Loss Of PTEN Expression Research Articles

Abstract 915: Expanded biomarker results from a phase I dose escalation study of GDC-0032, a beta isoform-sparing PI3K inhibitor

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Preclinical data indicate that GDC-0032 has increased activity against PIK3CA mutant cancer cell lines. As presented by Juric et al., 34 patients were enrolled in this study and dose escalation has been completed (Juric D. et al. AACR 2013, Abstract LB-64). Metabolic partial responses via FDG-PET were observed in 6 out of 13 patients assessed, and clinical partial responses (PRs) were observed in 6 patients, with 5 of these patients having PIK3CA mutant cancers. Tumor tissue was obtained from 30 out of 34 patients enrolled onto the study. The most frequently recruited cancer type was breast cancer (41%) followed by colorectal (15%) and lung cancer (15%). Fourteen patients enrolled onto the study had PIK3CA mutant tumors (41%) as determined using a TaqMan-based PCR assay. Three patients had total loss of PTEN expression, as assessed by immunohistochemical staining, and a further 3 patients had reduced PTEN expression based on a H-score assessment. Out of the 6 patients that had reduced PTEN expression, 2 patients had coexisting mutations with PIK3CA. Where known, 4 out of 5 patients that had a PR showed intact PTEN expression, with the fifth patient having reduced PTEN expression. Samples were further analyzed using an in-house developed PCR-based multiplexed assay that detects activating mutations within an additional ten oncogenes. PIK3CA mutations were largely mutually exclusive with mutations in the Ras pathway, however 3 out of the 14 PIK3CA mutant patients had a coexisting mutation within KRas. A further 2 patients had KRas mutations, 1 patient had a NRas mutation and 2 patients had EGFR mutations. Preliminary analysis suggests lack of benefit in patients with KRas mutations treated with GDC-0032 single agent. Optional on-study biopsies were collected from 2 patients and demonstrated pharmacodynamic inhibition of the PI3K pathway as assessed by reverse phase protein array for approximately 45 endpoints, including 1 patient at the lowest dose. In conclusion, our preliminary data indicates that GDC-0032 demonstrates single agent activity in patients with PIK3CA mutations tumors with unaltered PTEN or MAP-kinase pathways. Citation Format: Timothy R. Wilson, Heidi Savage, Carol O'Brien, Sandra Sanabria, Ray S. Lin, Marie-Claire Wagle, Yibing Yan, Mark R. Lackner, Hema Parmar, Jerry Y. Hsu, Dejan Juric, Ian E. Krop, Ramesh K. Ramanathan, Daniel D. Von Hoff, Jose Baselga. Expanded biomarker results from a phase I dose escalation study of GDC-0032, a beta isoform-sparing PI3K inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2014-915

