Amy Mulick Cassidy

Clinical and Biochemical Consequences of CYP17A1 Inhibition with Abiraterone Given with and without Exogenous Glucocorticoids in Castrate Men with Advanced Prostate Cancer

CONTEXT Abiraterone acetate is a small-molecule cytochrome P450 17A1 (CYP17A1) inhibitor that is active in castration-resistant prostate cancer. OBJECTIVE Our objective was to determine the impact of abiraterone with and without dexamethasone treatment on in vivo steroidogenesis. DESIGN AND METHODS We treated 42 castrate, castration-resistant prostate cancer patients with continuous, daily abiraterone acetate and prospectively collected blood and urine before and during abiraterone treatment and after addition of dexamethasone 0.5 mg daily. RESULTS Treatment with single-agent abiraterone acetate was associated with accumulation of steroids with mineralocorticoid properties upstream of CYP17A1. This resulted in side effects, including hypertension, hypokalemia, and fluid overload, in 38 of 42 patients that were generally treated effectively with eplerenone. Importantly, serum and urinary androgens were suppressed by more than 90% from baseline. Urinary metabolites of 17-hydroxypregnenolone and 17-hydroxyprogesterone downstream of 17α-hydroxylase remained unchanged. However, 3α5α-17-hydroxypregnanolone, which can be converted via the backdoor pathway toward 5α-dihydrotestosterone, increased significantly and correlated with levels of the major 5α-dihydrotestosterone metabolite androsterone. In contrast, urinary metabolites of 11-deoxycortisol and active glucocorticoids declined significantly. Addition of dexamethasone to abiraterone acetate significantly suppressed ACTH and endogenous steroids, including 3α5α-17-hydroxypregnanolone. CONCLUSION CYP17A1 inhibition with abiraterone acetate is characterized by significant suppression of androgen and cortisol synthesis. The latter is associated with a rise in ACTH that causes raised mineralocorticoids, leading to side effects and incomplete 17α-hydroxylase inhibition. Concomitant inhibition of 17,20-lyase results in diversion of 17-hydroxyprogesterone metabolites toward androgen synthesis via the backdoor pathway. Addition of dexamethasone reverses toxicity and could further suppress androgens by preventing a rise in substrates of backdoor androgen synthesis.

Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

Improved Survival in a Cohort of Trial Participants with Metastatic Castration-resistant Prostate Cancer Demonstrates the Need for Updated Prognostic Nomograms

BACKGROUND Median overall survival (OS) in men with metastatic castration-resistant prostate cancer (CRPC) was 13-16 mo in the predocetaxel era. Prognostic nomograms for survival estimation in CRPC were constructed prior to the introduction of docetaxel and other novel treatments. OBJECTIVE To examine whether prognostic models still accurately reflect survival in a large cohort of trial participants. DESIGN, SETTING, AND PARTICIPANTS Survival analysis of 442 men with CRPC sequentially treated in clinical trials at our institution from June 2003 to December 2011. OUTCOME MEASURES AND STATISTICAL ANALYSIS Predicted survival by Halabi and Smaletz nomograms was compared to observed survival. Cox model multivariate analysis (MVA) used variables at referral, including performance status (PS); levels of prostate-specific antigen (PSA), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), haemoglobin (Hb), and albumin; presence of visceral disease, and metastatic disease at diagnosis. RESULTS AND LIMITATIONS From point of referral, chemotherapy-naïve patients had a median OS of 30.6 mo (95% confidence interval [CI], 27.6-36.5 mo). In contrast, predicted survival using the Halabi and Smaletz models was 21 and 18 mo, respectively. In these patients, poor PS, lower Hb level, and increasing LDH level were the strongest predictors in the MVA. In patients referred after chemotherapy, survival from referral was 17.5 mo (95% CI, 16.0-19.5 mo) and increasing LDH level and presence of visceral metastases were the strongest predictors of survival. Median OS from diagnosis of CRPC was 40.7 mo in the overall cohort (95% CI, 36.8-44.0 mo). Clinical trial participation was safe, with low mortality rate. This cohort of men participated in phase 1, 2 and 3 trials and expanded access programs; their data may not reflect survival in all CRPC patients. CONCLUSIONS Due to the impact of highly effective novel therapies on survival, prognostic nomograms in current use require revalidation regarding their ability to predict survival in CRPC.

