Correlation of integrated ERG/PTEN assessment with biochemical recurrence in prostate cancer.

INTRODUCTION: Prostate cancer is a heterogeneous disease, with a complex molecular landscape that evolves throughout disease progression. Common alterations in genes such as ERG and PTEN have been attributed to worse prognosis. This study aimed to further examine the clinical relevance of PTEN and ERG expression in a cohort of patients with prostate cancer post radical prostatectomy. METHODS: Tissue microarrays were constructed from 132 patients with prostate cancer from the Irish Prostate Cancer Research Consortium and University Hospital of Orebro, Sweden. Patients were divided into three groups - Group 1: biochemical recurrence, Group 2: no biochemical recurrence and Group 3: immediate progression after surgery. PTEN and ERG immunohistochemical analysis was performed and the association between expression levels and clinical parameters were compared. RESULTS: Pathological stage pT3 tumours were more common at borderline significantly higher levels amongst patients who biochemically recurred when compared to patients who did not recur after radical prostatectomy (p = 0.05). ERG and PTEN expression levels were compared separately and concurrently across all three patient groups. Lack of ERG expression was strongly associated with immediate progression after surgery (p = 0.029). Loss of/low PTEN trended towards an association with immediate progression, however this was not statistically significant (p = 0.066). CONCLUSION: In this study, negative ERG expression was strongly associated with immediate biochemical progression after radical prostatectomy. Moreover, a trend towards a relationship between aberrant PTEN expression and progression was observed. Additional studies with long-term follow up data may provide further clinical insight into the genomic heterogeneity in this population.

Assessment of MYC/PTEN Status by Gene-Protein Assay in Grade Group 2 Prostate Biopsies.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non-organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74-24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

Risk Stratification of Prostate Cancer Through Quantitative Assessment of PTEN Loss (qPTEN).

BACKGROUND: Phosphatase and tensin homolog (PTEN) loss has long been associated with adverse findings in early prostate cancer. Studies to date have yet to employ quantitative methods (qPTEN) for measuring of prognostically relevant amounts of PTEN loss in postsurgical settings and demonstrate its clinical application. METHODS: PTEN protein levels were measured by immunohistochemistry in radical prostatectomy samples from training (n = 410) and validation (n = 272) cohorts. PTEN loss was quantified per cancer cell and per tissue microarray core. Thresholds for identifying clinically relevant PTEN loss were determined using log-rank statistics in the training cohort. Univariate (Kaplan-Meier) and multivariate (Cox proportional hazards) analyses on various subpopulations were performed to assess biochemical recurrence-free survival (BRFS) and were independently validated. All statistical tests were two-sided. RESULTS: PTEN loss in more than 65% cancer cells was most clinically relevant and had statistically significant association with reduced BRFS in training (hazard ratio [HR] = 2.48, 95% confidence interval [CI] = 1.59 to 3.87; P < .001) and validation cohorts (HR = 4.22, 95% CI = 2.01 to 8.83; P < .001). The qPTEN scoring method identified patients who recurred within 5.4 years after surgery (P < .001). In men with favorable risk of biochemical recurrence (Cancer of the Prostate Risk Assessment - Postsurgical scores <5 and no adverse pathological features), qPTEN identified a subset of patients with shorter BRFS (HR = 5.52, 95% CI = 2.36 to 12.90; P < .001) who may be considered for intensified monitoring and/or adjuvant therapy. CONCLUSIONS: Compared with previous qualitative approaches, qPTEN improves risk stratification of postradical prostatectomy patients and may be considered as a complementary tool to guide disease management after surgery.

Synthetic Essentiality of Metabolic Regulator PDHK1 in PTEN-Deficient Cells and Cancers.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.

Assessment of epigenetic alterations and in silico analysis of mutation affecting PTEN expression among Indian cervical cancer patients.

