PTEN Protein Loss Research Articles

PTEN loss is a context-dependent outcome determinant in obese and non-obese endometrioid endometrial cancer patients

Endometrial cancer incidence is increasing, due in part to a strong association with obesity. Mutations in the phosphatidylinositol 3-kinase (PI3K) pathway, the central relay pathway of insulin signals, occur in the majority of endometrioid adenocarcinomas, the most common form of endometrial cancer. We sought to determine the impact of PI3K pathway alterations on progression free survival in a cohort of endometrioid endometrial cancers. Prognostic utility of PIK3CA, PIK3R1, and PTEN mutations, as well as PTEN protein loss by immunohistochemistry, was explored in the context of patient body mass index. Reverse-phase protein arrays were utilized to assess protein expression based on PTEN status. Among 187 endometrioid endometrial cancers, there were no statistically significant associations between PFS and PIK3CA, PIK3R1, PTEN mutation or loss. When stratified by body mass index, PTEN loss was associated with improved progression free survival (P < 0.006) in obese (body mass index ≥ 30) patients. PTEN loss resulted in distinct protein changes: Canonical PI3K pathway activation was observed only in the non-obese population while decreased expression of β-CATENIN and phosphorylated FOXO3A was observed in obese patients. These data suggest the impact of PTEN loss on tumor biology and clinical outcomes must be interpreted in the context of body mass index, and provide a potential explanation for discrepant reports on the effect of PTEN status and obesity on prognosis in endometrial cancer. This reveals a clinically important interaction between metabolic state and tumor genetics that may unveil the biologic underpinning of obesity-related cancers and impact ongoing clinical trials with PI3K pathway inhibitors.

PI3K mutations in breast cancer: prognostic and therapeutic implications.

The PI3K pathway is the most frequently enhanced oncogenic pathway in breast cancer. Among mechanisms of PI3K enhancement, PIK3CA mutations are most frequently (∼30%) observed, along with protein loss of PTEN. Since the first discovery of PIK3CA mutations in solid malignancies in 2004, numerous studies have revealed the prognostic and therapeutic implications of these mutations. Although many issues remain unconfirmed, some have been carved in stone by the level of consistency they have shown among studies: 1) PIK3CA mutations are most likely to be observed in ER-positive/HER2-negative tumors, and are associated with other good prognostic characters; 2) PIK3CA mutations can coexist with other PI3K-enhancing mechanisms, such as HER2 amplification and PTEN protein loss; 3) PIK3CA mutations are potentially a good prognostic marker; 4) PIK3CA may predict a poorer tumor response to trastuzumab-based therapies, but its impact on disease-free survival and overall survival is uncertain; and 5) based on reports of early clinical trials, PIK3CA mutations do not guarantee a dramatic response to PI3K inhibitors. Collectively, there is currently no sufficient evidence to recommend routine genotyping of PIK3CA in clinical practice. Given that PIK3CA-mutant breast cancer appears to have a distinct tumor biology, development of more individualized targeted therapies based on the PIK3CA genotype is awaited.

Abstract P2-05-03: Anastrozole and everolimus in hormone receptor-positive metastatic breast cancer: Safety profile, activity and associations of molecular alterations in the PI3K/AKT/mTOR pathway