Abstract 448: Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study

Abstract Introduction There are numerous causes triggering CRC. 25-80% of CRC shown a deregulation in Epidermal Growth Factor Receptor (EGFR) pathway. Two signaling pathways downstream of the EGFR are dysregulated in CRC the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K) pathway. Activating mutations in KRAS and BRAF (MAPK pathway) and PIK3CA affect prognosis and/or response to anti-EGFR MoAb. PTEN is a downstream effector of EGFR pathway and is involved in PI3K pathway. Loss of PTEN protein expression can occur through epigenetic silencing and mutation or allelic loss. Immunohistochemistry (IHC) is the most effective way to assay for loss of PTEN expression. IHC lack of reproducibility and this has lead to discordant results regarding the concordant in paired CRC metastases and primary tumors.The aim of the work was to asses the feasibility of PTEN gene expression analysis from FFPE tissue in CRC samples. Materials and methods We selected a series of 33 patients, already tested for KRAS mutational status and resulted wild-type. At the time of the study mutational status of NRAS gene has not a role as predictive marker, so was not performed. A retrospective analysis on CRC FFPE tissue was performed. As control samples we have selected specimen of colorectal surgery not tumor-associated (diverticulitis). On these samples BRAF mutational analysis and PTEN gene expression was performed. For BRAF analysis, DNA was extracted by means of QIAamp DNA FFPE tissue kit (Qiagen, USA). BRAF mutational status was assed through direct automated sequencing (3100 Genetic Analyzer, Applied Biosystems) and TaqMan mutation detection assay (ABI PRIM 7900HT, Applied Biosystems). For PTEN gene expression, total RNA was obtained through the miRNeasy FFPE tissue kit (Qiagen, USA), according to manufacturer instructions, and reverse transcribed. PTEN expression was assessed by means of TaqMan probes. Results According to BRAF mutational status we have found 10/33 (30%) mutated samples. PTEN expression levels were evaluated by the comparison of tumor samples vs diverticula samples, the data obtained were then integrated with those from BRAF mutational analysis. Interestingly we found that all the BRAF mutated samples present also a down-regulation of PTEN. Statistical analyses were conducted through the X2 test with the Yatesha correction (p=0.00023). The p-value was also calculated Exact Fisher Test (p= 0.00021) to confirm the results. Conclusion The determination of PTEN gene expression level is feasible with real-time PCR using TaqMan probe technology. BRAF analysis might be used as prognostic and/or predictive marker in CRC patients’ treatment. The identification of a validated method for PTEN analysis would establish it as a molecular marker meriting further investigation in large numbers of available primary tumors from patients with CRC. Further analyses are requested to confirm these data. Citation Format: Christian D. Rolfo, Marta Castiglia, Daniela Cabibi, Valentina Calo', Florinda Di Piazza, Viviana Bazan, Fabio Brocco, Stefano Caruso, Loredana Bruno, Konstantinos Papadimitriou, Francesco Passiglia, Marc Peeters, Patrick Pauwels, Antonio Russo. Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2014-448

Abstract 4720: Evaluation of a genomic classifier (Decipher®) in subsets of primary tumors with common prostate cancer genomic alterations

Abstract Background ETS gene fusions and PTEN loss are the most common genomic alteration events in prostate cancer that stratify patients into molecularly distinct subtypes. Recently, a commercially available genomic classifier (GC, DecipherTM) has been validated to predict metastasis in patients with high-risk prostate cancer. In this study we evaluated the robustness of the GC model in the light of prostate cancer molecular heterogeneity. Methods: Analysis of PTEN expression loss, ERG overexpression and GC scores were performed on a previously reported genome-wide microarray dataset (Karnes et al., 2013). Microarray probesets mapping to exons of PTEN were benchmarked to identify a representative exon whose loss of expression was highly correlated to FISH analysis for PTEN deletion (PTEN-loss) performed on an independent dataset. ERG overexpression (ERG+) was determined similarly and the microarray probesets that best correlate with the FISH status (in independent dataset) were used to annotate the ERG status of the samples in this study. Receiver operating characteristic area under the curve (AUC), and multivariable regression analysis were used to assess GC performance for predicting metastasis in subsets of patient tumors with genomic alterations. Results Using the microarray method 42% of the tumors had ERG overexpression, 23% had PTEN-loss and 11% had both events. ERG+ and PTEN-loss were not on their own predictive of metastasis with AUCs of 0.52 (CI:0.44-0.6) and 0.47(CI 0.39-0.56), respectively. In ERG+ tumors GC had an AUC of 0.82 (p=4e-7) and high GC score ERG+ patients had a hazard ratio (HR) of 100 (p=6e-7) for metastasis. Similarly, in PTEN-loss tumors GC had an AUC of 0.83 (p=7.9e-5) and high GC score PTEN-loss patients had an HR of 62 (p=1.8e-4) for metastasis. When GC was evaluated in patient tumors with both ERG+ and PTEN-loss the AUC was 0.85 (p=2e-15) and high GC score patients had an HR of 95 (p=4e-4) for metastasis. In multivariable analysis, Decipher was the only significant risk factor with an HR of 55 (CI 12.2-249, p=1.8e-7) for metastasis after adjusting for ERG+, PTEN-loss, Gleason score, PSA, tumor stage, margins and adjuvant treatment. Conclusion The Decipher genomic classifier is a powerful prognostic marker that retains its significance for predicting metastasis in subsets of patients with tumors that harbor common genomic alterations. Patients with high GC scores and these alterations may represent an extremely high-risk subset of prostate cancer. Citation Format: Mohammed Alshalalfa, Ismael Vergara, Nicholas Erho, Elai Davicioni, Robert Jenkins, Kollmeyer Thomas. Evaluation of a genomic classifier (Decipher®) in subsets of primary tumors with common prostate cancer genomic alterations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2014-4720