Genetic and epigenetic anomalies accountable for genetic dysregulation are the most common aberrations that determine the underlying heterogeneity of the tumor cells. Currently, phosphatase and tensin homolog (PTEN) incongruity has emerged as potent and persuasive malfunctioning in varied human malignancies. In this study, we have analysed the promoter hypermethylation and expression status of PTEN. We identified different mutations in the exonic region of PTEN. Functional consequences of these mutations were explored using in silico techniques. Promoter hypermethylation of PTEN was detected using methylation-specific polymerase chain reaction (MS-PCR), expression analysis was performed with immunohistochemistry (IHC) and mutation by direct sequencing in a total of 168 uterine cervix tumor cases. The findings were statistically correlated with the clinical parameters. In addition, the effect of nonsynonymous mutations was studied with molecular dynamics simulations. PTEN promoter hypermethylation (45.8%) was found to be significantly associated with the of PTEN loss (57.14%, P < 0.0001). Tumor stages, tumor size, lymph node (LN) were found to be significantly correlated with both PTEN promoter hypermethylation and PTEN loss. Histological grade, however, showed a significant association with only PTEN loss. In total, 11.76% of tumors exhibited mutations in exon 5 and 7, out of which E150K of exon 5 showed the highest deviations in the crystal structure of PTEN by in silico analysis. This study provides valuable insights into oncology and paves the path in the development of efficient biomarker and/or imperative therapeutic tool for cervical cancer treatment.

PTEN status assessment in the Johns Hopkins active surveillance cohort.

BACKGROUND: Up to half of men with Gleason score 6 (GS6) prostate cancers initially managed with active surveillance (AS) will eventually require definitive therapy, usually due to tumor grade reclassification during follow-up. We examined the association between PTEN status on biopsy and subsequent clinicopathologic outcomes in men with GS6 cancers who enrolled in AS. METHODS: We performed a case-control study of men enrolled in the Johns Hopkins AS cohort with diagnostic biopsy tissue available for immunohistochemical (IHC) staining. IHC was performed for PTEN using genetically validated protocols for all patients. Cases included men who underwent grade reclassification to GS ≥ 3 + 4 = 7 on biopsy within 2 years of follow-up (i.e., early reclassification) or reclassification to GS ≥ 4 + 3 = 7 on biopsy or radical prostatectomy during follow-up (i.e., extreme reclassification). Control patients were diagnosed with GS6 cancer and monitored on AS for at least 8 years without undergoing biopsy reclassification. RESULTS: Among 67 cases with adequate tissue, 31 men underwent early reclassification and 36 men underwent extreme reclassification. Cases were compared to 65 control patients with adequate tissue for assessment. On initial prostate biopsy, cases were older (median age 67 vs. 65, p = 0.024) and were less likely to meet very-low-risk criteria (64 vs 79%, p = 0.042) as compared to controls. Although not statistically significant, PTEN loss was observed in only 1 (1.5%) of 65 controls as compared to 6 (9%) of 67 cases (p = 0.062). CONCLUSIONS: PTEN loss was rare among men with GS6 prostate cancer enrolled in AS at Johns Hopkins. Despite this, PTEN loss was more frequent among men who underwent early or extreme reclassification to higher-grade cancer as compared to controls. Additional studies in larger low-risk cohorts may better elucidate a potential role for PTEN in selecting patients for AS.

A novel quantitative method of pten expression assessment in tumor tissue.

Phosphatase and Tensin Homolog deleted on chromosome 10 (PTEN) gene is one of the most important tumor suppressor genes which is involved in the regulation of many signaling cascades (AKT/PKB and MAPK). Subtle changes in its activity lead to cancer susceptibility or aggressive tumor behaviour. Despite the diversity of mechanisms leading to PTEN inactivation, it is frequently associated with a decreased or complete loss of protein expression. About 20% decrease in PTEN expression could lead to the development of cancer. There have been no objective, quantitative methods of PTEN expression assessment that allow to measure the subtle variations of the protein concentration in a tissue-contextual manner. A new quantitative algorithm of immunostaining evaluation based on combination of color deconvolution and relative chromogen signal intensity was used in the study. The proposed algorithm was implemented in the popular ImageJ image analysis software and positively verified in cancer cell lines and tissue models as well as in the tissue samples of colorectal cancer (CRC) patients. The proposed quantitative method of PTEN expression assessment creates an alternative to currently available subjective methods and forms the basis for inter-case and inter-tissue comparisons. Using the algorithm it would be possible to identify three groups of patients with advanced colorectal cancer which could significantly differ in the overall survival. The research should be continued.

