David J. VanderWeele

Low‐grade prostate cancer diverges early from high grade and metastatic disease

Understanding the developmental relationship between indolent and aggressive tumors is central to understanding disease progression and making treatment decisions. For example, most men diagnosed with prostate cancer have clinically indolent disease and die from other causes. Overtreatment of prostate cancer remains a concern. Here we use laser microdissection followed by exome sequencing of low‐ and high‐grade prostate cancer foci from four subjects, and metastatic disease from two of those subjects, to evaluate the molecular relationship of coincident cancer foci. Seventy of 79 (87%) high‐confidence somatic mutations in low‐grade disease were private to low‐grade foci. In contrast, high‐grade foci and metastases harbored many of the same mutations. In cases in which there was a metastatic focus, 15 of 80 (19%) high‐confidence somatic mutations in high‐grade foci were private. Seven of the 80 (9%) were shared with low‐grade foci and 65 (82%) were shared with metastatic foci. Notably, mutations in cancer‐associated genes and the p53 signaling pathway were found exclusively in high‐grade foci and metastases. The pattern of mutations is consistent with early divergence between low‐ and high‐grade foci and late divergence between high‐grade foci and metastases. These data provide insights into the development of high‐grade and metastatic prostate cancer.

Quantitative characterization of androgen receptor protein expression and cellular localization in circulating tumor cells from patients with metastatic castration-resistant prostate cancer

BackgroundMany current therapies for metastatic castration-resistant prostate cancer (mCRPC) are aimed at AR signaling; however, resistance to these therapies is inevitable. To personalize CRPC therapy in an individual with clinical progression despite maximal AR signaling blockade, it is important to characterize the status of AR activity within their cancer. Biopsies of bone metastases are invasive and frequently fail to yield sufficient tissue for further study. Evaluation of circulating tumor cells (CTCs) offers an alternative, minimally invasive mechanism to characterize and study late-stage disease. The goal of this study was to evaluate the utility of CTC interrogation with respect to the AR as a potential novel therapeutic biomarker in patients with mCRPC.MethodsFifteen mL of whole blood was collected from patients with progressive, metastatic mCRPC, the mononuclear cell portion was isolated, and fluorescence-activated cell sorting (FACS) was used to isolate and evaluate CTCs. A novel protocol was optimized to use ImageStreamX to quantitatively analyze AR expression and subcellular localization within CTCs. Co-expression of AR and the proliferation marker Ki67 was also determined using ImageStreamX.ResultsWe found inter-patient and intra-patient heterogeneity in expression and localization of AR. Increased AR expression and nuclear localization are associated with elevated co-expression of Ki-67, consistent with the continued role for AR in castration-resistant disease. Despite intra-patient heterogeneity, CTCs from patients with prior exposure to abiraterone had increased AR expression compared to CTCs from patients who were abiraterone-naïve.ConclusionsAs our toolbox for targeting AR function expands, our ability to evaluate AR expression and function within tumor samples from patients with late-stage disease will likely be a critical component of the personalized management of advanced prostate cancer. AR expression and nuclear localization varies within patients and between patients; however it remains associated with markers of proliferation. This supports a molecularly diverse AR-centric pathobiology imparting castration-resistance.

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti‐apoptotic gene Bcl‐xL. Anti‐apoptotic Bcl‐2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N‐acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl‐xL did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H2O2 exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death. Copyright

Mammalian target of rapamycin promotes vincristine resistance through multiple mechanisms independent of maintained glycolytic rate.

Deregulation of the phosphoinositide 3-kinase-Akt pathway is a major contributor to oncogenesis and resistance to cancer therapy. Recent work has shown mammalian target of rapamycin (mTOR) to be a major target downstream of Akt that contributes to both transformation and therapeutic resistance. Although inhibitors of Akt are not yet clinically available, rapamycin, a mTOR-specific inhibitor, has long been used as an immunosuppressant, and several rapamycin analogues are now in clinical trials in oncology. Recent data indicate that a mTOR complex phosphorylates Akt, and this complex is insensitive to rapamycin. We show that dominant-negative mTOR diminishes phosphorylation of endogenous Akt and exogenous myristoylated Akt (mAkt), that prolonged exposure to rapamycin also inhibits Akt activation, and that this inhibition is dependent on new protein synthesis. These data suggest that mTOR facilitates Akt activation through mechanisms other than direct phosphorylation. A constitutively active mTOR mutant that fails to enhance Akt phosphorylation nevertheless promotes resistance to multiple antimicrotubule agents, indicating that mTOR also mediates survival independent of Akt. Although Akt- and mTOR-mediated survival has been linked to regulation of cellular metabolism, we also show that survival and metabolic control are separable. The hexokinase inhibitor 5-thioglucose markedly inhibits glycolytic rate but does not diminish vincristine resistance mediated by mAkt or mTOR, and it has only a minor effect on mTOR- or mAkt-mediated resistance to growth factor withdrawal, suggesting that Akt-mTOR-mediated resistance is largely independent of maintenance of glycolytic rate. We conclude that mTOR activity can promote resistance through multiple mechanisms independent of maintained glycolytic rate.

