Complete Loss Of PTEN Expression Research Articles

Immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies; their correlation with histological subtypes and one-year survival.

Gastric carcinoma is a major cause of cancer-related morbidity and mortality worldwide. Gastric neoplasms arise from genetic and epigenetic changes in various genes. Present study evaluates the immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies and correlates the expression of these proteins with clinicopathological features. Adequate endoscopic biopsies of 27 cases of gastric carcinoma were evaluated for World Health Organization (WHO) and Lauren's classification subtypes along with HER2/neu, PTEN, and Ki-67 immunoexpression. HER2/neu immunostaining was scored as proposed in the Trastuzumab for gastric cancer (ToGA) trial while PTEN staining and downregulation were assessed using an immunoreactive score. The cut-off for Ki-67 expression was taken as 90th percentile of the values in adjacent non-neoplastic tissue. All statistical analysis was done at 5% level of significance with SPSS v22 statistical software. Tubular adenocarcinoma was the commonest WHO histological subtype and 56% of cases were of intestinal type as per Lauren's classification. 55.6% of cases showed a complete loss of PTEN expression in neoplastic tissue. 17 of the 19 cases with adjacent non-neoplastic tissue showed PTEN downregulation in neoplastic tissue. 81.5% of cases had a high Ki-67 index and HER2/neu overexpression was noted in 36% of cases. All the four cases who died had high Ki-67 proliferation indices; 3 patients had loss of PTEN expression and HER2/neu overexpression. We conclude that these immunomarkers can play important role in the behavior of gastric carcinomas and can be targeted for new therapies.

IMMUNOHISTOCHEMICAL STUDY ON PTEN AND CYCLIN D1 IN NON-NEOPLASTIC AND NEOPLASTIC ENDOMETRIAL LESIONS

Abstract
 Introduction: Endometrial carcinoma (EC) is the most common gynaecological malignancy in developed countries and has been classified into two groups, type 1 and type 2. Type 1 or endometrioid endometrial carcinomas (EECA) accounts for 80% of EC and are thought to develop following a continuum of premalignant lesions ranging from endometrial hyperplasia without atypia (EH) and atypical hyperplasia (AH). PTEN (phosphatase and tensin homolog), a tumor suppressor gene is commonly inactivated in 83 % of endometrioid carcinoma and 55% of precancerous lesions. Cyclin D1, a cell cycle regulator is overexpressed in about 40% of endometrial carcinomas.
 Aim: To study the expression of PTEN (Phosphatase and tensin homolog) and Cyclin D1 in non-neoplastic and neoplastic endometrial lesions by immunohistochemistry (IHC).
 Methods: A 2 year cross-sectional study (September 2017 to August 2019) on 115 endometrial samples was done in the Department of Pathology, RIMS. Histomorphological features and IHC expression of PTEN and Cyclin D1 in the various endometrial lesions were studied and evaluated, data collected in IBM SPSS Statistics 21 was statistically analyzed using Chi - square and Fisher’s Exact test.
 Results: Out of the 115 cases, 47(40.9%) were diagnosed as benign proliferative endometrium, 20(17.4%) benign secretory endometrium, 21(18.3%) hyperplasia without atypia, 15(13.0%) atypical hyperplasia and 12(10.4%) endometrial carcinoma with an age group spanning from 26-68 years (mean age = 46.4). Following IHC staining, 91.7%(11/12) and 83.3%(10/12) cases of EC and 80%(12/15) and 73.3%(11/15) cases of AH showed complete loss of PTEN expression and Cyclin D1 overexpression, respectively when compared to other benign lesions and was statistically significant (p < .001).
 Conclusion: Loss of PTEN and Cyclin D1 overexpression was seen in a significant number of EECA and AH, suggesting both as an early event in endometrial carcinogenesis. Therefore, we propose the use of PTEN and Cyclin D1 immunostaining as an adjunct to histopathological diagnosis as it may be informative in the identification and further management of premalignant endometrial lesions that are likely to progress to carcinoma
 Keywords: PTEN, Cyclin D1, endometrial hyperplasia, endometrial carcinoma, endometrioid endometrial carcinomas.

SO-10 An open-label, multi-centre, phase Ib/II study of PI3Kβ selective inhibitor GSK2636771 administered in combination with paclitaxel in patients with advanced gastric cancer having alterations in PI3K/Akt pathway