In situ assessment of PI3K and PTEN alterations in mycosis fungoides: correlation with clinicopathological features.

Deregulated signalling through phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in tumour initiation and progression. We have already shown that AKT is activated in skin lesions in Mycosis Fungoides (MF) and we herein further investigate the frequency and clinical significance of PTEN and PI3K at the protein and at the DNA level as well as the presence of AKT1 mutations in skin lesions from 50 patients with MF clinical stages I-IV in relation to clinicopathological features. Increased p-AKT expression correlated with poor prognosis in plaques (P = 0.0198), whereas p-AKT was an independent predictor of poor survival in the entire cohort (P = 0.017, HR = 1.012). PTEN cytoplasmic expression was found low or absent in all 77.3% of cases and inversely correlated with advanced clinical stages (P = 0.0744). Molecular analysis showed no AKT1 mutation, no PI3KCA copy number gain, only 1 case with PI3KCA mutation in exon 9 and 3 cases with PTEN mutations (7%) in exons 7, 8 and 5. The latter correlated with disease (P = 0.0253) and progression (P < 0.0001) free survival in tumour stage. Although activation of PI3K/AKT signalling pathway due to PTEN alterations is rarely attributed to abnormalities in PTEN, PI3K, and AKT1 genes, PTEN mutations exert a negative effect on patients' prognosis with tumours.

Resveratrol increases brown adipose tissue thermogenesis markers by increasing SIRT1 and energy expenditure and decreasing fat accumulation in adipose tissue of mice fed a standard diet.

PURPOSE: Adipose tissue is central to the regulation of energy balance. Two functionally different fat pads are present in mammals: white adipose tissue, the primary site of triglyceride storage, and brown adipose tissue (BAT), which is specialized in heat production. In this context, new strategies capable of modulating the development and function of white and BAT become relevant. In the present study, we analyzed the influence of resveratrol (sirtuin activator) on energy balance and the expression of thermogenesis markers. METHODS: Mice were divided into two groups: standard diet (ST) and standard diet plus resveratrol (ST + RSV). RESULTS: After 2 months of treatment, ST + RSV mice presented significantly decreased fat accumulation in adipose tissue, with diminished total cholesterol and glucose plasma levels. Additionally, increased oxygen consumption was observed in ST + RSV group. Analyses of mRNA of thermogenesis-related genes showed significant increase in UCP1, SIRT1, PTEN and BMP-7 expression in BAT. CONCLUSION: Our data suggest that improved metabolism produced by oral administration of resveratrol is, at least in part, associated with increased thermogenesis followed by high expression of UCP1 and SIRT1, which can mediate higher energy expenditure and decreased fat accumulation in adipose tissue.

Best practice for PTEN gene and protein assessment in anatomic pathology.

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.

Assessment of genetic markers and glioblastoma stem-like cells in activation of dendritic cells.

Glioblastoma (GBM) is the most common and aggressive intraparenchymal primary brain tumor in adults. The principal reasons for the poor outcomes of GBM are the high rates of recurrence and resistance to chemotherapy. The aim of this study was to determine the role of tailored cellular therapy for GBM with a poor prognosis and compare the activity of dendritic cells (DCs) that have encountered GBM cells. Detecting the correlations between methylation and expression of MGMT and PTEN genes and GBM cancer stem cells (CSCs) markers after co-cultures with a mononuclear cell cocktail are also aims for this study. Allogenic umbilical cord blood (UCB)-derived DCs were labeled with the CD11a and CD123 for immature DCs, and CD80 and CD11c for mature DCs. CD34, CD45, and CD56 cells were isolated from allogenic UCB for using in DCs maturation. GBM CSCs were detected with CD133/1 and CD111 antibodies after co-culture studies. DC activation was carried out via GBM cells including CD133 and CD111 cells and a mononuclear cells cocktail including CD34, CD45, and CD56 natural killer cells. Real-time PCR was performed to detect the expression and promoter methylation status of PTEN and MGMT genes. The expression of CSCs markers was found in all GBM cases, and a statistically significant correlation was found among them after co-culture studies. The most pronounced affinity of DCs to GBM cells was observed at dilutions between 1/4 and 1/256 in co-cultures. There was a statistically significant correlation between cellularity and granularity ratios for CD123 and CD11c. PTEN and MGMT gene expression and methylation values were evaluated with respect to CSCs expression and no statistical significance was found. Activation of DCs might associate with CSCs and the mononuclear cells cocktail including CD34, CD45, and CD56 cells which were obtained from allogenic UCB.