Quantitative Multiparametric MRI Features and PTEN Expression of Peripheral Zone Prostate Cancer: A Pilot Study.

OBJECTIVE The objective of our study was to investigate associations between quantitative image features of multiparametric MRI of the prostate and PTEN expression of peripheral zone prostate cancer. MATERIALS AND METHODS A total of 45 peripheral zone cancer foci from 30 patients who had undergone multiparametric prostate MRI before prostatectomy were identified by a genitourinary pathologist and a radiologist who reviewed histologic findings and MR images. Histologic sections of cancer foci underwent immunohistochemical analysis and were scored according to the percentage of tumor-positive cells expressing PTEN as negative (0-20%), mixed (20-80%), or positive (80-100%). Average and 10th percentile apparent diffusion coefficient (ADC) values, skewness of T2-weighted signal intensity histogram, and quantitative perfusion parameters (i.e., forward volume transfer constant [K(trans)], extravascular extracellular volume fraction [ve], and reverse reflux rate constant between the extracellular space and plasma [k(ep)]) from the Tofts model were calculated for each cancer focus. Associations between the quantitative image features and PTEN expression were analyzed with the Spearman rank correlation coefficient (r). RESULTS Analysis of the 45 cancer foci revealed that 21 (47%) were PTEN-positive, 12 (27%) were PTEN-negative, and 12 (27%) were mixed. There was a weak but significant negative correlation between Gleason score and PTEN expression (r = -0.30, p = 0.04) and between k(ep) and PTEN expression (r = -0.35, p = 0.02). There was no significant correlation between other multiparametric MRI features and PTEN expression. CONCLUSION This preliminary study of radiogenomics of peripheral zone prostate cancer revealed weak-but significant-associations between the quantitative dynamic contrast-enhanced MRI feature k(ep) and Gleason score with PTEN expression. These findings warrant further investigation and validation with the aim of using multiparametric MRI to improve risk assessment of patients with prostate cancer.

Circulating tumor cells capture disease evolution in advanced prostate cancer

BackgroundGenetic analysis of advanced cancer is limited by availability of representative tissue. Biopsies of prostate cancer metastasized to bone are invasive with low quantity of tumor tissue. The prostate cancer genome is dynamic, however, with temporal heterogeneity requiring repeated evaluation as the disease evolves. Circulating tumor cells (CTCs) offer an alternative, “liquid biopsy”, though single CTC sequencing efforts are laborious with high failure rates.MethodsWe performed exome sequencing of matched treatment-naïve tumor tissue, castrate resistant tumor tissue, and pooled CTC samples, and compared mutations identified in each.ResultsThirty-seven percent of CTC mutations were private to CTCs, one mutation was shared with treatment-naïve disease alone, and 62% of mutations were shared with castrate-resistant disease, either alone or with treatment-naïve disease. An acquired nonsense mutation in the Retinoblastoma gene, which is associated with progression to small cell cancer, was identified in castrate resistant and CTC samples, but not treatment-naïve disease. This timecourse correlated with the tumor acquiring neuroendocrine features and a change to neuroendocrine-specific therapy.ConclusionsThese data support the use of pooled CTCs to facilitate the genetic analysis of late stage prostate cancer.

Sustained Complete Response to Cytotoxic Therapy and the PARP Inhibitor Veliparib in Metastatic Castration-Resistant Prostate Cancer - A Case Report.

Solid tumors harboring BRCA1 or BRCA2 mutations have been shown to respond to PARP inhibitors. These responses are partial and transient. In this case report, we describe an 82-year-old male with poorly differentiated prostate cancer with metastases to the lung, liver, abdomen, and bowel. Molecular testing demonstrated alterations in BRCA2, ERG, and TP53. Based on this result, he was enrolled in a therapeutic trial and received carboplatin, gemcitabine, and veliparib, to which he had a partial response. He continued to respond while on veliparib maintenance alone, and after 38 cycles he had a sustained complete response. A sustained complete response to PARP inhibitor-based therapy has not previously been described for prostate cancer. This case suggests that cytotoxic therapy in combination with PARP inhibitors may yield exceptional responses, and molecular studies may help guide patient selection for these therapies.

