PTEN Immunohistochemistry Research Articles

Loss of PTEN expression as diagnostic marker of endometrial precancer: A systematic review and meta-analysis.

Endometrial hyperplasia may be either a benign proliferation or a premalignant lesion. In order to differentiate these two conditions, two possible histologic classifications can be used: the World Health Organization (WHO) classification and the endometrial intraepithelial neoplasia (EIN) classification. The 2017 European Society of Gynaecological Oncology guidelines recommend the use of immunohistochemistry for tumor suppressor protein phosphatase and tensin homolog (PTEN) to improve the differential diagnosis. Nonetheless, its diagnostic accuracy has never been defined. We aimed to assess the diagnostic accuracy of immunohistochemistry for PTEN in the differential diagnosis between benign and premalignant endometrial hyperplasia. Electronic databases were searched from their inception to May 2018 for studies assessing immunohistochemical expression of PTEN in endometrial hyperplasia specimens. PTEN status ("loss" or "presence") was the index test; histological diagnosis ("precancer" or "benign") was the reference standard. Sensitivity, specificity, positive and negative likelihood ratios (LR+, LR-), diagnostic odds ratio (DOR), and area under the curve (AUC) on summary receiver operating characteristic curves were calculated (95% CI), with a subgroup analysis based on the histologic classification adopted (WHO vs EIN). Twenty-seven observational studies with 1736 cases of endometrial hyperplasia were included. Pooled estimates showed low diagnostic accuracy: sensitivity 54% (95% CI 50%-59%), specificity 66% (63%-69%), LR+ 1.55 (1.29-1.87), LR- 0.72 (0.62-0.83), DOR 3.56 (2.02-6.28), AUC 0.657. When the WHO subgroup was compared with the EIN subgroup, higher accuracy (AUC 0.694 vs. 0.621), and higher heterogeneity in all analyses, were observed. Immunohistochemistry for PTEN showed low diagnostic usefulness in the differential diagnosis between benign and premalignant endometrial hyperplasia. In the absence of further evidence, the recommendation about its use should be reconsidered.

Opposite Prognostic Impact of Single PTEN-loss and PIK3CA Mutations in Early High-risk Breast Cancer.

PTEN-loss and PIK3CA mutations have been addressed as markers of PI3K activation in breast cancer. We evaluated these markers in early high-risk breast cancer (EBC) focusing on PTEN immunohistochemistry (IHC) issues, particularly in HER2-positive disease. We examined PTEN-loss and PIK3CA mutations in 1265 EBC patients treated with adjuvant chemotherapy within two clinical trials. Two different methods for the evaluation of PTEN IHC were used, one upfront binary (loss; no-loss) and the other initially multi-scale allowing for the classification of "grey zone" tumors with low and very low PTEN protein expression. PTEN-loss (33.4% and 22.1%, depending on the IHC method) and PIK3CA mutations (29.6%) were associated with ER/PgR/HER2-negative and ER/PgR-positive disease, respectively. Concordance of the two IHC methods was moderate (Cohen's kappa 0.624). PTEN-loss discrepancy and intra-tumor heterogeneity concerned "grey zone" tumors that were prevalent among HER2-positive cancers. PTEN-loss independently conferred higher risk for relapse and death. Compared to single PIK3CA mutations,single PTEN-loss was independently associated with increased risk for relapse and death. Depending on the evaluation method, in HER2-positive cancer, PTEN-loss was without- or of marginal unfavorable prognostic significance. In EBC, PTEN-loss is an independent predictor of poor outcome. When occurring singly, PTEN-loss and PIK3CA mutations have opposite prognostic impact. In HER2-positive disease, assessment of PTEN-loss by IHC appears unreliable and the marker is without clear prognostic significance.

Iatrogenic endometriosis harbors somatic cancer-driver mutations.