Abstract Background Combining aromatase inhibitors with PI3K/AKT/mTOR inhibitors in patients with hormone receptor (HR)-positive metastatic breast cancer has demonstrated clinical efficacy. There is limited data on associations between molecular signatures and activity. Patients and Methods We evaluated the combination of anastrozole and everolimus in 56 patients with HR-positive, metastatic breast cancer. The primary objective was to establish safety and maximum tolerated dose (MTD). Dose limiting toxicities (DLTs) were defined as serious grade 3 or 4 toxicities related to treatment that occurred during cycle 1. Dose level 1 was anastrozole 1mg PO QD and everolimus 5 mg PO QD and dose level 2 was anastrozole 1 mg PO QD and everolimus 10 mg PO QD (a dose level -1 included everolimus 2.5 mg PO QD). Secondary endpoints included evaluation of antitumor activity and molecular associations with response. When tissue was available, Next Generation Sequencing (NGS) was performed using genomic libraries selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of &amp;gt;500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA). An analysis was then performed for all classes of genomic alterations. Results The median age was 59 (range, 37-82) and the median number of prior therapies in the metastatic setting was 3 (range, 0-13). The initial oral daily dose of anastrozole 1 mg oral and everolimus 10 mg PO daily was well tolerated. Five dose-limiting toxicities (DLTs) were seen at full doses, including grade 3 thrombocytopenia (1 patient), grade 3 neutropenia (1 patient), grade 3 increased liver enzymes (1 patient), grade 3 hyperglycemia (1 patient) and, grade 3 mucositis (1 patient). The most common grade 3 or 4 treatment-related toxicities were neutropenia (5%), increased liver enzymes (5%), and hyperbilirubinemia (3%). Of the 56 patients on study, 36 were tested for at least one molecular alteration in the PI3K/AKT/mTOR pathway. Twelve of these 36 patients had NGS analysis of their tumor tissue. Eighteen of 36 patients (50%) tested had at least one alteration in the pathway, including mutations in PIK3CA (n=16), PIK3R1 (n=1), and AKT1 (n=2); PTEN protein loss (n=1); and, AKT3 amplification (n=1). Sixteen of 56 evaluable patients (29%) achieved stable disease (SD) /partial response (PR)/complete response (CR) ≥ 6 months (n = 3 (5%) with PR/CR). Thirteen of the 16 patients who achieved SD/PR/CR ≥ 6 months were tested for a genetic alteration in PI3K/AKT/mTOR pathway and 7 of these patients (54%) had at least one alteration in the pathway, including mutations in PIK3CA (n=6), PIK3R1 (n=1), and AKT1 (n=1); PTEN loss (n=1); and AKT3 amplification (n=1). Conclusions Combination anastrozole 1 mg and everolimus 10 mg is well tolerated and is active in heavily-pretreated patients with HR-positive breast cancer. The presence of a molecular alteration in the PI3K/AKT/mTOR pathway did not predict for clinical activity of this combination. Citation Format: Jennifer J Wheler, Filip Janku, Stacy L Moulder, Aung Naing, Sarina A Piha-Paul, Gerald S Falchook, Ralph G Zinner, Apostolia M Tsimberidou, Siqing Fu, David S Hong, Johnique T Atkins, Roman Yelensky, Vince Miller, Philip J Stephens, Vincente Valero, Funda Meric-Bernstam, Razelle Kurzrock. Anastrozole and everolimus in hormone receptor-positive metastatic breast cancer: Safety profile, activity and associations of molecular alterations in the PI3K/AKT/mTOR pathway [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-03.

MP1-17 PTEN STATUS DETERMINATION IN PROSTATE CANCER: COMPARISON OF IHC AND FISH IN A LARGE MULTI-CENTER COHORT

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2015MP1-17 PTEN STATUS DETERMINATION IN PROSTATE CANCER: COMPARISON OF IHC AND FISH IN A LARGE MULTI-CENTER COHORT Tamara L. Lotan, Carlos L. Morais, Wei Wei, Tamara Jamaspishvili, Ziding Feng, Jesse McKenney, Jeff Simko, Dean Troyer, Lawrence True, James D. Brooks, Jeremy Squire, and CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) Tamara L. LotanTamara L. Lotan More articles by this author , Carlos L. MoraisCarlos L. Morais More articles by this author , Wei WeiWei Wei More articles by this author , Tamara JamaspishviliTamara Jamaspishvili More articles by this author , Ziding FengZiding Feng More articles by this author , Jesse McKenneyJesse McKenney More articles by this author , Jeff SimkoJeff Simko More articles by this author , Dean TroyerDean Troyer More articles by this author , Lawrence TrueLawrence True More articles by this author , James D. BrooksJames D. Brooks More articles by this author , Jeremy SquireJeremy Squire More articles by this author , and CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT)CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.180AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES PTEN loss is a promising prognostic and potentially predictive biomarker in prostate cancer. Because PTEN loss in prostate cancer occurs most commonly via gene deletion, we developed and validated an inexpensive immunohistochemical (IHC) assay to detect PTEN protein loss. Here, we studied the sensitivity and specificity of this assay relative to fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in prostate cancer. METHODS 1275 primary prostate tumors from the Canary Retrospective Prostate Cancer Tissue Microarray (TMA) cohort were analyzed for PTEN loss by IHC. PTEN IHC was performed by autostaining on the Ventana Discovery Ultra platform using a rabbit monoclonal antibody (Cell Signaling, clone D4.3) and scored by two independent reviewers with very good agreement (96.4% agreement over 2783 spots; κ = 0.905; 95% CI=0.887-0.923). FISH was previously performed in the same cohort using a four color probe set (CymogenDx) yielding measurable results for 612 tumors. RESULTS By IHC, 8% (108/1275) of tumors had homogeneous PTEN loss in all sampled tumor glands, 12% (150/1275) had heterogeneous PTEN loss in a subset of sampled tumor glands, 66% (837/1275) had intact PTEN and 14% (180/1275) had ambiguous or missing immunostaining results. 551 tumors had both IHC and FISH data available for comparison. Intact PTEN immunostaining was 91% specific for absence of PTEN gene deletion by FISH, with 413/454 tumors with 2 copies of PTEN showing intact PTEN IHC. PTEN IHC loss was 98% sensitive for homozygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 48/49 homozygous tumors. PTEN IHC was 62% sensitive for detection of hemizygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 30/48 tumors with hemizygous deletion. Other inactivating mutations of PTEN (not detectable by FISH) may lead to PTEN protein loss in tumors with normal or hemizygous PTEN copy number. CONCLUSIONS Our validated and automated PTEN immunostaining protocol is a simple and relatively inexpensive test that is 91% specific and 98% sensitive for detection of homozygous PTEN gene deletions in a large multi-center study of prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e7-e8 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tamara L. Lotan More articles by this author Carlos L. Morais More articles by this author Wei Wei More articles by this author Tamara Jamaspishvili More articles by this author Ziding Feng More articles by this author Jesse McKenney More articles by this author Jeff Simko More articles by this author Dean Troyer More articles by this author Lawrence True More articles by this author James D. Brooks More articles by this author Jeremy Squire More articles by this author CAnary Pathology Team Investigating Tissue Biomarkers (CAPIT) More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP1-19 A NOVEL LIVE CELL MICROFLUIDIC DIAGNOSTIC USING PHENOTYPIC BIOMARKERS WITH OBJECTIVE ALGORITHMIC ANALYSIS FOR PROSTATE CANCER RISK STRATIFICATION.