Contemporary Population-Based Comparison of Localized Ductal Adenocarcinoma and High-Risk Acinar Adenocarcinoma of the Prostate

OBJECTIVE To compare pathological characteristics, treatment patterns, and survival in patients with ductal adenocarcinoma (DC) compared to those with acinar adenocarcinoma (AC). MATERIALS AND METHODS Using the National Cancer Database, we identified patients diagnosed with clinically localized (cN0, cM0) pure DC (n = 1328) and AC (n = 751,635) between 1998 and 2011. High-risk AC was defined as Gleason 8-10. Demographic, treatment, pathological, and survival characteristics of patients were compared. RESULTS Compared to patients with Gleason 8-10 AC, those with DC presented with lower mean prostate-specific antigen (10.3 vs 16.2 ng/mL, P <.001), had similar rates (11.7% vs 11.5%, P = .8) of clinical extra-capsular extension (stage ≥ cT3), and were more likely to undergo prostatectomy (54% vs 36%, P <.001). Compared to patients with Gleason 8-10 AC undergoing prostatectomy, those with DC had more favorable pathology: stage ≥ T3 (39% vs 52%, P <.001), fewer positive lymph nodes (4% vs 11%, P <.001), and fewer positive margins (25% vs 33%, P <.001). On Kaplan-Meier analysis, patients with DC had similar 5-year survival (75.0%, 95% confidence interval [CI] [71.7-78.9]) compared to those with Gleason 8-10 AC (77.1%, 95% CI [76.6%-77.6%], P = .2). On Cox multivariable analysis, patients with Gleason 8-10 AC had a similar risk of death compared to those with DC (hazards ratio = 0.92, 95% CI [0.69-1.23], P = 6). CONCLUSION In this large contemporary population-based series, patients with DC of the prostate presented with lower prostate-specific antigen, had more favorable pathological features, and similar overall survival compared to men with Gleason 8-10 AC.

Abstract 5420: Nuclear PTEN directly regulates androgen receptor activity in prostate cancer

The deletion of PTEN, a negative regulator of the PI3K pathway, is among the most frequent genomic alterations in prostate cancer. Attempts to target this alteration using PI3K pathway inhibitors, however, have yielded little clinical benefit for patients, and the function of PTEN loss in prostate cancer progression is not fully understood. Prostate cancers lacking PTEN are less responsive to therapy targeting the Androgen Receptor (AR), a dominant driver of prostate cancer growth, but how PTEN function interacts with AR activity is not clear. We used immunoblotting, immunoprecipitation, proximity ligation assay, chromatin immunoprecipitation, and transcriptome sequencing in cells with endogenous PTEN or PTEN knocked down to evaluate the interactions between these major factors in prostate cancer progression. We demonstrate that PTEN interacts with the Androgen Receptor in the nucleus, and PTEN can bind chromatin at known Androgen Response Elements. Stimulation of cells with androgen and other media components leads to accumulation of phosphorylated (inactive) PTEN, loss of nuclear PTEN, and decreased PTEN-AR interaction. Transcriptome analysis of cells with PTEN knocked down demonstrates increased expression of AR pathway genes, even in the absence of androgen stimulation. These results suggest nuclear PTEN may play a direct role in negative regulation of the Androgen Receptor, with implications for management of PTEN-negative prostate cancers. Citation Format: Yen T. Nguyen, Jose A. Rodriguez-Nieves, Qi Yang, David VanderWeele. Nuclear PTEN directly regulates androgen receptor activity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5420.

Integrative Genomic Analysis of Coincident Cancer Foci Implicates CTNNB1 and PTEN Alterations in Ductal Prostate Cancer

BACKGROUND Ductal adenocarcinoma of the prostate is an aggressive subtype, with high rates of biochemical recurrence and overall poor prognosis. It is frequently found coincident with conventional acinar adenocarcinoma. The genomic features driving evolution to its ductal histology and the biology associated with its poor prognosis remain unknown. OBJECTIVE To characterize genomic features distinguishing ductal adenocarcinoma from coincident acinar adenocarcinoma foci from the same patient. DESIGN, SETTING, AND PARTICIPANTS Ten patients with coincident acinar and ductal prostate cancer underwent prostatectomy. Laser microdissection was used to separately isolate acinar and ductal foci. DNA and RNA were extracted, and used for integrative genomic and transcriptomic analyses. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Single nucleotide mutations, small indels, copy number estimates, and expression profiles were identified. Phylogenetic relationships between coincident foci were determined, and characteristics distinguishing ductal from acinar foci were identified. RESULTS AND LIMITATIONS Exome sequencing, copy number estimates, and fusion genes demonstrated coincident ductal and acinar adenocarcinoma diverged from a common progenitor, yet they harbored distinct alterations unique to each focus. AR expression and activity were similar in both histologies. Nine of 10 cases had mutually exclusive CTNNB1 hotspot mutations or phosphatase and tensin homolog (PTEN) alterations in the ductal component, and these were absent in the acinar foci. These alterations were associated with changes in expression in WNT- and PI3K-pathway genes. CONCLUSIONS Coincident ductal and acinar histologies typically are clonally related and thus arise from the same cell of origin. Ductal foci are enriched for cases with either a CTNNB1 hotspot mutation or a PTEN alteration, and are associated with WNT- or PI3K-pathway activation. These alterations are mutually exclusive and may represent distinct subtypes. PATIENT SUMMARY The aggressive subtype ductal adenocarcinoma is closely related to conventional acinar prostate cancer. Ductal foci contain additional alterations, however, leading to frequent activation of two targetable pathways.