Does incisional endometriosis (IE) harbor somatic cancer-driver mutations? We found that approximately one-quarter of IE cases harbor somatic-cancer mutations, which commonly affect components of the MAPK/RAS or PI3K-Akt-mTor signaling pathways. Despite the classification of endometriosis as a benign gynecological disease, it shares key features with cancers such as resistance to apoptosis and stimulation of angiogenesis and is well-established as the precursor of clear cell and endometrioid ovarian carcinomas. Our group has recently shown that deep infiltrating endometriosis (DE), a form of endometriosis that rarely undergoes malignant transformation, harbors recurrent somatic mutations. In a retrospective study comparing iatrogenically induced and endogenously occurring forms of endometriosis unlikely to progress to cancer, we examined endometriosis specimens from 40 women with IE and 36 women with DE. Specimens were collected between 2004 and 2017 from five hospital sites in either Canada, Germany or the Netherlands. IE and DE cohorts were age-matched and all women presented with histologically typical endometriosis without known history of malignancy. Archival tissue specimens containing endometriotic lesions were macrodissected and/or laser-capture microdissected to enrich endometriotic stroma and epithelium and a hypersensitive cancer hotspot sequencing panel was used to assess for presence of somatic mutations. Mutations were subsequently validated using droplet digital PCR. PTEN and ARID1A immunohistochemistry (IHC) were performed as surrogates for somatic events resulting in functional loss of respective proteins. Overall, we detected somatic cancer-driver events in 11 of 40 (27.5%) IE cases and 13 of 36 (36.1%) DE cases, including hotspot mutations in KRAS, ERBB2, PIK3CA and CTNNB1. Heterogeneous PTEN loss occurred at similar rates in IE and DE (7/40 vs 5/36, respectively), whereas ARID1A loss only occurred in a single case of DE. While rates of detectable somatic cancer-driver events between IE and DE are not statistically significant (P > 0.05), KRAS activating mutations were more prevalent in DE. Detection of somatic cancer-driver events were limited to hotspots analyzed in our panel-based sequencing assay and loss of protein expression by IHC from archival tissue. Whole genome or exome sequencing, or epigenetic analysis may uncover additional somatic alterations. Moreover, because of the descriptive nature of this study, the functional roles of identified mutations within the context of endometriosis remain unclear and causality cannot be established. The alterations we report may be important in driving the growth and survival of endometriosis in ectopic regions of the body. Given the frequency of mutation in surgically displaced endometrium (IE), examination of similar somatic events in eutopic endometrium, as well as clinically annotated cases of other forms of endometriosis, in particular endometriomas that are most commonly linked to malignancy, is warranted. This study was funded by a Canadian Cancer Society Impact Grant [701603, PI Huntsman], Canadian Institutes of Health Research Transitional Open Operating Grant [MOP-142273, PI Yong], the Canadian Institutes of Health Research Foundation Grant [FDN-154290, PI Huntsman], the Canadian Institutes of Health Research Project Grant [PJT-156084, PIs Yong and Anglesio], and the Janet D. Cottrelle Foundation through the BC Cancer Foundation [PI Huntsman]. D.G. Huntsman is a co-founder and shareholder of Contextual Genomics Inc., a for profit company that provides clinical reporting to assist in cancer patient treatment. R. Aguirre-Hernandez, J. Khattra and L.M. Prentice have a patent MOLECULAR QUALITY ASSURANCE METHODS FOR USE IN SEQUENCING pending and are current (or former) employees of Contextual Genomics Inc. The remaining authors have no competing interests to declare. Not applicable.

Effects of Molecular Heterogeneity on Survival of Patients With BRAFV600-Mutated Melanoma Treated With Vemurafenib With or Without Cobimetinib in the coBRIM Study.