PTEN IHC was 62% sensitive for detection of hemizygous PTEN deletion by FISH, with homogeneous or heterogeneous PTEN protein loss in 30/48 tumors with hemizygous deletion. Other inactivating mutations of PTEN (not detectable by FISH) may lead to PTEN protein loss in tumors with normal or hemizygous PTEN copy number. CONCLUSIONS: Our validated and automated PTEN immunostaining protocol is a simple and relatively inexpensive test that is 91% specific and 98% sensitive for detection of homozygous PTEN gene deletions in a large multi-center study of prostate cancer.

Prostate adenocarcinomas aberrantly expressing p63 are molecularly distinct from usual-type prostatic adenocarcinomas

We have described a rare group of prostate adenocarcinomas that show aberrant expression of p63, a protein strongly expressed in prostatic basal cells and absent from usual-type acinar prostate cancers. The partial basal-like immunophenotype of these tumors is intriguing in light of the persistent debate surrounding the cell-of-origin for prostate cancer; however, their molecular phenotype is unknown. We collected 37 of these tumors on radical prostatectomy and biopsy and assessed subsets for a diverse panel of molecular markers. The majority of p63-expressing tumors were positive for the ΔNp63 isoform (6/7) by immunofluorescence and p63 mRNA (7/8) by chromogenic in situ hybridization. Despite p63 positivity, these tumors uniformly expressed luminal-type cytokeratin proteins such as CK18 (13/13), CK8 (8/8), and markers of androgen axis signaling commonly seen in luminal cells, including androgen receptor (10/11), NKX3.1 (8/8), and prostein (12/13). Conversely, basal cytokeratins such as CK14 and CK15 were negative in all cases (0/8) and CK5/6 was weakly and focally positive in 36% (4/11) of cases. Pluripotency markers including β-catenin, Oct4, and c-kit were negative in p63-expressing tumors (0/11). Despite nearly universal expression of androgen receptor and downstream androgen signaling targets, p63-expressing tumors lacked ERG rearrangements by fluorescence in situ hybridization (0/14) and ERG protein expression (0/37). No tumors expressed SPINK1 or showed PTEN protein loss (0/19). Surprisingly, 74% (14/19) of p63-expressing tumors expressed GSTP1 protein at least focally, and 33% (2/6) entirely lacked GSTP1 CpG island hypermethylation by bisulfite sequencing. In contrast to usual prostatic adenocarcinomas, prostate tumors with p63 expression show a mixed luminal/basal immunophenotype, uniformly lack ERG gene rearrangement, and frequently express GSTP1. These data strongly suggest that p63-expressing prostate tumors represent a molecularly distinct subclass and further study of this rare tumor type may yield important insights into the role of p63 in prostatic biology and the prostate cancer cell-of-origin.