Loss of PTEN expression accompanied with activation of PI3K/AKT pathway commonly occurs in gastric cancer. GSK2636771 is a potent, orally bioavailable, adenosine triphosphate-competitive selective inhibitor of PI3Kβ with minimal off-target effects. Here, we conducted a dose-finding study with safety and efficacy analysis of GSK2636771 combined with paclitaxel in patients with advanced gastric cancer (AGC) who progressed from first-line chemotherapy. In this multi-center phase Ib/II study, patients with PTEN-deficient AGC who failed to respond to first-line chemotherapy with fluoropyrimidine and platinum were eligible. PTEN expression was evaluated by immunohistochemistry and the PTEN-deficient was defined by < 100 of PTEN histoscore (PTEN H-score). A standard 3+3 design included two dose levels of GSK2636771 (300mg once daily and 200mg once daily) in combination with paclitaxel (80mg/m2 on days 1, 8, and 15) during phase Ib study to determine the recommended phase II dose (RP2D). Phase II study was subsequently conducted based on the RP2D derived from phase Ib study to assess safety and antitumor activity. Primary objectives included defining RP2D and assessment of progression-free survival (PFS) in patients administered with RP2D. Secondary objectives included overall survival (OS), objective response rate (ORR), disease control rate (DCR), safety, and biomarker analysis incorporating investigation of PTEN H-score (0 vs. 1-99) and somatic alteration of cancer by next-generation sequencing (NGS). This trial is registered with ClinicalTrials.gov, number NCT02615730. A total of 42 patients were enrolled, median age was 59 years old (range: 32-88 years old) and most patients were male (34/42). During phase Ib study, grade 3 hypocalcemia was observed in 2 out of 5 patients with GSK2636771 300mg once daily combined with paclitaxel. The RP2D was GSK2636771 200mg once daily combined with paclitaxel (80mg/m2 on days 1, 8, and 15). The median follow-up duration of 29.9 months [95% confidential interval (CI), 21.5-38.3 weeks]. Among 37 patients treated with RP2D with and 33.4 weeks (95% CI, 26.2-40.7 weeks), median PFS and OS were 12.1 (95% CI, 11.1-13.2 weeks) and 33.4 weeks (95% CI, 26.2-40.7 weeks), respectively. Among 28 patients with measurable disease and administered with RP2D, confirmed response and disease control was observed in 5 patients (ORR=17.9%) and 19 patients (DCR=67.9%), respectively. Patients with PTENnull tumors (H-score 0) exhibited favorable PFS (18.9 vs. 11.6 weeks, P=0.026) compared to those with PTENpartial loss tumors (H-score 1-99). Most common any grade of adverse events (AEs) were neutropenia (38.1%), peripheral neuropathy (28.8%), anorexia (21.4%), diarrhea (16.7%), and myalgia (14.3%). No treatment related death or unexpected AEs resulting in treatment cessation were observed with the RP2D. GSK2636771 combined with paclitaxel revealed manageable toxicities and antitumor activity in patients with PTEN-deficient AGC who progressed after first-line chemotherapy. Complete loss of PTEN expression may be associated with clinical benefit from GSK2636771 and paclitaxel. Further analysis of the association with biomarkers and clinical outcomes is underway.

Abstract B22: Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo approaches

Abstract PTEN negatively regulates the Class I PI 3-kinases by metabolizing phosphatidylinositol(3,4,5)trisphosphate (PIP3) and acts as a tumor suppressor. Heterozygous germline PTEN mutations in humans manifest into a complex multiorgan disorder known as PTEN hamartoma tumor syndrome (PHTS). Patients present with hamartomatous skin lesions, benign GI polyps, have an increased risk of developing certain types of cancer (breast, colon, endometrium, thyroid, GI tract and kidney) and have macrocephaly and autism spectrum disorders (ASD). There is huge variability in disease symptoms in PHTS patients, the cause of which is unclear. It has been proposed that this correlates with the nature of the aberration in PTEN and its impact on PTEN expression and function, allowing to discriminate the following cancer-risk groups in PHTS patients: 1. High cancer risk: Patients expressing a catalytically inactive but stable PTEN protein (mis-sense PTEN mutations such as the C124S, G129R, R130Q etc.) have a severe phenotype, with benign tumors and aggressive malignant cancers at a young age of one of more tissues. 2. Medium risk: Patients with complete loss of PTEN expression due to insertions, deletions and truncations in the PTEN gene present with an intermediate severity with benign and malignant tumors. 3. Low risk: Patients with PTEN protein characteristics that do not fall into Group 1 or 2 but with some detectable suppression of AKT signaling. This covers a range of PTEN mutants, with often a mix of characteristics such as being unstable but active or even overactive, stable but partially inactive or no apparent activity defect. These patients have a mild phenotype, mainly related to a broad range of ASD symptoms such as macrocephaly, developmental delay and mental retardation. Some patients are severely autistic. Patients may have benign tumors and skin hamartomas, but malignant cancer is rare. Here we expand our analysis by characterizing a range of additional PHTS PTEN mutations using biochemical methods. Of the several mutations characterized, the R173C mutation leads to a PTEN enzyme that retains its activity but becomes partially unstable, putting it in Group 3. This mutation was of particular interest because the R173 site in PTEN is also the third most commonly somatically mutated PTEN site in cancer (COSMIC database; accessed September 2018). Mouse models such as the heterozygous PTEN heterozygous mice (PTEN +/- mice) and PTEN +/C124S represent Groups 1 and 2, and their characteristics coincide with the predicted phenotypes. However, no clinically relevant mouse models for the low-risk PHTS group have been reported. We have generated and characterized a PTEN +/R173C PHTS mouse model. We show that the PTEN +/R173C mice have macrocephaly, fewer tumors and a significantly longer tumor-free survival compared to the PTEN +/-, thus providing further evidence that the above-proposed PTEN genotype-phenotype correlation holds true in PHTS. Acknowledgment: This work has been supported by PTEN Research. Citation Format: Wayne Pearce, Nicoletta Kessaris, Nicholas R. Leslie, Bart Vanhaesebroeck, Priyanka Tibarewal, Gala Classen, Virginia A Garcia, Victoria Rathbone, Nisha Kriplani, Georgia Constantinou. Investigation of PTEN genotype-phenotype correlations in the PTEN hamartoma tumor syndrome (PHTS) using in vitro and in vivo approaches [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B22.