The treatment of advanced BRAFV600-mutated melanomas with BRAF inhibitors (BRAFi) has improved survival, but the efficacy of BRAFi varies among individuals and the development of acquired resistance to BRAFi through reactivation of mitogen-activated protein kinase (MAPK) signaling is common. We performed an exploratory, retrospective analysis to investigate the effects of BRAFV600 allelic balance, coexisting oncogene mutations, cell proliferation signaling levels, and loss of PTEN expression on progression-free survival (PFS) in patients in the phase III coBRIM study, which compared the combination of the MEK inhibitor cobimetinib with the BRAFi vemurafenib versus vemurafenib as monotherapy. Baseline tumor samples from the intention-to-treat population were analyzed by targeted deep sequencing at a median coverage of 3,600× and by immunohistochemistry for cell proliferation markers, BRAFV600E, and PTEN. The association of these biomarkers with PFS was assessed by Cox proportional hazards modeling. Gene expression in relation to loss of PTEN was profiled by RNA sequencing in 205 patient samples and 42 BRAFV600-mutated melanoma cell lines. Neither BRAFV600 allelic balance nor coexisting mutations in the RAS/RAF/RTK pathway affected PFS in either treatment group. Increased baseline MAPK signaling and cell proliferation did not affect PFS in patients treated with cobimetinib combined with vemurafenib. PTEN loss was associated with reduced PFS in patients treated with vemurafenib alone but not in patients treated with cobimetinib combined with vemurafenib. Deeper inhibition of the MAPK pathway through targeting of both MEK and BRAF may override the effects of tumor heterogeneity and improve PFS in all patients with advanced BRAFV600 melanoma.

Effect of Preanalytic Variables on an Automated PTEN Immunohistochemistry Assay for Prostate Cancer.

Phosphatase and tensin homolog (PTEN) is a promising prognostic and potentially predictive biomarker in prostate cancer. To assess the effects of preanalytic variables on an analytically validated and fully automated PTEN immunohistochemistry assay. PTEN immunohistochemistry was performed on Ventana immunostaining systems. In benign prostate tissues, immunostaining intensity across variable conditions was assessed by digital image analysis. In prostate tumor tissues, immunostaining was scored visually. Delay of fixation for 4 hours or longer at room temperature or 48 hours or longer at 4°C and duration of formalin fixation did not significantly alter immunostaining intensity. Intensity of staining was highest in 10% formalin compared with other fixatives. Tumor tissues with PTEN loss processed using protocols from 11 academic institutions were all evaluable and scored identically. PTEN immunostaining of needle biopsies where tissue blocks had been stored for less than 10 years was more frequently scored as nonevaluable compared with blocks that had been stored for 10 years or longer. This effect was less evident for radical prostatectomy specimens, where low rates of nonevaluable staining were seen for 23 years or more of storage. Storage of unstained slides for 5 years at room temperature prior to immunostaining resulted in equivalent scoring compared with freshly cut slides. Machine-to-machine variability assessed across 3 Ventana platforms and 2 institutions was negligible in 12 tumors, and platform-to-platform variability was also minor comparing Ventana and Leica instruments across 77 tumors (κ = 0.926). Automated PTEN immunostaining is robust to most preanalytic variables in the prostate and may be performed on prostate tumor tissues subjected to a wide range of preanalytic conditions. These data may help guide assay development if PTEN becomes a key predictive biomarker.