407P - Pik3Ca Mutations and Loss of Pten Expression in Circulating Tumor Cells (Ctcs) in Patients with Metastatic Breast Cancer (Mbc)

ABSTRACT Aim: Currently, several methods are available to detect CTCs in peripheral blood; the enumeration of CTCs has emerged as a promising biomarker. CTCs shed from metastatic cancers may allow the non-invasive analysis of the evolution of tumor genomes during treatment and disease progression through “liquid biopsies” 1. PIK3CA mutations and loss of PTEN expression are being analyzed in many studies in breast cancer. Frequent deregulation and aberration of this pathway have been implicated not only in breast cancer development and progression, but also in breast cancer therapy resistance, and as a potential predictor of response to new treatments 2,3. The objective of this study is to analyze genomic alterations of the PTEN gene and PIK3CA (exon 20) in CTCs in patients with MBC. 1. Cristofanilli M, et al. N Engl J Med 2004;351:781-91. 2. Sarah-Jane Dawson, et al. N Engl J Med 2013;368:1199-1209. 3. Cancer Genome Atlas Network. Nature 2012;490:61-70. Methods: We carried out a descriptive study in patients with MBC currently undergoing treatment at Hospital Universitario de Salamanca (Spain). CTCs were quantified by means an automated fluorescence method (MetaFer-MetaCyte of MetaSystem), allowing selection of cells by size and genetic alterations. We analyzed genomic alterations in PTEN gene in CTCs by FISH and High Resolution Melting (HRM) was performed to measure PIK3CA mutations in circulating tumor DNA. Finally, we studied the association between PTEN deletion and PIK3CA mutations. Results: In this preliminary analysis, a total of 30 samples have been obtained. CTCs (size >7 µm) were detected in 27 of 30 women. Of 27 samples, there were 12 cases (44,4%) of PTEN loss detected by FISH, and there were 10 cases (37,1%) in which PIK3CA mutations were detected by HRM. In 5 samples, there were association between PTEN deletion and PIK3CA mutations. These 5 cases were Luminal (A and B) tumors. Conclusions: Monitoring and isolation of CTCs represents an opportunity to study specific genomic alterations. In this study, we carried out the analysis of two genes involved in the same signaling pathway: PTEN and PIK3CA; which may have additive or synergistic effects through non-canonical functional pathways. Disclosure: All authors have declared no conflicts of interest.

Constitutive AKT activation in follicular lymphoma

BackgroundThe phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.MethodsTo clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exon 9 and 20 using direct sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls.ResultsAKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed low level of pAKT. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) FL cases showed loss of PTEN expression. 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample.ConclusionsThese data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

Loss of PTEN expression and AKT activation in HER2-positive breast carcinomas.

To examine the expression of AKT and PTEN in a series of HER2-positive primary invasive breast tumors using immunohistochemistry, and to associate these expression profiles with classic pathologic features such as tumor grade, hormone receptor expression, lymphatic vascular invasion, and proliferation. A total of 104 HER2-positive breast carcinoma specimens were prepared in tissue microarrays blocks for immunohistochemical detection of PTEN and phosphorylated AKT (pAKT). Original histologic sections were reviewed to assess pathological features, including HER2 status and Ki-67 index values. The associations between categorical and numeric variables were identified using Pearson's chi-square test and the Mann-Whitney, respectively. Co-expression of pAKT and PTEN was presented in 59 (56.7%) cases. Reduced levels of PTEN expression were detected in 20 (19.2%) cases, and these 20 tumors had a lower Ki-67 index value. In contrast, tumors positive for pAKT expression [71 (68.3%)] were associated with a higher Ki-67 index value. A role for AKT in the proliferation of HER2-positive breast cancers was confirmed. However, immunohistochemical detection of PTEN expression did not correlate with an inhibition of cellular proliferation or control of AKT phosphorylation, suggesting other pathways in these mechanisms of control.