Combined loss of TFF3 and PTEN is associated with lethal outcome and overall survival in men with prostate cancer.

Trefoil Factor 3 (TFF3) has been implicated in Prostate Cancer (PCa) progression. However, its prognostic value and association with other biomarkers have not been fully explored. We assessed the combined value of TFF3 and PTEN in two cohorts: one is managed surgically for localized PCa and the second is managed non-surgically by androgen deprivation therapy for advanced disease. 228 radical prostatectomies (RP) and 318 transurethral resection of prostate (TURP) samples were assessed by immunohistochemistry (IHC) for TFF3 and by IHC and fluorescent in situ hybridization (FISH) for PTEN. Results of biomarkers expression were correlated with various pathological and clinical outcome parameters including biochemical recurrence (BCR) in the RP cohort and cancer-specific mortality (PCSM) and overall survival (OS) in the TURP cohort. TFF3 expression was detected in 131/226 (57.9%) RP samples and 148/318 (46.5%) of TURP cases. In general, TFF3 positivity was less frequently observed with advanced Gleason Groups. TFF3 expression was also assessed in relation to PTEN expression. Only 15-16% of TFF3-expressed cases were present in association with complete loss of PTEN expression in the TURP and localized cohorts, respectively. Loss of TFF3 expression was not related to BCR after RP, but was prognostic in the non-surgical cohort and associated with decrease OS and PCSM (HR 2.31, CI: 1.67-3.18, p < 0.0001) and (HR 3.99, CI: 2.43-6.56; p < 0.0001), respectively. Adjusting for Gleason score, combined loss of TFF3/PTEN was most associated with OS (HR 2.33, CI: 1.49-3.62; p < 0.0001) and PCSM (HR = 3.44, CI: 1.75-6.78, p < 0.0001). The study documents for the first time significant association for combined status of TFF3 expression and PTEN loss in OS and PCSM in patients not managed by surgical intervention. Prospective assessment of PTEN and TFF3 may provide further insight into molecularly subtyping PCa and aid in stratifying patients at risk for lethal disease.

PTEN Genetic and Epigenetic Alterations Define Distinct Subgroups in North Indian Breast Cancer Patients

Background:Breast cancer is a heterogeneous disease that can be subdivided on the basis of histopathological features, genetic alterations, and gene-expression profiles. PTEN gene is considered an established tumor suppressor gene in different types of cancer including breast cancer. However, the role of PTEN alterations in north Indian breast cancer has not been explored especially in defining a group with distinct histological factors.Methodology:181 sporadic breast cancer and their adjacent normal tissues were included in the present study. We analyzed methylation and LOH through MS-PCR and microsatellite markers respectively. While, for PTEN protein expression, we used immunohistochemistry. All the molecular findings were correlated with the clinicopathological parameters of the patients to underline clinical relevance.Results:We found that LOH and methylation of the PTEN promoter were significantly associated with loss of PTEN protein expression, while, PTEN mutation was a rare event. Furthermore, out of 46 double hit cases (i.e., having both methylation and LOH), 70% (32/46) cases showed complete loss of PTEN expression (P= 0.0249). Both LOH and PTEN promoter methylation were associated significantly with age and clinical stage, while, methylation and loss of PTEN expression were associated with high grade and Her-2 negativity. In addition, a quadruple (ER/PR/Her-2 and PTEN) negative group with distinct features was found.Conclusion:The pattern of PTEN expression and its correlation with the clinical parameters indicates that loss of PTEN expression defines a clinical group with distinct features. Hence, PTEN expression provides differential therapeutic strategies for north Indian breast cancer.