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

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

PTEN Protein Loss and Clinical Outcome from Castration-resistant Prostate Cancer Treated with Abiraterone Acetate

BackgroundLoss of the tumor suppressor phosphatase and tensin homolog (PTEN) occurs frequently in prostate cancers. Preclinical evidence suggests that activation of PI3K/AKT signaling through loss of PTEN can result in resistance to hormonal treatment in prostate cancer. ObjectiveTo explore the antitumor activity of abiraterone acetate (abiraterone) in castration-resistant prostate cancer (CRPC) patients with and without loss of PTEN protein expression. Design, setting, and participantsWe retrospectively identified patients who had received abiraterone and had hormone-sensitive prostate cancer (HSPC) and/or CRPC tissue available for PTEN immunohistochemical analysis. Outcome measurements and statistical analysisThe primary end point was overall survival from initiation of abiraterone treatment. Relationship with outcome was analyzed using multivariate Cox regression and log-rank analyses. Results and limitationsA total of 144 patients were identified who had received abiraterone post-docetaxel and had available tumor tissue. Overall, loss of PTEN expression was observed in 40% of patients. Matched HSPC and CRPC tumor biopsies were available for 41 patients. PTEN status in CRPC correlated with HSPC in 86% of cases. Loss of PTEN expression was associated with shorter median overall survival (14 vs 21 mo; hazard ratio [HR]: 1.75; 95% confidence interval [CI], 1.19–2.55; p=0.004) and shorter median duration of abiraterone treatment (24 vs 28 wk; HR: 1.6; 95% CI, 1.12–2.28; p=0.009). PTEN protein loss, high lactate dehydrogenase, and the presence of visceral metastases were identified as independent prognostic factors in multivariate analysis. ConclusionsOur results indicate that loss of PTEN expression was associated with worse survival and shorter time on abiraterone treatment. Further studies in larger and prospective cohorts are warranted. Patient summaryPTEN is a protein often lost in prostate cancer cells. In this study we evaluated if prostate cancers that lack this protein respond differently to treatment with abiraterone acetate. We demonstrated that the survival of patients with loss of PTEN is shorter than patients with normal PTEN expression.

MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

BackgroundEpithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis.MethodsWe identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach.Results17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3′-untranslated region of PTEN mRNA using a dual-luciferase reporter assay.ConclusionsMiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-799) contains supplementary material, which is available to authorized users.

Emergence, Involution, and Progression to Carcinoma of Mutant Clones in Normal Endometrial Tissues

Sporadic somatic inactivation of genes such as PTEN within histologically normal endometrium (latent precancers) is an early step in endometrial carcinogenesis. We have used clone-specific mutations of PTEN to determine the fate of latent precancers over time in women who do (high risk) and do not (low risk) develop endometrial neoplasia. PTEN immunohistochemistry was performed on 45 occurrences of endometrial neoplasia and their paired antecedent benign biopsies, along with age matched sample pairs from 167 patients who did not develop a neoplasm. When PTEN-deficient cells were present at both time points, DNA sequencing was performed to determine whether they were single or multiple independent events. Loss of PTEN protein in isolated glands was common in the initial normal biopsies of high- and low-risk groups (42% and 27%, respectively, P = 0.066). Protein-deficient glands have a tendency to disappear over time in low-risk women (P = 0.047) and, even when "persistent," are infrequently (19%, 3/16) confirmed to be the same clone. Similarly, only a small proportion (6.7%, 1/15) of latent precancers seen in high-risk women are the direct progenitors of subsequent neoplasia. There is a high rate of latent precancer turnover in both low- and high-risk patients, with rare long-term persistence of unique clones, which may or may not progress to a histologic lesion. The temporal dynamics of clonal emergence, persistence, and involution are sufficiently complex that in the individual patient, the presence of a latent precancer has an unknown contribution to long-term cancer risk.

PTEN Regulates BCRP/ABCG2 and the Side Population through the PI3K/Akt Pathway in Chronic Myeloid Leukemia

A small population of cancer stem cells named the “side population” (SP) has been demonstrated to be responsible for the persistence of many solid tumors. However, the role of the SP in leukemic pathogenesis remains controversial. The resistance of leukemic stem cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ATP-binding cassette sub-family G member 2 (ABCG2), is well known as a specific marker of the SP and could be controlled by several pathways, including the PI3K/Akt pathway. Our data demonstrated that compared with wild-type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated phosphatase and tensin homologue deleted on chromosome -10 (PTEN) and activated phosphorylated-Akt (p-Akt). PTEN and p-Akt downregulation could be abrogated by both the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin. Moreover, in CML patients in the accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression was accompanied by the loss of PTEN protein and the up-regulation of p-Akt expression. These results suggested that the expression of ABCG2 and the SP may be regulated by PTEN through the PI3K/Akt pathway, which would be a potentially effective strategy for targeting CML stem cells.