838P - PI3K/AKT pathway deleterious mutations in lethal prostate cancer

Background: PI3K/AKT signaling is commonly hijacked in prostate cancer (PC), associating with poorer prognosis and worse response to next generation hormonal treatment (NGHT) including abiraterone and enzalutamide. We studied deleterious pathway mutations in metastatic castration resistant PC (mCRPC) patients correlating these to prognosis and response to NGHT in a case-control study. Methods: Patients with mCRPC and available treatment-naïve and/or CRPC tumor samples were evaluated. Mutation analyses involved MiSeqTM based targeted next generation sequencing (NGS); PTEN was assessed by immunohistochemistry (IHC) with the control cohort having no PTEN IHC loss. Correlations between pathway mutations and outcomes and response to NGHT utilized Cox and Kaplan-Meier analyses. Results: Overall 418 samples (from Feb/15 to Dec/17) were sequenced by targeted NGS; 46 (11%) had mutated pathway genes of which 26 (6%) (PIK3CA, n = 18; AKT1, n = 6; PIK3R1, n = 2) were pathogenic pathway activating mutations. Among these 25 were previously described, while an undescribed PIK3R1 mutation (R543*), located in the iSH2 domain, was also identified. We then randomly selected 56 tumour samples with normal PTEN expression by IHC as a control group without detected pathway aberrations. Overall, 43% (10/23) of patients with mutated cancers had NGHT in pre-, and 57% (13/23) in post-chemotherapy settings, while 3 were NGHT naïve. In the control group, 14 patients (25%) were pre-chemotherapy while 42 (75%) post-chemotherapy. Deleterious pathway mutations were associated with a shorter median overall survival (2.8 vs 4.3 years; HR: 2.73; p < 0.001) and median duration of NGHT (5.9 vs 10.0 mo; p < 0.001), despite a higher proportion of post-chemotherapy patients in the control group. Conclusions: PI3K/AKT pathway deleterious mutations impart a poor prognosis from mCRPC and are associated with shorter responses to NGHT. We envision that these data can impact treatment selection in a targeted treatment approach for mCRPC. Legal entity responsible for the study: The Institute of Cancer Reserach. Funding: CRUK, Movember, PCF, PCUK. Disclosure: All authors have declared no conflicts of interest.

Computer-Based Intensity Measurement Assists Pathologists in Scoring Phosphatase and Tensin Homolog Immunohistochemistry — Clinical Associations in NSCLC Patients of the European Thoracic Oncology Platform Lungscape Cohort

IntroductionPhosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3-kinase activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. MethodsAfter an external quality assessment using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n = 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. ResultsAll three antibodies differentiated six PTEN+ versus six PTEN- cases on external quality assessment. For 138G6 and SP218, high sensitivity and specificity was found for all H-score threshold values including prospectively defined 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Because of over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cutoff values, PTEN- was associated with smoking history, squamous cell histology, and higher tumor stage (p < 0.001). In adenocarcinomas, PTEN- was associated with poor survival. ConclusionCalibration of immunoreactivity intensities by pathologists following computerized H-score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies.

Genomic Profiling of HER2-Positive Gastric Cancer: PI3K/Akt/mTOR Pathway as Predictor of Outcomes in HER2-Positive Advanced Gastric Cancer Treated with Trastuzumab.

HER2-positive gastric cancer (GC) affects 7%-34% of patients with GC. Trastuzumab-based first-line treatment has become the standard of care for HER2-positive advanced gastric cancer (AGC). However, there are no clinically validated biomarkers for resistance to HER2-targeted therapies. Upregulation of PI3K pathway and tyrosine kinase receptor (TKR) alterations have been noted as molecular mechanisms of resistance in breast cancer. Our study aimed to perform a molecular characterization of HER2-positive AGC and investigate the role of PI3K/Akt/mTOR signaling pathway activation and TKR gene copy number (GCN) gains as predictive biomarkers in HER2-positive AGC treated with trastuzumab. Forty-two HER2-positive GC samples from patients treated with trastuzumab-based first-line chemotherapy were selected. DNA samples were sequenced. PTEN and MET immunohistochemistry were also performed. Concurrent genetic alterations were detected in 97.1% of HER2-positive AGC. We found activation of PI3K/Akt/mTOR pathway in 52.4% of patients and TKR GCN gains in 38.1%. TKR GCN gains did not correlate with overall survival (OS) or progression-free survival (PFS). Multivariate Cox models showed that PI3K/Akt/mTOR activation negatively affects the effectiveness of trastuzumab-based chemotherapy in terms of OS and PFS. Our results provide for the first time a detailed molecular profile of concurrent genetic alterations in HER2-positive AGC. PI3K pathway activation could be used as a predictive marker of worse outcome in this patient population. In addition, gains in copy number of other TKR genes in this subgroup may also influence the survival benefit obtained with trastuzumab. This article reports, for the first time, a detailed molecular profile of genomic alterations in patients with HER2-positive advanced gastric cancer (AGC). PI3K/Akt/mTOR signaling pathway activation seems to have a differentially negative effect on overall survival and progression-free survival in AGC treated with trastuzumab-based chemotherapy. Combining different targeted agents could be a successful therapeutic strategy to improve the prognosis of HER2-positive AGC.

