Filipe L.F. Carvalho

PTEN loss is associated with upgrading of prostate cancer from biopsy to radical prostatectomy

When distinguishing between indolent and potentially harmful prostate cancers, the Gleason score is the most important variable, but may be inaccurate in biopsies due to tumor under-sampling. This study investigated whether a molecular feature, PTEN protein loss, could help identify which Gleason score 6 tumors on biopsy are likely to be upgraded at radical prostatectomy. Seventy one patients with Gleason score 6 tumors on biopsy upgraded to Gleason score 7 or higher at prostatectomy (cases) were compared with 103 patients with Gleason score 6 on both biopsy and prostatectomy (controls). A validated immunohistochemical assay for PTEN was performed, followed by fluorescence in situ hybridization (FISH) to detect PTEN gene deletion in a subset. PTEN protein loss and clinical–pathologic variables were assessed by logistic regression. Upgraded patients were older than controls (61.8 vs 59.3 years), had higher pre-operative PSA levels (6.5 vs 5.3 ng/ml) and a higher fraction of involved cores (0.42 vs 0.36). PTEN loss by immunohistochemistry was found in 18% (13/71) of upgraded cases compared with 7% (7/103) of controls (P=0.02). Comparison between PTEN immunohistochemistry and PTEN FISH showed the assays were highly concordant, with 97% (65/67) of evaluated biopsies with intact PTEN protein lacking PTEN gene deletion, and 81% (13/16) of the biopsies with PTEN protein loss showing homozygous PTEN gene deletion. Tumors with PTEN protein loss were more likely to be upgraded at radical prostatectomy than those without loss, even after adjusting for age, preoperative PSA, clinical stage and race (odds ratio=3.04 (1.08–8.55; P=0.035)). PTEN loss in Gleason score 6 biopsies identifies a subset of prostate tumors at increased risk of upgrading at radical prostatectomy. These data provide evidence that a genetic event can improve Gleason score accuracy and highlight a path toward the clinical use of molecular markers to augment pathologic grading.

Notch signaling in prostate cancer: a moving target.

By regulating cell fate, proliferation, and survival, Notch pathway signaling provides critical input into differentiation, organization, and function of multiple tissues. Notch signaling is also becoming an increasingly recognized feature in malignancy, including prostate cancer, where it may play oncogenic or tumor suppressive roles.

Inconsistency in albuminuria predictors in type 2 diabetes: a comparison between neural network and conditional logistic regression.

Albuminuria is a sensitive marker to predict future cardiovascular events in patients with type 2 diabetes mellitus. However, current studies only use conventional regression models to discover predictors of albuminuria. We have used 2 different statistical models to predict albuminuria in type 2 diabetes mellitus: a multilayer perception neural network and a conditional logistic regression. Neural network models were used to predict the level of albuminuria in patients with type 2 diabetes mellitus, which include a matched case-control study for the population. For each case, we randomly selected 1 control matched by age and body mass index (BMI). The input variables were sex, duration of diabetes, systolic and diastolic blood pressure, glomerular filtration rate, high-density lipoprotein, low-density lipoprotein, triglyceride, high-density lipoprotein/triglyceride ratio, cholesterol, fasting blood sugar, and glycated hemoglobin. Age and BMI were included only in the neural network model. This model included 4 hidden layers and 1 bias. Relative error of predictions was 0.38% in the training group, 0.52% in the testing group, and 1.20% in the holdout group. The most robust predictors of albuminuria were high-density lipoprotein (21%), cholesterol (14.4%), and systolic blood pressure (9.7%). Using the conditional logistic regression model, glomerular filtration rate, time of onset to diabetes, and sex were significant indicators in the onset of albuminuria. Using a neural network model, we show that high-density lipoprotein is the most important factor in predicting albuminuria in type 2 diabetes mellitus. Our neural network model complements the current risk factor models to improve the care of patients with diabetes.