Rb Loss Is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma

Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide the diagnosis and therapy of this aggressive tumor. We examined the status of RB1, TP53, and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy-number alteration analysis, and sequencing of formalin-fixed paraffin-embedded specimens. We found retinoblastoma (Rb) protein loss in 90% of small cell carcinoma cases (26 of 29) with RB1 allelic loss in 85% of cases (11 of 13). Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3 of 7) showed Rb protein loss. In contrast, only 7% of primary high-grade acinar carcinomas (10 of 150), 11% of primary acinar carcinomas with neuroendocrine differentiation (4 of 35), and 15% of metastatic castrate-resistant acinar carcinomas (2 of 13) showed Rb protein loss. Loss of PTEN protein was seen in 63% of small cell carcinomas (17 of 27), with 38% (5 of 13) showing allelic loss. By IHC, accumulation of p53 was observed in 56% of small cell carcinomas (14 of 25), with 60% of cases (6 of 10) showing TP53 mutation. Loss of RB1 by deletion is a common event in prostatic small cell carcinoma and can be detected by a validated IHC assay. As Rb protein loss rarely occurs in high-grade acinar tumors, these data suggest that Rb loss is a critical event in the development of small cell carcinomas and may be a useful diagnostic and potential therapeutic target.

Association of PTEN protein loss with upgrading of prostate cancer from biopsy to radical prostatectomy.

127 Background: Active surveillance is increasingly recommended for men with low-risk Gleason Score 3+3=6 (GS 6) prostate cancer. Yet, approximately one-third of patients with GS 6 cancer on biopsy are upgraded to higher GS at radical prostatectomy (RP). Previous studies have shown that clinical-pathologic parameters (age, prostate-specific antigen [PSA], prostate volume, extent of disease) are weak predictors of GS6 tumor upgrade. Our goal was to investigate the utility of PTEN as a molecular marker to predict upgrading in GS 6 biopsies. Methods: In a retrospective case-control study, 71 patients with GS 6 tumors on needle biopsy that were upgraded to GS 7 or higher cancer at RP (cases) were compared to 103 patients whose GS 6 tumors on needle biopsy were not upgraded at RP (controls). The most extensively involved needle core biopsy from each case was immunostained and scored for PTEN protein loss using a previously validated immunohistochemical (IHC) assay and binary scoring system. Confirmatory fluorescence in situ hybridization (FISH) was used to assess for PTEN gene deletion in biopsies with PTEN protein loss. The correlation of upgrading with PTEN loss and with clinical-pathologic variables was assessed by logistic regression. Results: Patients with upgraded cancers were older than controls (61.8 vs. 59.3 years), had higher mean pre-operative PSA levels (6.53 vs. 5.26 ng/mL), and a higher fraction of biopsy cores involved by tumor (0.42 vs, 0.36). However, of all pathologic variables, PTEN protein loss by IHC was most predictive of upgrading. Overall, PTEN protein loss was found in 18.3% (13 out of 71) of upgraded cases compared to 6.8% (7 out of 103) of controls (p=0.02). In the cases with PTEN protein loss, FISH confirmed homozygous PTEN deletion in 90% (9 out of 10) of upgraded tumors compared to 67% (4 out of 6) of interpretable not upgraded controls. On multivariate analyses, even after adjusting for age, preoperative PSA, clinical stage and race, GS 6 tumors with PTEN protein loss on biopsy were significantly more likely to be upgraded at RP compared to those without PTEN loss with odds ratio (OR) = 3.04 (1.08-8.55; p=0.035). Conclusions: In prostate needle biopsies, PTEN IHC may help distinguish men with low risk cancer from men with intermediate or higher risk cancers.