Regulation of the tumor suppressor PLZF and prostate cancer prognosis.

137 Background: PLZF ( ZBTB16) is an androgen-regulated tumor suppressor gene. Homozygous PLZF deletions are seen in a proportion of localized as well as castration-resistant prostate cancers (PC). Since PTEN has been shown to regulate PLZF expression in vitro, we hypothesized that PTEN status impacts PLZF expression in men with PC. We further hypothesized that low PLZF is associated with a poorer PC prognosis. Methods: We studied patients diagnosed with PC during prospective follow-up of the Health Professionals Follow-up Study (HPFS; n = 254) and the Physicians’ Health Study (PHS; n = 150). We performed whole transcriptome profiling of tumor specimens from cancer diagnosis and, in a subset of patients ( n = 253), a genomically-validated immunohistochemistry for PTEN. Patients were followed for metastases and PC-specific mortality (lethal cancer) after primary treatment for a median of 14 years. Univariable linear and multivariable logistic regression models for cancer outcomes, adjusted for age and calendar year, were used. For validation, the association of PTEN copy number variations and PLZF mRNA expression was assessed in The Cancer Genome Atlas (TCGA) primary PC cohort (n = 333). Results: Loss of PTEN protein expression was associated with lower PLZF expression (–0.72 standard deviations [SD]; 95% CI, –0.38 to –1.06; p &lt; 0.001; HPFS and PHS cohorts), as were copy number variations in PTEN (TCGA cohort; –0.46 SD for deep deletions vs. diploid status; 95% CI, –0.13 to –0.79; p = 0.006) . PLZF expression did not differ significantly by Gleason grade in any cohort (all p &gt; 0.09). In both the HPFS and PHS cohorts, the 25% of patients with the lowest PLZF expression were significantly more likely than those with higher expression to have progression to lethal cancer. This association was independent from Gleason grade, PTEN loss, and a signature of androgen receptor signaling (adjusted odds ratio for lethal cancer, 2.69; 95% CI, 1.18 to 6.14; p = 0.019). Conclusions: Loss of PTEN expression is associated with lower expression of PLZF amongst primary PC not exposed to ADT. In line with its action as a tumor suppressor, low PLZF expression is associated with lethal PC. Whether further suppression of PLZF with ADT worsens outcomes in advanced PC needs further study.

Abstract OT3-06-06: A phase I/II study of preoperative letrozole, everolimus, and TRC105 in women with newly diagnosed local or locally advanced potentially resectable hormone-Receptor positive and Her2 negative breast cancer