A HIF-Regulated VHL-PTP1B-Src Signaling Axis Identifies a Therapeutic Target in Renal Cell Carcinoma

Signaling through the VHL-PTP1B-Src pathway in renal cell carcinomas may determine sensitivity to Src inhibitors and provide a basis for treatment planning. Detecting Sensitivity to Src Inhibitors Typically, tumor suppressors are welcome tenants in cells, protecting them from becoming cancerous. But in a twist of fate, 40% of patients with renal cell carcinoma (RCC) regard the presence of a functional tumor suppressor—the von Hippel-Lindau (VHL) protein—as bad news. And for good reason. In contrast to the other 60% of RCC patients whose tumors are driven by the loss of intact VHL, the tumor-suppressor positive cancers are more likely to be resistant to immunotherapy and chemotherapy, and because researchers do not know what drives tumorigenesis, no rational targeted therapies exist. Not content to wait for another twist of fate, Suwaki et al. have delved deeply into these kidney cancers and found that VHL functions as part of an activated signaling pathway that renders the cells sensitive to anticancer agents that target the Src oncoprotein. The presence of VHL and other markers of this pathway can flag those RCC patients who may benefit from drugs that block Src kinase activity. Enhanced activity of the Src tyrosine kinase has been implicated in cancer development and is the target of the anticancer drug dasatinib. By measuring the extent of phosphorylation of critical proteins in a pair of cell lines with and without functional VHL, the authors saw that Src and its substrates were activated only when VHL was present. And in 215 RCCs, the presence of VHL tended to be associated with highly active Src kinase. Dasatinib inhibited DNA synthesis and cell growth only in cells with VHL, whether they were grown in culture or as xenografts in mice. The authors also replicated the well-known ability of VHL to negatively regulate the hypoxia-sensitive transcription factor HIF, which is activated indirectly by Src. As expected, expression of HIF in VHL-containing cancer cells conferred resistance to dasatinib. An independent analysis of this pathway in tumor samples from an additional 131 patients with RCC confirmed the positive correlation between VHL and Src and its associated pathway proteins. Here, the authors used quantitative phosphoproteomics and immunohistochemical profiling to show the correlation. Because they used automated digital image analysis and an unsupervised hierarchical clustering of the tumors on the basis of the expression of VHL, Src, pFAK and PTP1B, this approach has the potential to be used in the clinic for tumor characterization. Personalized approaches to cancer treatment require an armamentarium of matched pairs of drugs and validated biomarkers that predict response to therapy. The markers for an activated Src pathway discerned by the authors could in theory be assessed in any solid cancer; in fact, a test in bladder carcinomas showed that, as in RCCs, the Src pathway proteins were activated. If such a test can be applied to many solid cancers, physicians could use it to predict whether a patient is likely to respond to anti-Src agents. Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

Unbiased and Automated Identification of a Circulating Tumour Cell Definition That Associates with Overall Survival

Circulating tumour cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC however remains subjective, as they are morphologically heterogeneous. We acquired digital images, using the CellSearch™ system, from blood of 185 castration resistant prostate cancer (CRPC) patients and 68 healthy subjects to define CTC by computer algorithms. Patient survival data was used as the training parameter for the computer to define CTC. The computer-generated CTC definition was validated on a separate CRPC dataset comprising 100 patients. The optimal definition of the computer defined CTC (aCTC) was stricter as compared to the manual CellSearch CTC (mCTC) definition and as a consequence aCTC were less frequent. The computer-generated CTC definition resulted in hazard ratios (HRs) of 2.8 for baseline and 3.9 for follow-up samples, which is comparable to the mCTC definition (baseline HR 2.9, follow-up HR 4.5). Validation resulted in HRs at baseline/follow-up of 3.9/5.4 for computer and 4.8/5.8 for manual definitions. In conclusion, we have defined and validated CTC by clinical outcome using a perfectly reproducing automated algorithm.

Sarcopenia and change in body composition following maximal androgen suppression with abiraterone in men with castration-resistant prostate cancer

Background:Standard medical castration reduces muscle mass. We sought to characterize body composition changes in men undergoing maximal androgen suppression with and without exogenous gluocorticoids.Methods:Cross-sectional areas of total fat, visceral fat and muscle were measured on serial CT scans in a post-hoc analysis of patients treated in Phase I/II trials with abiraterone followed by abiraterone and dexamethasone 0.5 mg daily. Linear mixed regression models were used to account for variations in time-on-treatment and baseline body mass index (BMI).Results:Fifty-five patients received a median of 7.5 months abiraterone followed by 5.4 months abiraterone and dexamethasone. Muscle loss was observed on single-agent abiraterone (maximal in patients with baseline BMI >30, −4.3%), but no further loss was observed after addition of dexamethasone. Loss of visceral fat was also observed on single-agent abiraterone, (baseline BMI >30 patients −19.6%). In contrast, addition of dexamethasone led to an increase in central visceral and total fat and BMI in all the patients.Interpretation:Maximal androgen suppression was associated with loss of muscle and visceral fat. Addition of low dose dexamethasone resulted in significant increases in visceral and total fat. These changes could have important quality-of-life implications for men treated with abiraterone.