Glucocorticoid Resistance in IKZF1-Deleted BCP-ALL: It Is PTEN Again

▪Single copy deletions of IKZF1, which occur in 10-15% of all B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, are associated with a poor outcome. We previously showed that loss of IKZF1 dictates resistance to glucocorticoids (GC) in BCP-ALL cell lines, a knockout mouse model and ex-vivo analysis of primary leukemic cells. When we analyzed the initial response to prednisolone therapy, we found that pediatric patients who suffer from an IKZF1 deleted leukemia are strongly enriched in the poor responder group (14 vs 7%, p<0.05). Although we were able to show that glucocorticoid receptor (GR)-mediated transcription regulation is attenuated in IKZF1 deleted cells, in remained unclear how loss of IKZF1 affects GR signaling.In T-ALL, GC resistance is frequently induced by genomic mutations that result in hyperactivation of AKT. AKT mediated phosphorylation of the GR results in suppression of GR function and resistance to GC treatment. Although the genomic loci that are frequently mutated in T-ALL resulting in hyperactivation of AKT, such as PTEN, are unaffected in BCP-ALL, we tested whether aberrant activation of AKT may also cause resistance to GC in BCP-ALL. Indeed, both CRISPR/Cas9 induced knockout and shRNA mediated knockdown of IKZF1 resulted in activation of AKT in human BCP-ALL cell lines. This hyperactivation was also observed in splenic B cells isolated from Ikzf1+/- mice. Active AKT in turn promotes phosphorylation of the GR on Ser143. This phosphorylation inhibits GR function, at least in part, by preventing shuttling of the ligand bound protein to the nucleus.In order to understand how a single copy loss of IKZF1 activates AKT, we tested expression of upstream regulators of AKT activity and found a near complete loss of PTEN expression, the phosphatase that inhibits PI3K and AKT signaling. Indeed, shRNA mediated knockdown of PTEN in wildtype cells resulted in activation of AKT and induced resistance to GCs. Furthermore, in IKZF1+/- cells we observed an upregulation of HES1, a transcriptional factor that is known to repress expression of the PTEN gene. Of note, HES1 was previously identified as a target of IKZF1 mediated transcriptional repression in erythroid and T cells.To test whether this AKT mediated mechanism of GC resistance could be reverted through pharmacological inhibition, we tested the effect of AKT inhibitors on prednisolone induced apoptosis of human BCP-ALL cell lines (figure A). In contrast to control cells, where inhibition of AKT had little to no effect on the sensitivity to GC, in IKZF1 deleted cells AKT inhibition reverted the resistance phenotype to a level comparable to control cells. Importantly, also in patient derived xenografts we observed that AKT inhibition was able to sensitize cells to GC treatment (Figure B).In conclusion, we identified hyperactivation of AKT, as a result of aberrant HES1 expression, as the mechanism causing GC resistance in IKZF1 deleted BCP-ALL. The availability of AKT inhibitors that are under clinical evaluation, may allow the development of combination therapies that restore the response to GCs in IKZF1-deleted BCP-ALL.Figures:A) IKZF1, PTEN, AKT, pAKT(Ser473), pGR(Ser134) and GR protein expression levels of NALM6 IKZF1+/- cell lysates were analyzed by western blot. Non-targeting controls were used as control and actin was used as a loading control.B) NALM6 parental, non-targeting controls or IKZF1+/- cells were treated for 48 hours with increasing concentrations of prednisolone with or without 0.5µM AKT inhibitor MK2206 and analyzed using an MTT based viability assay. All values were normalized to untreated NALM6 cells. Error bars represent ± standard error of the mean (SEM).(C) hTERT immortalized MSCs were seeded in a 96 wells format and allowed to settle for 24 hours prior to the addition of BCP-ALL patient-derived xenografts (PDX). PDX cells were allowed to settle for 24 hours before prednisone was added in increasing concentrations with or without AKT inhibitor MK2206. After 3 days, sensitivity to treatment was measured by flowcytometry using an amine staining to test for membrane integrity as a measure of cell viability. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B138: Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer

Abstract Introduction: For recurrent and metastatic gastric cancer, systemic chemotherapy is used for palliative purposes, and a paclitaxel- or irinotecan-based regimen is typically used in the second-line setting. Recent advances in research including the TCGA project now enable gastric cancer to be divided into four molecular subgroups, yet whether there are differences in chemotherapy response for each molecular subgroup remain largely unknown. Our aim in this study is to retrospectively compare the efficacy of paclitaxel as second-line chemotherapy according to molecular subtype and to evaluate prognostic value for these markers. Method: A total of 235 metastatic gastric cancer (mGC) patients who have been treated with second-line paclitaxel-containing regimen (paclitaxel monotherapy, FL/paclitaxel) between 2005-2017 were assessed. Archived formalin-fixed, paraffin-embedded tissue blocks obtained before the start of paclitaxel treatment were examined for EGFR, HER2, and PTEN status using immunohistochemistry (IHC). HER2 positivity was defined as either IHC 3+ or 2+ with amplification confirmed by FISH/SISH, and EGFR positivity was defined as IHC 3+. PTEN loss was defined as having complete loss of PTEN expression. MSI-H and EBV-positive group was excluded in this study. Treatment response was based on CT images taken every 2 to 3 cycles of chemotherapy and evaluated according to the RECIST criteria. Overall survival (OS) and progression-free survival (PFS) were calculated based on the starting point of second-line chemotherapy until death or disease progression, respectively. Those negative for all three markers (HER2, EGFR, PTEN loss) were categorized as genome stable (GS)-like molecular group. Result: Patients were classified into subgroups based on HER2, EGFR, and PTEN expression; 19.6% (46/235) were HER2 positive, 15.0% (26/173) were EGFR positive, and PTEN loss was observed in 11.6% (20/173). Expression status for the three markers showed tendency towards mutual exclusivity. When coexpression rates were taken into account, 35.3% (61/173) showed positivity in at least one of the markers, and 64.7% (112/173) were negative for all three. This latter group, categorized as GS-like molecular group, showed a tendency towards poor overall survival compared with patients positive for at least one molecular marker, with median survival of 5.7 vs 8.8 months (P=0.012). There were no significant differences in OS or PFS in terms of HER2 and EGFR status. However, patients with PTEN loss had significantly longer overall survival than those with intact PTEN expression, with median survival time of 12.0 months (95% confidence interval [CI], 7.0 - 21.2) and 6.2 months (95% CI, 4.7-7.2), respectively (P=0.003). Conclusion: In mGC patients treated with second-line paclitaxel chemotherapy, HER2 and EGFR status had no predictive or prognostic value. Contrary to the general association of PTEN loss with poor prognosis in many solid cancers including gastric cancer, PTEN loss group showed better survival outcome for second-line paclitaxel treatment in our study. Indirect association of PTEN with other pathways of multidrug resistance or cell cycle-related molecules that ultimately affect the PI3K/Akt pathway may play a role. Further investigation is needed for specific mechanisms and factors that lead to increased paclitaxel sensitivity in the PTEN loss group. Citation Format: Kyoo Hyun Kim, Jeong Hyun Jo, Sejung Park, Minkyu Jung, Woo Sun Kwon, Jee Hung Kim, Hyo Song Kim, Seung-Hoon Beom, Hyun Cheol Chung, Sun Young Rha. Prognostic value of PTEN loss for second-line paclitaxel treatment efficacy in metastatic gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B138.