Improved risk assessment of endometrial cancer by combined analysis of MSI, PI3K-AKT, Wnt/ß-catenin and P53 pathway activation.

OBJECTIVE: To investigate if analysis of genetic alterations in the main pathways involved in endometrioid type carcinogenesis (PI3K-AKT, Wnt/ß-catenin, P53-activation and MSI) improves the current risk assessment based on clinicopathological factors. METHODS: Formalin fixed paraffin embedded (FFPE) primary tumor samples of 65 patients with FIGO-stage I endometrioid type endometrial cancer (EEC) were selected from the randomized PORTEC-2 trial. Tumors were stained by immunohistochemistry for P53, PTEN and ß-catenin. Tumor DNA was isolated for sequence analysis of TP53 (exons 4 to 8), hotspot mutation analysis of KRAS (exon 1) and PI3K (exon 9 and 20) and microsatellite-instability (MSI) analysis including MLH1 promotor-methylation status. Univariate and multivariate analyses for disease-free survival (DFS) using Cox regression models were performed. RESULTS: P53 status (HR 6.7, 95%CI 1.75-26.0, p=0.006) and MSI were the strongest single genetic prognostic factors for decreased DFS, while high PI3K-AKT pathway activation showed a trend and ß-catenin was not prognostic. The combination of multiple activated pathways was the most powerful prognostic factor for decreased DFS (HR 5.0; 95%CI 1.59-15.6 p=0.006). Multiple pathway activation, found in 8% of patients, was strongly associated with aggressive clinical course. In contrast, 40% of patients had no alterations in the investigated pathways and had a very low risk of disease progression. CONCLUSIONS: Activation of multiple oncogenic pathways in EEC was the most powerful prognostic factor for decreased DFS, resulting in an individual risk assessment superior to the current approach based on clinicopathological factors.

Pten positively regulates brown adipose function, energy expenditure, and longevity.

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpß form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.

Clinical assessment of PTEN loss in endometrial carcinoma: immunohistochemistry outperforms gene sequencing.

PTEN (phosphatase and tensin homolog) is a tumor suppressor that negatively regulates the PI3K-AKT signaling pathway, which is implicated in the pathogenesis of endometrial carcinoma. Sanger sequencing has been considered to be the gold standard for detection of PTEN sequence abnormalities. However, this approach fails to address the epigenetic mechanisms that contribute to functional PTEN loss. Using a study cohort of 154 endometrioid and non-endometrioid endometrial carcinomas, we performed full-length PTEN sequencing and PTEN immunohistochemistry on each tumor. PTEN sequence abnormalities were detected in a significantly lower proportion of cases (43%) than PTEN protein loss (64%, P=0.0004). Endometrioid tumors had a significantly higher proportion of PTEN sequence abnormalities and PTEN protein loss than non-endometrioid tumors. Within the latter group, PTEN sequence abnormalities and PTEN protein loss were most frequent in undifferentiated carcinomas, followed by mixed carcinomas; they were least frequent in carcinosarcomas. Overall, at least one PTEN sequence abnormality was detected in each exon, and the greatest number of sequence abnormalities was detected in exon 8. Pure-endometrioid tumors had a significantly higher frequency of sequence abnormalities in exon 7 than did the non-endometrioid tumors (P=0.0199). Importantly, no mutational hotspots were identified. While PTEN protein loss by immunohistochemistry was identified in 89% of cases with a PTEN sequence abnormality, PTEN protein loss was detected by immunohistochemistry in 44% of cases classified as PTEN wild type by sequencing. For the first time, we demonstrate that PTEN immunohistochemistry is able to identify the majority of cases with functional PTEN loss. However, PTEN immunohistochemistry also detects additional cases with PTEN protein loss that would otherwise be undetected by gene sequencing. Therefore, for clinical purposes, immunohistochemistry appears to be a preferable technique for identifying endometrial tumors with loss of PTEN function.