Loss of ARID1A expression and its relationship with PI3K-Akt pathway alterations and ZNF217 amplification in ovarian clear cell carcinoma

AT-rich interactive domain 1A (ARID1A) is a subunit of switch/sucrose non-fermentable (SWI/SNF) complex. Recently, alterations of ARID1A gene, phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) pathway and zinc-finger protein 217 (ZNF217) gene have been identified as frequent molecular genetic changes in ovarian clear cell carcinoma. The relationships between these events have not been studied and integrated in the same cohort. This study was aimed at determining the correlation between these molecular events and other clinicopathological factors, including the prognostic impacts of these clinicopathological factors. A total of 68 ovarian clear cell carcinoma cases were collected and subjected to immunohistochemistry testing for ARID1A, SMARCA2, SMARCA4, SMARCB1 and phosphatase and tensin homolog (PTEN), mutation analysis for phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and fluorescence in situ hybridization for ZNF217 amplification. The correlations between ARID1A expression, PI3K-Akt pathway, ZNF217 amplification and other clinicopathological factors were analyzed. Loss of ARID1A expression was present in 35 cases (52%) and loss of SMARCA2 expression occurred in 1 case. SMARCA4 and SMARCB1 expressions were preserved in all cases. PIK3CA mutations were present in 23 cases (34%) and loss of PTEN expression occurred in 8 cases (12%). Alterations in the PI3K-Akt pathway (PIK3CA mutations or loss of PTEN expression) were found in 42 cases (62%). ZNF217 amplification was detected in 21 cases (31%). Loss of ARID1A expression was significantly related to younger patient age (P=0.048), PI3K-Akt pathway activation (P=0.046) and ZNF217 amplification (P=0.028). All of the clinicopathological factors were not prognostic factors for ovarian clear cell carcinoma after multivariate analysis, except International Federation of Gynecology and Obstetrics staging (P=0.001). Our results showed that loss of ARID1A expression usually coexisted with PI3K-Akt pathway activation and/or ZNF217 amplification. Synergic effects of loss of ARID1A and PI3K-Akt pathway activation as well as ZNF217 amplification may be related to the development of ovarian clear cell carcinoma.

Phase II, single stage, cohort expansion study of MK-2206 in recurrent endometrial serous cancer.

5515 Background: Although PTEN mutations or loss of PTEN expression are uncommon in endometrial serous carcinomas, approximately half of these tumors harbor PIK3CA mutations and/or amplification of...

The Search Continues: Looking for Predictive Biomarkers for Response to Mammalian Target of Rapamycin Inhibition in Endometrial Cancer

ObjectivePI3K/mammalian target of rapamycin (mTOR) pathway aberrations occur in 40% to 80% of endometrial cancer. Prior studies suggest KRAS mutations are associated with resistance to mTOR inhibitors in solid tumors. The objective of this study was to determine if biomarker expression in the PI3K/mTOR pathway or KRAS mutations would predict response to therapy with everolimus, an mTOR inhibitor.MethodsSpecimens from a phase II study of everolimus in recurrent endometrioid endometrial cancer were utilized. The primary end point was clinical benefit rate (CBR: objective response and nonprogression at 20 weeks). Correlative studies evaluating PTEN expression and phospho-S6 ribosomal protein (pS6rp) status by immunohistochemistry and KRAS mutational analysis were performed.ResultsSix of 28 evaluable patients achieved prolonged stable disease (SD) at 20 weeks (CBR, 21%). Loss of PTEN expression did not predict CBR (P = 0.62) with a positive predictive value (PPV) of 0.13. Five (83%) of 6 patients with SD maintained PTEN expression. Neither pS6rp expression (P = 0.65) nor KRAS mutation (P = 0.99) predicted CBR; the PPV was 0.14 for each. Eighty percent (4/5) of those with SD were KRAS wild type. Combined analysis of pS6rp expression and KRAS mutation provided 100% PPV (95% confidence interval, 39.6%–100%), suggesting no chance of CBR for these individuals with 100% specificity (95% confidence interval, 46.3%–100%).ConclusionsS6rp phosphorylation, loss of PTEN expression, and presence of KRAS mutations alone did not correlate with CBR. However, positive pS6rp staining combined with KRAS mutation performed with 100% PPV and specificity to predict nonresponse. Identifying patients who will not benefit from mTOR inhibitors can direct therapy and reduce exposure to agents that add toxicity without clinical benefit.

