following radical prostatectomy (post-RP RT) is unclear. We hypothesized that a genomic classifier (GC) would provide prognostic and predictive insight into the development of clinical metastases in men receiving post-RP RT and inform decision-making. Methods: GC scores were calculated from 188 patients with pT3 or margin positive PCa, who received post-RP RT at Thomas Jefferson University and Mayo Clinic, between 1990 and 2009. The primary endpoint was clinical metastasis. Prognostic accuracy of the models were tested using c-index and decision curve analysis. Cox regression tested the relationship between GC and metastasis. Results: The cumulative incidence of metastasis at 5 years post-RT was 0%, 9%, and 29% for low, average, and high GC scores, respectively (p=0.002). In multivariable analysis, GC and pre-RP PSA were independent predictors of metastasis (both p<0.01). Within the low GC score (<0.4), there were no differences in the cumulative incidence of metastasis comparing those who received adjuvant or salvage RT (p=0.79). However, for patients with higher GC scores (≥0.4) cumulative incidence of metastasis at 5-year was 6% vs. 23% for patients treated with adjuvant vs. salvage RT (p<0.01). Conclusions: In patients treated with post-RP RT, GC is prognostic for the development of clinical metastasis beyond routine clinical/pathologic features. Though preliminary, patients with low GC are best treated with salvage radiation, while those with high GC benefit from adjuvant therapy. These findings provide the first rationale selection of timing of post-RP RT.

12 Background: Cholesterol is needed in prostate cancer cells for cell proliferation and for androgen biosynthesis. Cholesterol-lowering statin drug use is associated with a lower risk of total and advanced prostate cancer and lower prostate cancer-specific mortality (PCSM). We hypothesized that tumor expression of cholesterol synthesis enzymes, transporters and regulators is associated with PCSM. Methods: We studied mRNA expression of 43 cholesterol metabolism genes in surgical specimens from 249 participants in the Health Professionals' Follow-up Study diagnosed with prostate cancer between 1986 and 2004. We included 81 cases of lethal prostate cancer and 168 indolent cases who survived >8 years without metastases. After multivariate dimensionality reduction for the synthesis, transport and regulator pathways using principal components analysis, we estimated odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression. We adjusted for age, year of diagnosis, body mass index, smoking status, family history, multivitamin use, high serum cholesterol and cholesterol-lowering drug use. Results: Reflecting the impact of the synthesis pathway and of the regulator pathway, respectively, the expression of squalene epoxidase (SQLE) and low-density lipoprotein receptor (LDLR) were significantly associated with lethal cancer in opposing directions. Compared to men with the mean level of SQLE expression, men with SQLE expression >1 standard deviation (SD) above the mean were 15.4 times more likely to have lethal cancer (95% CI: 3.31–71.53). For LDLR, expression >1 SD above the mean was associated with 96% lower odds of lethal cancer (95% CI: 0.01–0.70). Higher SQLE expression and lower LDLR expression were associated with higher Gleason grades (Fisher's test, p < 0.001 and p = 0.036, respectively) and were also predictive of PCSM independently from grade and tumor stage. Conclusions: These results suggest that the local cholesterol metabolism of the prostate tumor is tightly linked with mortality. Lethal cancers have higher activity of the second rate-controlling enzyme of cholesterol synthesis and are less dependent on cholesterol uptake.