A new immunohistochemistry prognostic score (IPS) for recurrence and survival in resected pancreatic neuroendocrine tumors (PanNET)

Pancreatic neuroendocrine tumor (PanNET) is a neoplastic entity in which few prognostic factors are well-known. Here, we aimed to evaluate the prognostic significance of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) by immunohistochemistry (IHC) and methylation analysis in 92 patients with resected PanNET and follow-up longer than 24 months. In multivariate analyses, ki-67 and our immunohistochemistry prognostic score (IPS-based on MGMT, NDRG-1 and PHLDA-3 IHC expression) were independent prognostic factors for disease-free-survival (DFS), while age and IPS were independent prognostic factors for overall survival (OS). Our IPS could be a useful prognostic biomarker for recurrence and survival in patients following resection for PanNET.

HES6 promotes prostate cancer aggressiveness independently of Notch signalling.

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high‐grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer‐specific up‐regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up‐ and down‐regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor‐independent manner. Using a Notch‐sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell‐autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression.

Abstract B56: Notch signaling in prostate cancer progression

Abstract Background: The Gleason grading system provides the most consistently valuable information on the lethal potential of prostate cancers. Therefore, molecular correlates of higher Gleason grade should serve both as biomarkers of aggressive disease and as therapeutic targets. A recently published meta-analysis indicated that members of the Notch signaling pathway, particularly the receptor NOTCH3 and the pathway target Hairy and Enhancer Split family (HES6), distinguish prostate cancers with high Gleason grade. Thus, Notch signaling may play a distinctive role in aggressive prostate cancers. Here we use prostate cancer cell lines and transgenic mouse models to explore the function Notch signaling in prostate cancer progression. Methods: Using a custom TaqMan real time reverse transcription–polymerase chain reaction (qRT-PCR) assay, we compared expression of all Notch pathway components in benign prostate epithelial cells to those in prostate cancer cell lines - 22Rv1, LNCaP, DU145, PC3. We further confirmed differential expression using additional qRT-PCR assays. To understand how NOTCH3 receptor regulates the expression of pathway9s targets HES1, HES4 and HES6, we up- or down-regulated NOTCH3 levels using plasmid-mediated gene or small interfering RNA (siRNA) transfer. We also studied how HES6 protein levels affected the expression of the oncogene c-MYC and its effects in cell proliferation and migration in a wound-healing assay. We performed immunohystochemical staining for HES6 in prostate tumors and metastasis from Hi-Myc and TRAMP transgenic mice, as well as in human benign, low-grade and high-grade tumors. Results: Three members of the pathway were significantly overexpressed in prostate cancer cells compared with benign prostate epithelial cells: the receptor NOTCH3 and the two target genes HES4 and HES6. siRNA-mediated NOTCH3 silencing did not change HES4 levels, but decreased the expression of HES1 and HES6, implicating these genes as Notch response genes in the prostate. However, compared to controls, PC3 and LNCaP cells overexpressing HES6 showed no change in proliferation but migrated dramatically faster. Contrary to previous studies in neural development, HES6 overexpression in most prostate cancer cell lines induced HES1 expression indicating that rather than inhibiting, HES6 stimulates Notch signaling. We confirmed the importance of HES6 in prostate cancer progression through immunohistochemical analysis on tissues from mouse models (Hi-Myc and TRAMP) and human patients with prostate cancer. In each case, we confirmed increased strong nuclear HES6 expression with increased cancer grade or stage. Conclusion: Our results confirm differential expression of Notch pathway members in aggressive prostate cancer and indicate a role for HES6, one target of the pathway, in cancer cell migration. This effect may result from a direct action of HES6 or cross-talk with other members of the Notch pathway. These data suggest that Notch signaling, and particularly HES6, may be useful in distinguishing indolent from lethal prostate cancers and that Notch pathway blockade may be a useful therapeutic strategy in those cases that require treatment. Citation Format: Filipe L.F. Carvalho, Brian Simons, David M. Berman. Notch signaling in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B56.