Mendelian Randomisation study of the influence of eGFR on coronary heart disease

Impaired kidney function, as measured by reduced estimated glomerular filtration rate (eGFR), has been associated with increased risk of coronary heart disease (CHD) in observational studies, but it is unclear whether this association is causal or the result of confounding or reverse causation. In this study we applied Mendelian randomisation analysis using 17 genetic variants previously associated with eGFR to investigate the causal role of kidney function on CHD. We used 13,145 participants from the UCL-LSHTM-Edinburgh-Bristol (UCLEB) Consortium and 194,427 participants from the Coronary ARtery DIsease Genome-wide Replication and Meta-analysis plus Coronary Artery Disease (CARDIoGRAMplusC4D) consortium. We observed significant association of an unweighted gene score with CHD risk (odds ratio = 0.983 per additional eGFR-increasing allele, 95% CI = 0.970–0.996, p = 0.008). However, using weights calculated from UCLEB, the gene score was not associated with disease risk (p = 0.11). These conflicting results could be explained by a single SNP, rs653178, which was not associated with eGFR in the UCLEB sample, but has known pleiotropic effects that prevent us from drawing a causal conclusion. The observational association between low eGFR and increased CHD risk was not explained by potential confounders, and there was no evidence of reverse causation, therefore leaving the remaining unexplained association as an open question.

Abstract 3072: Prospective study of genetic mutations in matched tumor and plasma specimens in advanced cancer patients referred to phase I trials

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Circulating free DNA (cfDNA) represents a minimally-invasive resource for detecting mutations in advanced cancer patients. The primary objective of this study was to determine if cfDNA is representative of tumor tissue for multiplex mutation detection utilizing Sequenom MassARRAY. Methods: Metastatic colorectal, breast, melanoma and ovary cancer patients referred to the Drug Development Unit at the Royal Marsden Hospital between 9/09-8/10 gave their consent to provide DNA from matched archival formalin fixed paraffin embedded (FFPE) tumor and plasma. Samples mutation genotyping using the Sequenom MassARRAY technology and the OncoCartaTM Panel allowed the parallel analysis of 238 simple and complex mutations across 19 selected key oncogenes. Dilutions (10ng/µl to 0.01ng/µl) of HCT116 DNA containing KRAS G13D mutation were used to determine assay sensitivity and specificity. Results: Serial dilution spiking experiments revealed that the KRAS G13D mutation was reproducibly detectable to 40ng/ml of HCT116 DNA; 74 patient samples were then analyzed. The median quantity of cfDNA was 305ng/ml (range 10-32000). Of the 25 colorectal patients, the median quantity of cfDNA was 361ng/ml (range 189-4603); KRAS, BRAF and PIK3CA mutations were detected in 8 (31%), 3 (12%) and 3 (12%) tumor specimens respectively; concordant data for matched plasma tumor DNA was found in 88% of patients for KRAS, 100% for BRAF mutations and 33% for PIK3CA (1 patient had a PIK3CA mutation in primary and plasma; one patient had a mutation detected only in the primary; one patient had no mutation in the primary or plasma, but a mutation detected in a metastatic liver deposit). The recently reported oncogenic AKT1 E17K mutation was detected in 1 colorectal patient in tissue and plasma. Of the 19 breast cancer patients, the median cfDNA concentration was 354ng/ml (range 10-32000); PIK3CA H1047R mutation was detected in 4 (21%) FFPE tumor specimens; concordance between matched FFPE and cfDNA was 75% (3 out of 4 patients). The AKT1 E17K mutation was detected in 1 patient in both tissue and plasma. In 15 melanoma patients the median cfDNA concentration was 141ng/ml (range 46-1002); BRAF V600E, NRAS codon 61 and MET R970C mutations were detected in 4 (27%), 3 (20%) and 1 (7%) FFPE tumor specimens respectively. Concordance between matched FFPE and cfDNA was 50% for BRAF, 75% for NRAS and 100% for MET mutations. Another MET mutation, T992I, was found in plasma but not in tissue for one patient. Of the 15 ovary patients, the median cfDNA concentration was 261ng/ml (range 79-957). Two KRAS mutations and 1 PIK3CA H1047R mutation were detected in FFPE, but no mutation was found in any plasma sample. Conclusions: A high concordance in detected mutations was observed between FFPE tumor and matched cfDNA. cfDNA is representative of tumor DNA and may be used for the prospective selection of cancer patients for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2011-3072