Activation of the PI3K/mTOR/AKT pathway and survival in solid tumors: systematic review and meta-analysis.

BackgroundAberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear.MethodsPubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model.ResultsAnalysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13).ConclusionActivation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway.

MP79-16 PTEN EXPRESSION LOSS IS ASSOCIATED WITH AN INCREASED RISK OF CANCER-SPECIFIC MORTALITY AMONG MEN WITH BIOCHEMICAL RECURRENCE AFTER RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2014MP79-16 PTEN EXPRESSION LOSS IS ASSOCIATED WITH AN INCREASED RISK OF CANCER-SPECIFIC MORTALITY AMONG MEN WITH BIOCHEMICAL RECURRENCE AFTER RADICAL PROSTATECTOMY Michael Gorin, Tamara Lotan, Bruce Trock, Elizabeth Platz, Elizabeth Humphreys, Angelo De Marzo, George Netto, Alcides Chaux, and Misop Han Michael GorinMichael Gorin More articles by this author , Tamara LotanTamara Lotan More articles by this author , Bruce TrockBruce Trock More articles by this author , Elizabeth PlatzElizabeth Platz More articles by this author , Elizabeth HumphreysElizabeth Humphreys More articles by this author , Angelo De MarzoAngelo De Marzo More articles by this author , George NettoGeorge Netto More articles by this author , Alcides ChauxAlcides Chaux More articles by this author , and Misop HanMisop Han More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2519AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Loss of the PTEN tumor suppressor gene has recently been shown to be associated with an increased risk of biochemical recurrence (BCR) among men with clinically localized prostate cancer treated with radical prostatectomy (RP). Little is known, however, regarding the influence of PTEN on the subsequent risk of disease progression and death among men who have experienced BCR. Thus, the objective of this study was to evaluate PTEN expression status as a risk factor for prostate cancer-specific mortality (PCSM) among men with BCR after RP. METHODS A tissue microarray constructed from men with BCR after RP was analyzed for PTEN expression status by immunohistochemistry. Using a validated scoring system, cases were dichotomized as either normal or markedly decreased/loss. Kaplan-Meier and proportional hazards regression analyses were performed to evaluate PTEN status as an independent predictor of PCSM, with survival measured from date of BCR. RESULTS PTEN expression status was evaluated for 346 men with BCR. During a median follow-up of 8 years (IQR 2-12) from BCR, 75 (21.7%) men died of prostate cancer. Actuarial prostate cancer-specific survival at 10 years after BCR was 78% for men with normal PTEN expression and 65% for men with PTEN expression loss (p < 0.001). After controlling for Gleason sum, pathologic stage, and salvage treatment, PTEN expression loss was associated with a hazard ratio for PCSM of 2.16 (95% CI 1.34-3.51, p = 0.002). CONCLUSIONS Among men with BCR, PTEN expression loss was independently associated with increased risk of PCSM. Because the prognosis for men with BCR is highly variable, PTEN expression, if validated, may prove to be useful to identify men who may benefit the most from salvage therapy. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e935-e936 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Michael Gorin More articles by this author Tamara Lotan More articles by this author Bruce Trock More articles by this author Elizabeth Platz More articles by this author Elizabeth Humphreys More articles by this author Angelo De Marzo More articles by this author George Netto More articles by this author Alcides Chaux More articles by this author Misop Han More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PTEN mutation, loss of heterozygosity, promoter methylation and expression in colorectal carcinoma: Two hits on the gene?