A threshold of clinically significant reduced PTEN expression in melanoma.

e21063Background: Current AJCC staging criteria provide limited prognostic value for stage III melanoma patients, who have a 50% chance of recurrence. Complete loss of PTEN expression is known to b...

A Transition Zone Showing Highly Discontinuous or Alternating Levels of Stem Cell and Proliferation Markers Characterizes the Development of PTEN-Haploinsufficient Colorectal Cancer

BackgroundStepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC.Methods and FindingsWe analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases.ConclusionsIn conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This bottleneck-like zone is usually followed by the emergence of invasive tumors with intact PTEN expression but dysregulated TP53 and uniformly high proliferation rates.

Abstracts of the Melanoma Bridge Meeting 2014, December 3-6, 2014, Naples, Italy.

Results Proteomic analysis of frozen melanoma clinical specimens and cell lines demonstrated that phosphorylation (activation) of AKT (P-AKT) correlated inversely with PTEN protein expression. Confirmatory analysis of PTEN expression by an immunohistochemical (IHC) assay on archival tumor samples identified a linear correlation between PTEN measurement by IHC and RPPA. Significantly increased expression of P-AKT was observed in melanomas with complete loss of PTEN protein expression compared to tumors with any other pattern of PTEN expression. Integrated analysis of BRAF/NRAS mutation status, PTEN expression, and clinical outcomes was performed using lymphadenectomy specimens from patients who underwent surgery for stage IIIB/C melanoma. Complete loss of PTEN expression correlated with shorter overall survival and time to brain metastasis on univariate and multivariate analyses. Testing of human melanoma cell lines demonstrated that cells with baseline or compensatory activation of the PI3K-AKT pathway were generally resistant to apoptosis induction following BRAF or MEK inhibition. Extended molecular profiling found that a subset of melanoma cell lines with de novo and acquired resistance to MEK inhibitors are characterized by increased expression of PGC1alpha and a metabolic phenotype of high oxidative phosphorylation (OxPhos). The resistant high OxPhos cell lines all demonstrated synergistic growth inhibition and apoptosis induction with combined targeting of MEK and mTORC1/2. mTORC1/2 inhibition decreased PGC1alpha expression in vitro and in vivo, and caused cytosolic localization of MITF. Treatment with a MEK inhibitor and an mTORC1/2 inhibitor demonstrated marked synergy in a BRAF-mutant, MEK inhibitor resistant human melanoma cell line in vivo.

Complete loss of PTEN protein expression correlates with shorter time to brain metastasis and survival in stage IIIB/C melanoma patients with BRAFV600 mutations.

Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAF(V600) mutations. The prevalence, pathologic features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event. Archival tissue from lymphadenectomy specimens among patients (n = 136) with stage IIIB or IIIC melanoma was assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined. The prevalence of BRAF(V600) mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathologic substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone metastasis. Analysis of PTEN in mutationally defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAF(V600) mutations. Loss of PTEN protein expression correlates significantly with decreased OS and time to MBM in stage IIIB/C melanoma patients with BRAF(V600) mutations. The findings add to evidence supporting a significant role for PTEN loss and the PI3K-AKT pathway in melanoma.

Regulation of the Tumor Suppressor PTEN through Exosomes: A Diagnostic Potential for Prostate Cancer

PTEN is a potent tumor-suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells. PTEN is not detected in exosomes derived from normal, noncancerous cells. We found that PTEN can be transferred to other cells through exosomes. In cells that have a reduction or complete loss of PTEN expression, the transferred PTEN is competent to confer tumor-suppression activity to acceptor cells. In PC patients, we show that PTEN is incorporated in the cargo of exosomes that circulate in their blood. Interestingly, normal subjects have no PTEN expression in their blood exosomes. Further, we found that the prostate-specific antigen (PSA) is incorporated in PC patients’ and normal subjects’ blood exosomes. These data suggest that exosomal PTEN can compensate for PTEN loss in PTEN deficient cells, and may have diagnostic value for prostate cancer.