Abstract Hypothesis: Pharmacologic inhibition of angiogenesis and mTOR pathway blockade will enhance the efficacy of aromatase inhibitors in the neoadjuvant setting in women with hormone receptor-positive and Her2-negative (HR+/Her2-) breast cancer. Background: In non-metastatic HR+/Her2- breast cancer, downstaging to stage I or 0 in the neoadjuvant setting has been associated with favorable prognosis. With chemotherapy yielding similar benefits with endocrine therapy, aromatase inhibitors have become the agents of choice in postmenopausal women. The elucidation of pathways that are operational in luminal breast cancer and compromise the efficacy of hormonal therapy along with preclinical and clinical evidence of successful combinations of endocrine therapy with targeted agents set the stage for more rational, effective, and equally well tolerated regimens. Proangiogenic signaling has been strongly associated with accelerated hormone-independent growth and resistance to endocrine therapies. Breast cancer cells with upregulated PI3K/AKT/mTOR pathway are resistant to hormonal therapy and this resistance has been restored by everolimus. Inhibiting mTOR, a downstream signaling node where multiple pathways converge has the potential to abrogate primary and escape signaling cascades that mediate resistance to endocrine therapies. Study Design: Phase I dose escalation: follows a 3+3 design. During the phase I, the Maximum Tolerated Dose (MTD) and Recommended Phase 2 Dose (RP2D) will be determined. Letrozole will be kept the same for all cohorts (2.5 mg PO qd). Everolimus will be escalated from 5 mg (cohort 1) to 10 mg PO qd (cohort 2). TRC105 will be 15 mg/kg q2 weeks (cohorts 1 and 2). Premenopausal women at the time of enrollment will receive goserelin 3.6 mg SC q4 weeks to achieve ovarian suppression. Duration of treatment is 24 weeks. Phase II dose expansion: will be initiated once the MTD and RP2D have been determined. Phase II follows a Gehan's two-stage design: 10 patients will enroll first, and if no patient has achieved downstaging, the study will close since it is unlikely (p=0.1) that 0/10 responses would occur if true response rate were &amp;gt;20%. If at least one patient has downstaging, 10 additional patients will enroll (total, 20). Primary Objectives Phase I: Determine the tolerability and feasibility of letrozole, everolimus, and TRC105 in women with newly diagnosed stage 2 and 3 HR+/Her2- breast cancer. Phase II: in the same patient population determine the efficacy, pharmacokinetic, and pharmacodynamic parameters of the combination. Correlative Studies: Immunohistochemistry for pAKT and PTEN, CD105, CD31/CD34, NG2, MCAM/CD146 on diagnostic tissue, research biopsy, and final surgical specimen. Surgical specimens that meet criteria for next-generation sequencing will undergo ribosome profiling Key Eligibility Criteria: Recent diagnosis of HR+Her2- breast cancer Stage 2 and 3 breast cancer Any histological grade No prior treatment specific for breast cancer Pre- and perimenopausal women are eligible if ovarian suppression is achieved with goserelin. Current Status: Cohort 1 of the Phase 1 complete. Cohort 2 of Phase 1 actively enrolling. Citation Format: Vaklavas C, Lamaster KV, Stringer EM, Falkson CI, Li Y, Forero A. A phase I/II study of preoperative letrozole, everolimus, and TRC105 in women with newly diagnosed local or locally advanced potentially resectable hormone-Receptor positive and Her2 negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-06-06.

Validation of Immunohistochemical Assays for Integral Biomarkers in the NCI-MATCH EAY131 Clinical Trial.

Biomarkers that guide therapy selection are gaining unprecedented importance as targeted therapy options increase in scope and complexity. In conjunction with high-throughput molecular techniques, therapy-guiding biomarker assays based upon immunohistochemistry (IHC) have a critical role in cancer care in that they inform about the expression status of a protein target. Here, we describe the validation procedures for four clinical IHC biomarker assays-PTEN, RB, MLH1, and MSH2-for use as integral biomarkers in the nationwide NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) EAY131 clinical trial. Validation procedures were developed through an iterative process based on collective experience and adaptation of broad guidelines from the FDA. The steps included primary antibody selection; assay optimization; development of assay interpretation criteria incorporating biological considerations; and expected staining patterns, including indeterminate results, orthogonal validation, and tissue validation. Following assay lockdown, patient samples and cell lines were used for analytic and clinical validation. The assays were then approved as laboratory-developed tests and used for clinical trial decisions for treatment selection. Calculations of sensitivity and specificity were undertaken using various definitions of gold-standard references, and external validation was required for the PTEN IHC assay. In conclusion, validation of IHC biomarker assays critical for guiding therapy in clinical trials is feasible using comprehensive preanalytic, analytic, and postanalytic steps. Implementation of standardized guidelines provides a useful framework for validating IHC biomarker assays that allow for reproducibility across institutions for routine clinical use. Clin Cancer Res; 24(3); 521-31. ©2017 AACR.