The phosphatase and tensin homologue (PTEN) gene is considered to be a tumour-suppressor gene in various types of cancer, colorectal carcinoma among them. According to the 'two-hit' tumour-suppressor gene concept, inactivation occurs by any combination of the following three pathogenetic processes: mutation, loss of one allele [i.e. loss of heterozygosity (LOH)] or promoter methylation. To determine the frequencies of PTEN tumour-suppressor gene features in colorectal carcinoma, we used DNA from colorectal carcinoma xenografts/primary tumour cell lines (N=22) or neoplastic glands isolated by laser-capture microdissection (N=20). Sequencing exons 1-9 of the gene revealed a total of 8 somatic mutations in 5 tumours (3 with high-degree microsatellite instability). In 1 tumour, a truncating mutation of one allele was combined with two missense mutations of the other allele. Polymorphic microsatellite marker analyses (D10S5412, D10S579 and D10S1765) showed complete loss of one allele (i.e. LOH sensu stricto) in 3 tumours, but combined LOH and mutation was found only once. Promoter methylation, tested by MethyLight technology, was found in only 1 of the tumours, not combined with mutation or LOH. In contrast, by immunohistochemistry (mAb 6H2.1), reduction or even loss of PTEN expression was found in 18 tumours. Taken together, PTEN downregulation is a fairly frequent event in colorectal carcinoma, but this apparently is not usually caused by two hits on the gene.

Loss of PTEN Expression Is Associated With IGFBP2 Expression, Younger Age, and Late Stage in Triple-Negative Breast Cancer

To investigate the association between PTEN loss and IGFBP2 expression in a series of triple-negative breast cancers and to relate this expression to basal cytokeratin expression and clinicopathologic features. One hundred and one formalin-fixed and paraffin-processed triple-negative breast cancer cases from the University of Malaya Medical Centre were tested immunohistochemically for cytokeratins 5/6 and 14, PTEN, and IGFBP2. The resulting slides were scored for proportion and intensity of staining. Loss of tumor nuclear and cytoplasmic staining for PTEN occurred in 48.3% of cases and was significantly associated with younger age at diagnosis (47 years compared with 57 years in those without PTEN loss; P = .005). Independent predictors of PTEN loss were late stage at presentation (P = .026), cytokeratin 5/6 positivity (P = .028), and IGFBP2 expression (P = .042). High levels of IGFBP2 expression were seen in 32% of cases; an independent predictor of high levels was cytokeratin 14 negativity (P = .005). PTEN loss and high levels of IGFBP2 expression were associated with poorer survival, but neither of these trends was significant. PTEN loss is a frequent event in triple-negative breast cancers and is significantly associated with younger age at onset of breast cancer, late stage, and IGFBP2 expression.

Immunohistochemical expression of the mammalian target of rapamycin pathway in penile squamous cell carcinomas: a tissue microarray study of 112 cases

The aim of this study was to evaluate the immunohistochemical expression of mammalian target of rapamycin (mTOR) pathway-related biomarkers in penile carcinomas, and to assess associations with histological type, histological grade, and human papillomavirus (HPV) infection. We built four tissue microarrays from 112 invasive penile squamous cell carcinomas, and evaluated the immunohistochemical expression of PTEN, phospho-AKT, phospho-mTOR, and phospho-S6. We found decreased or loss of PTEN expression in 87% of cases. Warty and/or basaloid carcinomas had a higher proportion of PTEN loss (P=0.02), whereas keratinizing tumours showed higher levels of phospho-S6 (P=0.009); phospho-AKT and phospho-mTOR levels were not significantly different between warty/basaloid and keratinizing carcinomas (P=0.75 and P=0.77, respectively). PTEN was not associated with histological grade (P=0.18). Expression levels of phospho-S6 were significantly higher in low-grade tumours (P=0.001), whereas expression levels of phospho-AKT and phospho-mTOR were slightly higher in high-grade tumours (P=0.01 and P=0.35, respectively). We did not find any association between HPV infection and mTOR markers (P≥0.2 in all cases). Our results provide evidence of dysregulation of the mTOR pathway in penile carcinomas independently of HPV infection. Future clinical studies should further evaluate the prognostic and predictive usefulness of these markers in patients with penile cancer.

WNT signaling and distant metastasis in colon cancer through transcriptional activity of nuclear β-Catenin depend on active PI3K signaling.