PTEN expression profiles in colorectal adenocarcinoma and its precancerous lesions

The aim of the study was to determine expression of the PTEN suppressor gene in colorectal adenocarcinoma and its precancerous lesions (adenomatous polyps) in correlation with common clinical and histopathological features. Forty-four patients with adenomatous polyps and 32 with primary adenocarcinoma of the colon or rectum were enrolled in the study. They underwent endoscopic removal of polyps or major surgery and postoperative adjuvant chemo- and radiotherapy depending on staging of the disease. No patient had received chemotherapy and/or radiotherapy before the surgery. PTEN expression was evaluated using immunohistochemical staining on paraffin-embedded specimens and compared to clinicopathological features of tumors. In colorectal cancers, PTEN expression was found to be significantly lower than in normal intestinal mucosa and adenomatous polyps. That was associated with complete loss of PTEN expression observed more frequently in colorectal cancer, contrary to reduction of PTEN expression occurring mostly in polyps. A correlation between polyp diameter and loss of PTEN was demonstrated as well as between tumor size and TNM advanced stage and PTEN expression. The obtained results suggest that the PTEN/PI3K/Akt pathway may play an important role in early stages of sporadic colorectal carcinogenesis and reduced PTEN expression in late oncogenesis is associated with some adverse clinical and pathological features.

High Levels of Phosphatase and Tensin Homolog Expression Are Associated With Tumor Progression, Tumor Recurrence, and Systemic Metastases in pT1 Urothelial Carcinoma of the Bladder: A Tissue Microarray Study of 156 Patients Treated by Transurethral Resection

To evaluate immunohistochemical expression of phosphatase and tensin homolog (PTEN) and mammalian target of rapamycin (mTOR) pathway members in pT1 urothelial carcinomas treated by transurethral resection and to determine if immunohistochemistry can be used to predict prognosis. Formalin-fixed, paraffin-embedded tissue samples from 156 patients with pT1 urothelial carcinoma treated by transurethral resection were used to build 5 tissue microarrays. Tissue microarray sections were stained for PTEN, phosphorylated (phos)-AKT, phos-mTOR, phos-S6, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), and phos-4EBP1. Patients were monitored after initial treatment (mean, 22.5; median, 16; range, 3-108 months) to detect tumor recurrence, tumor progression, or systemic metastases. During follow-up, 101 patients (65%) showed tumor recurrence, 57 showed tumor progression (37%), and 18 showed systemic metastases (12%). Patients with ≥2 lesions at the initial workup had higher proportions and higher hazard ratios of tumor recurrence, tumor progression, and systemic metastases. Complete loss of PTEN expression was observed in 6 patients (4%), and >80% of the mTOR pathway members showed at least focal positivity. Proportions of tumors with higher levels of PTEN immunohistochemical expression were higher in patients with tumor recurrence (P=.001), tumor progression (P=.05), and systemic metastases (P=.001). Proportions of tumors with lower phos-S6 and low phos-4EBP1 levels were higher in patients with tumor recurrence (P≤.05). Proportions were similar for the remaining biomarkers. Higher levels of PTEN immunohistochemical expression were associated with higher rates of tumor recurrence, tumor progression, and systemic metastases in patients with pT1 urothelial carcinomas treated by transurethral resection.

The impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen treatment in breast cancer patients

Tamoxifen is a standard therapeutical treatment in patients with estrogen receptor positive breast carcinoma. However, less than 50% of estrogen receptor positive breast cancers do not respond to tamoxifen treatment whereas 40% of tumors that initially respond to treatment develop resistance over time. The underlying mechanisms for tamoxifen resistance are probably multifactorial but remain largely unknown. The primary aim of this study was to investigate the impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen by analyzing loss of heterozygosity (LOH) and immunohystochemical expression of PTEN in 49 primary breast carcinomas of patients treated with tamoxifen as the only adjuvant therapy. The effect of PTEN inactivation on breast cancer progression and disease outcome was also analyzed. Reduced or completely lost PTEN expression was observed in 55.1% of samples, while 63.3% of samples displayed LOH of PTEN gene. Inactivation of PTEN immunoexpression significantly correlated with the PTEN loss of heterozygosity, suggesting LOH as the most important genetic mechanism for the reduction or complete loss of PTEN expression in primary breast carcinoma. Most importantly, LOH of PTEN and consequential reduction of its immunoexpression showed significant correlation with the recurrence of the disease. Besides, our study revealed that LOH of PTEN tumor suppressor was significantly associated with shorter disease free survival, breast cancer specific survival and overall survival. In summary, our results imply that LOH of PTEN could be used as a good prognostic characteristic for the outcome of breast cancer patients treated with tamoxifen.

PTEN assessment and PI3K/mTOR inhibitors: Importance of simultaneous assessment of MAPK pathway aberrations.