Abstract P4-12-28: HER2-related biomarkers in HER2+ breast cancer patients in Asia Pacific

Abstract Recent findings suggest that HER2-related molecular markers such as PTEN deletion or downregulation, PIK3CA mutation, truncated HER2 receptor (p95HER2), and tumor HER2 mRNA levels, have the potential to predict anti-HER2 treatment response. We evaluated the distribution of these biomarkers at the time of primary diagnosis and their relationship to responsiveness to lapatinib treatment in the metastatic setting in HER2+ breast cancer patients. We conducted an observational study of female HER2+ breast cancer patients who were initiated on lapatinib treatment following recurrence or metastases in five Asia Pacific countries. Patients were enrolled between August 2010 and December 2012. Eligible patients had a tumor biopsy specimen available from their primary breast cancer diagnosis or before they started on any anti-HER2 treatment, had not been exposed to more than two lines of anti-HER2 treatment in the metastatic setting or other experimental anti-HER2 treatment, and had no other primary tumor. Biomarkers levels at primary diagnosis were measured; PTEN levels were assessed by immunohistochemistry and PIK3CA mutations were detected by a mass spectroscopy-based approach. The primary endpoint was progression-free survival (PFS) from the initiation of first lapatinib-based regimen given in metastatic setting to disease progression from that regimen or death from any cause. PFS analysis was conducted with a data cut-off date of 31 December 2012. A total of 162 patients were included in this study and 96% have confirmed HER2+ breast cancer primary tumor. The mean age was 52±10 years and 97% had metastases at study entry, with bone being the most common site of metastasis (48%). About a quarter had PTEN protein loss (24%), 30% had PIK3CA mutation, and 7% had both at primary diagnosis. No significant association was observed between both biomarkers or between each biomarker and estrogen receptor status or HER2 status. Table 1. Relationship between PTEN and PIK3CA PIK3CA mutation, n (%)PIK3CA wild-type, n (%)p-valuePTEN status 0.674Loss11 (29)27 (71) Normal33 (33)68 (67) Patients with altered PTEN expression, or PIK3CA mutation showed comparable PFS with lapatinib-based treatment as those with normal PTEN or PIK3CA expression at analysis cut-off date (median PFS 7.5 and 8.5 months respectively vs. 8.9 and 9.0 months respectively; p = 0.502 and p = 0.268 respectively). There remained no significant difference in PFS after having adjusted for significant confounders (HR 1.2 and 1.1 respectively; 95% CI 0.7–1.9 and 0.7–1.8 respectively; p = 0.481 and p = 0.730 respectively). The distribution of p95HER2 expression and tumor HER mRNA levels and their association with PFS will be included at the time of presentation. Our preliminary findings suggest that PTEN alteration, or PIK3CA mutation may not be predictive of clinical response to lapatinib treatment in HER2+ breast cancer patients. The final PFS results with additional markers will provide more clues regarding their relationship to treatment response. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-28.

PI3K Pathway Activation Provides a Novel Therapeutic Target for Pediatric Ependymoma and Is an Independent Marker of Progression-Free Survival

Currently, there are few effective adjuvant therapies for pediatric ependymoma outside confocal radiation, and prognosis remains poor. The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in cancer. PI3Ks transduce signals from growth factors and cytokines, resulting in the phosphorylation and activation of AKT, which in turn induces changes in cell growth, proliferation, and apoptosis. PI3K pathway status was analyzed in ependymoma using gene expression data and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein expression and the mutation status of PI3K catalytic subunit α-isoform gene (PIK3CA) was investigated. Genes in the pathway displayed significantly higher expression in supratentorial than in posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway activation, was seen in 72% of tumors (n = 169) and P-AKT expression was found to be an independent marker of a poorer progression-free survival. A significant association between PI3K pathway activation and cell proliferation was identified, suggesting that pathway activation was influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations were identified in PIK3CA. Our results suggest that the PI3K pathway could act as a biomarker, not only identifying patients with a worse prognosis but also those that could be treated with therapies targeted against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

PTEN Regulated BCRP/ABCG2 and Side Population Through PI3K/Akt Pathway In Chronic Myeloid Leukemia

A small population of cancer stem cells named as “side population (SP)” has been demonstrated to be responsible for many solid tumor maintenance. However, the role of SP in leukemic pathogenesis is still controversial. The resistance of leukemic stem cells in response to targeted therapies such as tyrosine kinase inhibitors (TKIs) results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ABCG2, is well-known as a specific marker of SP and could be controlled by several pathways including PI3K/Akt. Our data demonstrated that compared to wild type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated PTEN and activated p-Akt. It could be abrogated by both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin. Moreover, in CML patients at accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression accompanied with loss of PTEN protein and up-regulation of p-Akt expression was observed. These results suggested that expression of ABCG2 and fraction of SP may be regulated by PTEN through PI3K/Akt pathway, which will be the potentially effective strategy for targeting CML stem cells. Disclosures:No relevant conflicts of interest to declare.