Diet and colorectal cancer: analysis of a candidate pathway using SNPS, haplotypes, and multi-gene assessment.

There is considerable biologic plausibility to the hypothesis that genetic variability in pathways involved in insulin signaling and energy homeostasis may modulate dietary risk associated with colorectal cancer. We utilized data from 2 population-based case-control studies of colon (n = 1,574 cases, 1,970 controls) and rectal (n = 791 cases, 999 controls) cancer to evaluate genetic variation in candidate SNPs identified from 9 genes in a candidate pathway: PDK1, RP6KA1, RPS6KA2, RPS6KB1, RPS6KB2, PTEN, FRAP1 (mTOR), TSC1, TSC2, Akt1, PIK3CA, and PRKAG2 with dietary intake of total energy, carbohydrates, fat, and fiber. We employed SNP, haplotype, and multiple-gene analysis to evaluate associations. PDK1 interacted with dietary fat for both colon and rectal cancer and with dietary carbohydrates for colon cancer. Statistically significant interaction with dietary carbohydrates and rectal cancer was detected by haplotype analysis of PDK1. Evaluation of dietary interactions with multiple genes in this candidate pathway showed several interactions with pairs of genes: Akt1 and PDK1, PDK1 and PTEN, PDK1 and TSC1, and PRKAG2 and PTEN. Analyses show that genetic variation influences risk of colorectal cancer associated with diet and illustrate the importance of evaluating dietary interactions beyond the level of single SNPs or haplotypes when a biologically relevant candidate pathway is examined.

Assessment of PTEN tumor suppressor activity in nonmammalian models: the year of the yeast.

Model organisms have emerged as suitable and reliable biological tools to study the properties of proteins whose function is altered in human disease. In the case of the PI3K and PTEN human cancer-related proteins, several vertebrate and invertebrate models, including mouse, fly, worm and amoeba, have been exploited to obtain relevant functional information that has been conserved from these organisms to humans along evolution. The yeast Saccharomyces cerevisiae is an eukaryotic unicellular organism that lacks a canonical mammalian-like PI3K/PTEN pathway and PIP3 as a physiological second messenger, PIP2 being essential for its life. The mammalian PI3K/PTEN pathway can be reconstituted in S. cerevisiae, generating growth alteration phenotypes that can be easily monitored to perform in vivo functional analysis of the molecular constituents of this pathway. Here, we review the current nonmammalian model systems to study PTEN function, summarize our knowledge of PTEN orthologs in yeast species and propose the yeast S. cerevisiae as a sensitive biological sensor of PI3K oncogenicity and PTEN tumor suppressor activity.

Protein expression and cellular localization in two prognostic subgroups of diffuse large B-cell lymphoma: higher expression of ZAP70 and PKC-beta II in the non-germinal center group and poor survival in patients deficient in nuclear PTEN.

Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) show varying responses to conventional therapy, and this might be contributed to the differentiation stage of the tumor B-cells. The aim of the current study was to evaluate a panel of kinases (ZAP70, PKC-beta I and II and phosphorylated PKB/Akt) and phosphatases (PTEN, SHP1 and SHP2) known to be frequently deregulated in lymphoid malignancies. De novo DLBCL cases were divided into two subgroups, the germinal center (GC) group (14/28) and the non-germinal center (non-GC) or activated B-cell (ABC) group (14/28). ZAP70 and PKC-beta II were expressed in a significantly higher percentage of tumor cells in the clinically more aggressive non-GC group compared with the prognostically favourable GC group. Also, the subcellular localization of PKC-beta I and II differed in DLBCL cells, with the PKC-beta I isoform being expressed in both the cytoplasm and nucleus, while PKC-beta II was found exclusively in the cytoplasm. Loss of nuclear PTEN correlated with poor survival in cases from both subgroups. In addition, five cell lines of DLBCL origin were analyzed for protein expression and for mRNA levels of PTEN and SHP1. For the first time, we show that ZAP70 is expressed in a higher percentage of tumor cells in the aggressive non-GC subgroup of DLBCL and that PKC-beta I and II are differently distributed in the two prognostic subgroups of de novo DLBCL.