10510 Background: Loss of PTEN function increases PI3K/mTOR signaling. Preclinical data suggest that PTEN aberrations can predict sensitivity to drugs targeting the PI3K/mTOR pathway. Methods: Tumors from 461 patients with diverse cancers referred to the Clinical Center for Targeted Therapy were tested for cytoplasmic immunohistochemical expression of PTEN using the following scoring system: negative (no staining); positive (intact staining); reduced staining, with stroma serving as an internal positive control. Whenever possible, tumors were also tested for mutations in the MAPK pathway (KRAS, NRAS, and BRAF). We analyzed outcomes of patients treated with PI3K /mTOR with respect to their PTEN status. Results: Of 461 patients, 47 (10%) had negative PTEN expression, 168 (36.5%) had positive PTEN expression, and 246 (53.5%) had reduced PTEN. In tumor types with &gt;10 patients tested, negative PTEN was most frequent in uterine (10/33, 33%), renal (3/11, 27%), salivary gland (2/10, 20%), colorectal (15/82, 18%), breast (2/15, 13%), pancreatobiliary (3/24, 13%), and prostate cancers (2/19, 11%). Of 206 patients tested for MAPK (KRAS, NRAS, and BRAF) pathway mutations, patients with either negative or reduced PTEN had more MAPK mutations than patients with positive PTEN expression (72/144, 50% vs. 20/62, 32%, p=0.02). A total of 153 (33%) patients received therapies with PI3K /mTOR inhibitors and 17 (11%, 95% CI 0.07-0.17) achieved a partial response (PR). Proportions of PRs were 2/13 (15%) for PTEN negative, 3/50 (6%) for PTEN positive, and 12/90 (13%) for reduced PTEN. There was no significant difference in PR rate amongst patients with positive PTEN expression compared to patients with negative or reduced PTEN (3/50, 6% vs. 14/103, 14%; p=0.27). Conclusions: Complete loss of PTEN expression was found in 10% of patients. Positive PTEN expression was found in 36.5% of patients. PTEN status did not predict response to PI3K/mTOR inhibitors, perhaps because patients with negative and reduced PTEN expression had a higher incidence of simultaneous MAPK (KRAS, NRAS, BRAF) mutations.

PD10-04: Exploration of the Relationship between Loss of PTEN and BRCA1 Expression in Triple Negative Breast Carcinoma.

Abstract Based on the mechanism of action of PARP inhibitors, BRCAness breast and ovarian cancers could be defined as the optimal group of tumours to target with such drugs. As the BRCAness phenotype is not fully defined at present, a surrogate phenotype of triple negative breast cancer (TNBC) has been proposed. Results of recent clinical trials involving PARP inhibitors in the context of TNBC do not demonstrate a significant improvement in overall survival or progression free survival, partly due to the inaccurate selection of certain tumours. Data from cell culture studies suggests that loss of PTEN expression could be a marker of PARP inhibitor sensitivity. Materials and Methods: A retrospective study of 160 TNBC resected at Institut Bergonie between 1989 and 2010 was undertaken. Inclusion criteria were: ER &amp; PR immunohistochemcial (IHC) negativity (&amp;lt;10% tumour cells positive), non-amplification of HER2 (IHC score 0, 1, or 2 with negative FISH analysis) and an invasive ductal carcinoma resected before chemotherapy. For some familial cases, germline BRCA1 mutation screening had been performed in a diagnostic setting and was accompanied by genetic counselling. PTEN IHC, point mutation and gross rearrangement screening, along with BRCA1 expression and promoter methylation analysis (through real-time PCR and bisulfite treatment, respectively) and 53BP1 IHC were undertaken on this series of TNBC. Results: Twenty percent of sporadic and familial TNBC demonstrated reduced BRCA1 expression with a greater than 7 fold reduction in expression. Almost half of tumours demonstrated a complete loss of PTEN expression as assessed by IHC, with a deleterious PTEN point mutation identified in 5% of cases. Although a slight correlation is observed between the complete loss of PTEN and a reduction in BRCA1 expression, a complete overlap of these two alterations was not observed; tumours with reduced BRCA1 expression do not always demonstrate complete negativity on PTEN IHC and certain tumours displaying complete negativity on PTEN IHC do not show a reduction in BRCA1 expression. Full immunohistochemical characterization of the tumour series (ER, PR, HER2, AR, CK5/6 &amp; EGFR) together with the results of PTEN, BRCA1 and 53BP1 analyses will be presented. Conclusion These results suggest that at present, loss of PTEN expression is not a suitable surrogate marker for tumours with reduced BRCA1 expression. This should be borne in mind for ancillary studies of clinical trials involving PARP inhibitors in the treatment of breast tumours and further investigation is warranted. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-04.