Molecular Profiling of Patients With Advanced Colorectal Cancer: Princess Margaret Cancer Centre Experience

BackgroundMolecular aberrations in KRAS, NRAS, BRAF, and PIK3CA have been well-described in advanced colorectal cancer. The incidences of other mutations are less known. We report results of molecular profiling of advanced colorectal cancer in an academic cancer center. Patients and MethodsPatients with advanced colorectal were enrolled in an institution-wide molecular profiling program. Profiling was performed on formalin-fixed paraffin embedded archival tissues using a customized MassArray panel (23 genes, 279 mutations) or the Illumina MiSeq TruSeq Cancer Panel (48 genes, 212 amplicons, ≥ 500× coverage) in a Clinical Laboratory Improvement Amendments-certified laboratory. PTEN was determined by immunohistochemistry. ResultsFrom March 2012 to April 2014, 245 patients were enrolled. At least one mutation was found in 54% (97/178) and 91% (61/67) of patients using MassArray or MiSeq platforms, respectively (P < .01). Of all patients, KRAS G12/13 mutation was identified in 39%, and non-G12/13 KRAS, BRAF, or NRAS mutations were present in 9%, 6%, and 4%, respectively. Other common mutations included TP53 (68.7%), APC (41.8%), and PIK3CA (13.5%). Co-mutation with KRAS, NRAS, or BRAF was found in 75% of patients with PIK3CA mutation. Of 106 patients with known PTEN immunohistochemistry status, 16% were negative. A higher average number of mutations were observed in right versus left colorectal cancer (P < .01), with 13 of 14 BRAF mutations located in right colon cancer. ConclusionMutations are common in advanced colorectal cancer. Right colon cancers harbor more genetic aberrations than left colon or rectal cancers. These aberrations may contribute to differential outcomes to anti-epidermal growth factor receptor therapy among patients with right colon, left colon, or rectal cancers.

Evaluation of endometrial carcinoma prognostic immunohistochemistry markers in the context of molecular classification.

Molecular subclassification of endometrial carcinoma (EC) with Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identifies four subtypes [DNA polymerase epsilon (POLE) mutant, mismatch repair‐deficient, p53 wild‐type (wt), and p53 abnormal]. The aim of this study was to evaluate additional EC biomarkers in the context of these subtypes. Tissue microarrays encompassing 460 previously characterized ECs were assessed for L1‐cell adhesion molecule (L1CAM), progesterone receptor (PR), estrogen receptor (ER) alpha, stathmin, and phosphatase and tensin homolog (PTEN), by immunohistochemistry (IHC). Associations with clinicopathological parameters, molecular subtype, and outcomes were determined. About 413 ECs (75% endometrioid, >15% serous) had complete data. L1CAM overexpression was found in 16%, associated with older age, lower body mass index (BMI), advanced stage, grade 3 (97%), non‐endometrioid histology (84%), deep myometrial invasion, lymphovascular space invasion (LVSI), and ER‐negative, PR‐negative status. Tumours overexpressing L1CAM were associated with poor outcomes {hazard ratio (HR) [95% confidence interval (CI)] 3.35 [2.10–5.23] for disease‐specific survival [DSS], p < 0.0001}. PR positivity was associated with younger women, higher BMI, early stage (77% stage I), low grade (61%), endometrioid histology (90%) without LVSI or nodal disease, ER positivity (90%), p53wt tumours (55%), and favourable outcomes [HR (CI) 0.39 (0.25–0.62) for DSS, p < 0.0001]. ER positive tumours were early stage (73%), low grade, endometrioid histology, with improved DSS. Stathmin and PTEN IHC were not associated with outcomes. There was minimal agreement between IHC and mutation status for PTEN. L1CAM overexpression was significantly associated with the p53 abnormal molecular subtype, which accounted for more than 70% of the tumours overexpressing L1CAM. PR expression also correlated with molecular subtype, with most PR negative tumours being p53 abnormal. Multivariable analysis demonstrated that only ProMisE subtype [overall survival (OS), DSS, and progression‐free survival] and age (OS only) maintained an association with outcomes. The prognostic significance of the single biomarkers tested could be explained based on their being covariable with the ProMisE molecular subtype.