We determined whether active PI3K signaling together with nuclear accumulation of β-Catenin is necessary to fully activate canonical WNT signaling and examined the association of both signaling pathways with colon cancer progression. Using reporter gene assays we examined the activation of β-Catenin mediated transcription upon PI3K inhibition with or without β-Catenin nuclear accumulation. Ectopically induced as well as constitutively active WNT signaling strictly required PI3K activity whereas PI3K inhibition had no effect on β-Catenin subcellular localization but impaired β-Catenin binding to WNT target gene promoters and decreased WNT target gene expression. Transcriptional activity of nuclear β-Catenin depended on active PI3K signaling as nuclear accumulation of β-Catenin failed to induce WNT reporter gene transcription upon PI3K inhibition. PI3K dependend transcriptional transactivation of β-Catenin relies on events beyond phosphorylation at the AKT target site serine 552, as S552D-phosphomimetic β-Catenin mutants were unable to restore WNT signaling when inhibiting PI3K. To study the prognostic value of PI3K pathway activation (activating PIK3CA mutations or loss of PTEN expression) and nuclear β-Catenin expression, both variables were determined in 55 matched pairs of primary right sided colon cancer cases with or without distant metastasis. Activating mutations in the PIK3CA gene or loss of PTEN expression did not correlate with distant metastasis while high nuclear β-Catenin expression combined with activation of the PI3K pathway identified cases in which distant metastasis had occurred. Activation of the PI3K pathway was not associated with nuclear β-Catenin expression. We conclude that the transcriptional activity of nuclear β-Catenin depends on PI3K activity. However, PI3K on its own does not affect β-Catenin subcellular localization. Both factors synergize for full WNT signaling activity and are associated with distant metastasis in colon cancer. Thus, the detection of high nuclear β-Catenin expression and simultaneous PI3K pathway activation identifies colon cancer patients with a high risk for distant metastasis.

Altered immunohistochemical expressions of phosphorylated AKT and PTEN and PIK3CA mutation in adenocarcinoma of the lung 3 cm or less in diameter

The phosphatidylinositol-3 kinase (PI3K)/AKT oncogenic signaling pathway is activated by alterations of key pathway components such as PTEN and AKT. In addition, PIK3CA gene encoding a catalytic subunit of the PI3K is mutated in various tumors, including lung cancer. This study was conducted to evaluate the significances of altered phosphorylated AKT (p-AKT) and PTEN expressions using immunohistochemistry and PIK3CA gene mutation by peptide-nucleic acid clamping PCR in 100 resected adenocarcinoma of lung 3 cm or less in diameter. p-AKT was expressed in 28 cases (28.0%), whereas loss of PTEN expression was observed in 30 cases (30.0%). p-AKT expression was inversely correlated with PTEN expression (p

Endometrial Cancer: Molecular and Cellular Basis of Tumor Development, Novel Biomarkers and Therapeutic Agents, and Innovative Research Approaches

Endometrial Cancer: Molecular and Cellular Basis of Tumor Development, Novel Biomarkers and Therapeutic Agents, and Innovative Research Approaches

PTEN expression is involved in the invasive properties of HNSCC: A key protein to consider in locoregional recurrence

Specific phenotypic effects of PTEN in head and neck squamous cell carcinoma (HNSCC) remain poorly defined without a direct causal connection between the loss of PTEN function and the progression of cancer. Here, we describe a potential role for PTEN in cancer progression. Using an shRNA targeting PTEN in HNSCC cells, we show that the loss of PTEN expression is associated with a decrease of cell adhesion, a reduction in E-cadherin expression while cell migration is promoted. Together with the tissue organization and molecular markers expressed in tumors derived from shPTEN cells invivo, this study indicates that HNSCC cells deficient in PTEN expression undergo an epithelial‑mesenchymal transition (EMT). Additionally, our results suggest that both the low levels of expression and subcellular localization of PTEN are involved in the EMT phenotype, and ultimately in possible locoregional reccurences. We hypothesize that the loss of PTEN expression as well as the subcellular localization could be of interest as a predictive marker of recurrence in HNSCC.