Molecular alterations of Ras‐Raf‐mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase‐Akt signaling pathways in colorectal cancers from a tertiary hospital at Kuala Lumpur, Malaysia

Molecular alterations in KRAS, BRAF, PIK3CA, and PTEN have been implicated in designing targeted therapy for colorectal cancer (CRC). The present study aimed to determine the status of these molecular alterations in Malaysian CRCs as such data are not available in the literature. We investigated the mutations of KRAS, BRAF, and PTEN, the gene amplification of PIK3CA, and the protein expression of PTEN and phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110α) by direct DNA sequencing, quantitative real-time PCR, and immunohistochemistry, respectively, in 49 CRC samples. The frequency of KRAS (codons 12, 13, and 61), BRAF (V600E), and PTEN mutations, and PIK3CA amplification was 25.0% (11/44), 2.3% (1/43), 0.0% (0/43), and 76.7% (33/43), respectively. Immunohistochemical staining demonstrated loss of PTEN protein in 54.5% (24/44) of CRCs and no significant difference in PI3K p110α expression between CRCs and the adjacent normal colonic mucosa (p = 0.380). PIK3CA amplification was not associated with PI3K p110α expression level, but associated with male cases (100% of male cases vs 56% of female cases harbored amplified PIK3CA, p = 0.002). PI3K p110α expression was significantly higher (p = 0.041) in poorly/moderately differentiated carcinoma compared with well-differentiated carcinoma. KRAS mutation, PIK3CA amplification, PTEN loss, and PI3K p110α expression did not correlate with Akt phosphorylation or Ki-67 expression. KRAS mutation, PIK3CA amplification, and PTEN loss were not mutually exclusive. This is the first report on CRC in Malaysia showing comparable frequency of KRAS mutation and PTEN loss, lower BRAF mutation rate, higher PIK3CA amplification frequency, and rare PTEN mutation, as compared with published reports.

PTEN loss as a context-dependent determinant of patient outcomes in obese and non-obese endometrioid endometrial cancer patients.

5521 Background: Aberrations in the PI3K pathway, the central relay pathway of insulin signals, occur in the majority of endometrioid endometrial cancers. We explored the prognostic utility of PIK3CA, PIK3R1, and PTEN mutations, as well as PTEN protein loss, in the context of patient weight. Methods: Patients (pts) treated for endometrial cancer at a single institution between 2000 and 2009 were identified. Tumor DNA was extracted and exome sequencing performed using a 454 platform with confirmation of hot spot mutations by Sequenom. PTEN protein expression was determined by immunohistochemistry and reverse phase protein array (RPPA). RPPA for 135 relevant proteins was performed using a GeneTAC arrayer to create spot arrays. Slides were scanned, analyzed, and quantitated using Microvigene software. Results: One hundred eighty seven endometrioid endometrial cancer specimens were included. Median age was 61 yrs and median body mass index (BMI) was 33.5 kg/m2. The majority of pts had early stage (I/II) disease (74%) and grade 2 tumors (66%). There were no statistically significant associations between progression free survival (PFS) and PIK3CA, PIK3R1, PTEN mutation or loss. However, when stratified by BMI, PTEN loss was associated with a significantly improved PFS (p&lt; 0.006) in obese (BMI &gt; 30 kg/m2) pts. In contrast, PTEN loss was associated with a worse PFS (p&lt;0.06) in non-obese (BMI &lt; 30 kg/m2) pts. Further, PTEN loss in obese and non-obese pts resulted in distinct protein changes by RPPA, with canonical PI3K pathway activation observed only in the non-obese PTEN loss cohort. PTEN loss in obese pts was associated with decreased expression of CATENIN and phosphorylated FOXO3A. Conclusions: These data suggest the impact of PTEN loss on tumor biology and clinical outcomes must be interpreted in the context of BMI and provide potential explanation for prior discrepant findings on effect of PTEN status on prognosis in endometrial cancer. These data describe a clinically important interaction between metabolic state and tumor genetics that could potentially unveil the biologic underpinning of obesity-related cancers and may be relevant to ongoing clinical trials with PI3K pathway inhibitors.