Evidence for an hMSH3 defect in familial hamartomatous polyps

Patients with hamartomatous polyposis syndromes have increased risk for colorectal cancer (CRC). Although progression of polyps to carcinoma is observed, pathogenic mechanisms remain unknown. The authors examined whether familial hamartomatous polyps harbor defects in DNA mismatch repair (MMR), and assayed for somatic mutation of PTEN, a gene inactivated in the germline of some hamartomatous polyposis syndrome patients. Ten hamartomatous polyposis syndrome patients were genotyped for germline mutations. Epithelial and nonepithelial polyp DNA were assayed for microsatellite instability (MSI) and PTEN frameshift mutation. DNA MMR and PTEN protein expression were assessed in all polyps by immunohistochemistry. In addition, 99 MSI-high sporadic CRCs and 50 each of hMLH1(-/-) and hMSH3(-/-) cell clones were examined for PTEN frameshifts. Twenty-five (58%) of 43 hamartomatous polyposis syndrome polyps demonstrated dinucleotide or greater MSI in polyp epithelium, consistent with hMSH3 deficiency. MSI domains lost hMSH3 expression, and PTEN expression was lost in polyps from germline PTEN patients; sporadic hamartomatous polyps did not show any of these findings. PTEN analysis revealed wild type exon 7 and 8 sequences suggestive of nonexistent or rare events for PTEN frameshifts; however, MSI-high sporadic CRC showed 11 (11%) of 99 frameshifts within PTEN, with 4 tumors having complete loss of PTEN expression. Subcloning hMLH1(-/-) and hMSH3(-/-) cells revealed somatic PTEN frameshifts in 4% and 12% of clones, respectively. Nondysplastic epithelium from hamartomatous polyposis syndrome polyps harbors hMSH3 defects, which may prime neoplastic transformation. Polyps from PTEN(+/-) patients lose PTEN expression, but loss is not a universal early feature of all hamartomatous polyposis syndrome. However, PTEN frameshifts can occur in hMSH3-deficient cells, suggesting that hMSH3 deficiency could drive hamartomatous polyposis syndrome tumorigenesis.

Reduced/Absent PTEN Expression Is More Common in HER2 Non-Amplified DCIS.

Abstract BACKGROUND: Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway is a common event in breast cancer and appears to correlate with specific biologic subsets of disease. Laboratory models demonstrate that PI3K activation is necessary for several aspects of transformation including accelerated proliferation and invasiveness. We hypothesized that this pathway may play a role in the transition from carcinoma in situ to invasive cancer. The aim of this study was to assess whether well characterized modes of PI3K activation (PI3K mutation, PTEN loss) are present in DCIS.METHODS: 97 cases of pure high grade DCIS were identified from the MSKCC breast service database (1999-2003). Archival formalin-fixed paraffin embedded (FFPE) blocks were retrieved and appropriate sections obtained for DNA extraction and immunohistochemistry (IHC). Manual microdissection was performed to isolate pure DCIS lesions for multiplex array (Sequenom®) genotyping for PIK3CA mutations. Following antibody optimization, IHC staining for ER, PR, HER2, PTEN and pAKT were performed on freshly cut sections. HER2 amplification was confirmed by FISH. Stained sections were scored by two reviewers. ER and PR positivity was defined as any positive nuclear staining. PTEN and pAKT expression were scored relative to staining in adjacent normal ductal epithelium. Fisher's exact test was used for all comparisons.RESULTS: Of 97 cases, 47 (48%) cases were ER positive and 56 (58%) cases were HER2 amplified. Reduced PTEN expression was found in 27/97 (28%) cases and complete loss of PTEN in 8/97 (8%) cases. Overexpression of pAKT was seen in 10/97 (10%) cases. PIK3CA hotspot mutations (H1047R and E545A) were identified in 2/20 (10%) cases studied. Reduced or absent PTEN expression was more common in HER2 non-amplified DCIS as compared to HER2 amplified DCIS (22/41 (54%) vs 13/56 (23%), p=0.003). Conversely, pAKT overexpression was more common in HER2 amplified DCIS, 12% vs 7%, although this did not reach statistical significance.Exploratory analysis using IHC as a surrogate for molecular subtype demonstrated that 34 (35%) of high grade DCIS cases were luminal A, 13 (14%) cases were luminal B, 43 (44%) cases were HER2 subtype, and 7 (7%) were basal subtype. Reduced or absent PTEN expression was seen with similar frequency in HER2 negative subtypes, Luminal A (ER+HER2-) 53% and basal subtype (ER-PR-HER2-) 56%. Among HER2 amplified subtypes, overexpression of pAKT was more common in luminal B than in HER2+ cases (23% vs 10%), although this comparison is limited by small numbers. Overexpression of pAKT was not seen in the basal subtype. The PIK3CA mutations (n=2) identified were both in HER2 subtype (ER- HER2+) cases with normal PTEN expression.CONCLUSIONS: The PI3K pathway is activated in high grade DCIS and the mechanism of activation appears to correlate with molecular subtype. Reduced or complete loss of PTEN expression is more common in HER2 non-amplified high grade DCIS and occurs in both ER positive and ER negative cases. Loss of PTEN expression did not correlate with overexpression of pAKT. Preliminary data suggests that PIK3CA mutations are also an early event in tumorigenesis. Further analyses to delineate the role of this pathway in breast cancer tumorigenesis are ongoing. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6148.