Loss of PTEN Expression Is Associated with Poor Prognosis in Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas

Previously, we reported PIK3CA gene mutations in high-grade intraductal papillary mucinous neoplasms (IPMN). However, the contribution of phosphatidylinositol-3 kinase pathway (PI3K) dysregulation to pancreatic carcinogenesis is not fully understood and its prognostic value unknown. We investigated the dysregulation of the PI3K signaling pathway in IPMN and its clinical implication. Thirty-six IPMN specimens were examined by novel mutant-enriched sequencing methods for hot-spot mutations in the PIK3CA and AKT1 genes. PIK3CA and AKT1 gene amplifications and loss of heterozygosity at the PTEN locus were also evaluated. In addition, the expression levels of PDPK1/PDK1, PTEN, and Ki67 were analyzed by immunohistochemistry. Three cases carrying the E17K mutation in the AKT1 gene and one case harboring the H1047R mutation in the PIK3CA gene were detected among the 36 cases. PDK1 was significantly overexpressed in the high-grade IPMN versus low-grade IPMN (P = 0.034) and in pancreatic and intestinal-type of IPMN versus gastric-type of IPMN (P = 0.020). Loss of PTEN expression was strongly associated with presence of invasive carcinoma and poor survival in these IPMN patients (P = 0.014). This is the first report of AKT1 mutations in IPMN. Our data indicate that oncogenic activation of the PI3K pathway can contribute to the progression of IPMN, in particular loss of PTEN expression. This finding suggests the potential employment of PI3K pathway-targeted therapies for IPMN patients. The incorporation of PTEN expression status in making surgical decisions may also benefit IPMN patients and should warrant further investigation.

Abstract P4-05-14: Molecular and immunohistochemical profile of invasive micropapillary breast carcinoma

Abstract Background: Invasive micropapillary carcinoma (IMPC) of the breast is an uncommon disease subtype that is generally considered aggressive. A variety of carcinomas can exhibit micropapillary histology including lung, bladder, and ovary. Immunohistochemical (IHC) and genomic studies at these other organ sites have identified potential therapeutic targets including BRAF mutations. The purpose of this study was to characterize the IHC and molecular profile of a cohort of IMPC that could suggest potentially useful targeted therapies. Design: Eight tumor blocks (from seven patients) of IMPC were identified from the archives at the University of Iowa Hospitals and Clinics from 1997-2012. IHC stains for ER, PR, HER2, PTEN (Dako; clone 6H2.1, 1:100) and NM23H1 (Santa Cruz Biotechnology, clone: Nm301, 1:50) were performed. ER, PR, and HER2 were score according to ASCO/CAP guidelines. PTEN and NM23H1 were scored using a 4-tier system: P: strong homogenous; H: heterogeneous (positive with negative foci); R: reduced in more than 50%; and L: lost in all or majority. Genomic DNA was isolated from the formalin fixed paraffin embedded tumor samples used for IHC. BRAF V600 mutational analysis was analyzed using a laboratory-developed primer extension assay. Massive parallel sequencing (next generation sequencing, NGS) was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA). Results: The patients were 50-83 years of age. Six had Elston-Ellis Grade II tumors and one was Elston-Ellis grade I. Four patients had axillary lymph node metastases. All cases examined showed strong and diffuse estrogen and progesterone receptor expression with no over-expression of HER2. Complete PTEN loss was observed in 4 cases including 3 with lymph node positive disease, three of these four cases had concurrent reduced expression of NM23H1. No mutations in exon 15 of the BRAF gene were detected. By NGS, one sample showed a KITc.A1621C (pM541) polymorphism, three had no mutations, and sequencing from the other three was unsatisfactory. Molecular and IHC Profile of Seven Patients with IMPC of the BreastHistologic GradeAxillary Lymph Node MetastasisER/PRHER2PTENNM23H1NGS2NoPositive/PositiveNegativePPUnsatisfactory1NoPositive/PositiveNegativeLRNegative2NoPositive/PositiveNegativeHPIntronic2YesPositive/PositiveNegativeHPUnsatisfactory2YesPositive/PositiveNegativeLRKIT c.A1621C (pM541)2YesPositive/PositiveNegativeLPNegative2YesPositive/PositiveNegativeLRUnsatisfactory Conclusions: Loss of PTEN expression was observed in 57% of cases (4/7) and was often associated with lymph node metastasis. A direct correlation between PTEN and NM23H1 expression was observed in six cases. No BRAF or other mutations were detected. PTEN was included in the NGS panel. Reduced expression observed in our study is most likely due to posttranscriptional modifications. Concurrent reduced expression of the metastatic suppressor NM23H1 may play a role in the clinical behavior of IMPC. The high frequency of PTEN loss may have implications for targeted therapy towards the PI3K pathway in this subgroup of patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-14.