Abstract IA17: Exploiting the synthetic lethal properties of selective PI3K-β inhibition in PTEN deficient cells with GSK2636771

Abstract The phosphoinositide 3-kinase (PI3K) pathway is among the most commonly mutated pathways in human cancer. The majority of first-generation PI3K inhibitors currently in clinical trials inhibit multiple PI3K isoforms. The PIK3CB gene encodes p110β isoform, which has been reported to be required for pathway activation in phosphatase and tensin homolog (PTEN) deficient cancer cells. The observed dependence of PTEN-deficient tumors on p110β provides an opportunity for identification of a novel isoform-selective therapeutic with a synthetic lethal effect. During an extensive knowledge-based lead identification and optimization effort, we identified substituted benzimidazole GSK2636771 as a potent, orally bioavailable, PI3Kβ-selective inhibitor. The observed enzymatic potency for PI3Kβ is approximately 1000 and 10 fold higher than the potency against PI3Kα and PI3δ respectively. Pre-clinical data demonstrate the synthetic lethal relationship between PTEN deficiencies and PI3K-β inhibition. PTEN-deficient cells dosed with GSK2636771 show concentration dependent decreases in cellular levels of phosphorylated AKT (pAKT), and PRAS40 both in-vitro and in-vivo. A 3-day proliferation assay performed on a large panel of cell lines (n=80) grown in-vitro demonstrates selective growth inhibition in PTEN-deficient cells at lower concentrations of GSK2636771. In-vivo studies in PTEN-deficient prostate cancer models also show dose dependent tumor growth delay. PTEN is commonly mutated, deleted, or epigenetically silenced in a wide range of solid tumors. Loss of PTEN protein is particularly frequent in prostate (50%), endometrial (57%), and triple negative breast cancers (30%). Prospective selection of patients with this protein loss serves as a development path through the course of study P3B115717 (NCT01458067), a Phase I/IIa first-time-in-human study with GSK2363771. This study is currently in dose-escalation.β Citation Format: Joel Greshock. Exploiting the synthetic lethal properties of selective PI3K-β inhibition in PTEN deficient cells with GSK2636771. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA17.

Abstract 39: Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods.

Abstract Background: PTEN is a tumor suppressor that negatively regulates the PI3K signaling pathway by dephosphorylating PIP3, converting it to PIP2. PTEN loss of function results in activation of Akt and downstream signaling pathways, and has been associated with numerous cancers, most commonly endometrial cancer, glioblastoma, melanoma and prostate cancer. PTEN loss can be a result of multiple different aberrations which include mutations in the coding region leading to frame-shift or stop codons, genomic deletions as well as promoter methylation leading to loss of PTEN protein. Determination of PTEN status, both at the DNA and protein levels, may be important for clinical decision-making because loss of PTEN is associated with resistance to various targeted therapies, while inactivating mutations may confer sensitivity to therapeutics targeting PI3K pathway members. We have developed Sanger sequencing, immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) assays to assess PTEN mutation status in a set of FFPE patient specimens. Methods: Our Sanger sequencing assay for PTEN was developed to detect known hotspot mutations in exons 5-8, which occur in the majority of PTEN mutations reported in the COSMIC database. The IHC and CISH assays were developed using commercially available reagents. All assays were validated using a combination of plasmids, cell lines and FFPE tissues for specificity, sensitivity, reproducibility and concordance. Results: 22 patient FFPE tissue specimens from a variety of tumor types including colon, lung, pancreas, uterus and prostate were evaluated for PTEN mutation and expression status by both Sanger sequencing and IHC methods. Sequencing results were: 9 wild-type, 12 heterozygous mutant, and 1 hemizygous mutant. 7 of the 9 specimens reported as wild-type by sequencing exhibited normal protein expression levels by IHC, while 2 were PTEN negative by IHC, suggesting that mechanisms other than mutations in exons 5-8 contributed to the loss of expression of PTEN protein. These 2 specimens were further analyzed by CISH to determine copy number. Of the 13 specimens reported as mutant by sequencing, IHC protein expression results indicated that 6 were PTEN positive and 7 were PTEN negative, underscoring the observation that exon 5-8 mutation status alone is not sufficient to predict PTEN expression levels. Data on CISH analysis of the specimen that was hemizygous mutant by sequencing will be reported to confirm the copy loss. Conclusion: The data suggest that additional mutations outside the exon 5-8 region as well as promoter methylation may result in loss of PTEN protein, and that accurate clinical assessment of PTEN mutation and protein expression status in FFPE tissues requires complimentary methods including Sanger sequencing, IHC and CISH methodologies. Citation Format: Kellen Sakrison, Jennifer Wright, Eric Bruening, Stephane Wong, Cynthia Spittle, Sabita Sankar, Chad Galderisi. Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2013-39