Investigating the role of the IGF axis as a predictor of biochemical recurrence in prostate cancer patients post-surgery.

Between 20% and 35% of prostate cancer (PCa) patients who undergo treatment with curative intent (ie, surgery or radiation therapy) for localized disease will experience biochemical recurrence (BCR). Alterations in the insulin-like growth factor (IGF) axis and PTEN expression have been implicated in the development and progression of several human tumors including PCa. We examined the expression of the insulin receptor (INSR), IGF-1 receptor (IGF-1R), PTEN, and AKT in radical prostatectomy tissue of patients who developed BCR post-surgery. Tissue microarrays (TMA) of 130 patients post-radical prostatectomy (65 = BCR, 65 = non-BCR) were stained by immunohistochemistry for INSR, IGF-1R, PTEN, and AKT using optimized antibody protocols. INSR, IGF1-R, PTEN, and AKT expression between benign and cancerous tissue, and different Gleason grades was assessed. Kaplan-Meier survival curves were used to examine the relationship between proteins expression and BCR. INSR (P < 0.001), IGF-1R (P < 0.001), and AKT (P < 0.05) expression was significantly increased and PTEN (P < 0.001) was significantly decreased in cancerous versus benign tissue. There was no significant difference in INSR, IGF-1R, or AKT expression in the cancerous tissue of non-BCR versus BCR patients (P = 0.149, P = 0.990, P = 0.399, respectively). There was a significant decrease in PTEN expression in the malignant tissue of BCR versus non-BCR patients (P = 0.011). Combinational analysis of the tissue proteins identified a combination of decreased PTEN and increased AKT or increased INSR was associated with worst outcome. We found that in each case, our hypothesized worst group was most likely to experience BCR and this was significant for combinations of PTEN+INSR and PTEN+AKT but not PTEN+IGF-1R (P = 0.023, P = 0.028, P = 0.078, respectively). Low PTEN is associated with BCR and this association is strongly modified by high INSR and high AKT expression. Measurement of these proteins could help inform appropriate patient selection for postoperative adjuvant therapy and prevent BCR.

PTEN loss detection in prostate cancer: comparison of PTEN immunohistochemistry and PTEN FISH in a large retrospective prostatectomy cohort.

PTEN deletion is an established prognostic biomarker in prostate cancer. We compared PTEN immunohistochemistry (IHC) and PTEN fluorescence in situ hybridization (FISH) in the largest existing radical prostatectomy cohort with clinical follow-up data. There was high concordance between IHC and FISH: 93% (3098/3330) of tumors with intact PTEN IHC showed absence of PTEN gene deletion and 66% (720/1087) of cases with PTEN protein loss by IHC showed PTEN gene deletion by FISH. 84% (447/533) of cases with PTEN homozygous gene deletion had PTEN protein loss by IHC. PTEN loss by IHC was associated with reduced PSA recurrence-free survival (RFS) in multivariable models (HR=1.3; 95% CI: 1.16-1.47). Among cases with either PTEN deletion or absence of PTEN deletion by FISH, PTEN loss by IHC was strongly associated with reduced RFS on univariable analysis (p=0.0005 and p<0.0001 respectively). Among cases with intact PTEN by IHC, homozygous (p=0.04) but not heterozygous (p=0.10) PTEN gene deletion was weakly associated with reduced RFS. Among cases with PTEN loss by IHC, both homozygous (p=0.0044) and heterozygous (p=0.0017) PTEN gene deletion were associated with reduced RFS. These data support the utility of PTEN IHC and PTEN FISH as complementary screening tools for PTEN loss in prostate cancer.

Abstract 4740: Loss of the tumor suppressor zinc finger protein-36 and risk of lethal prostate cancer

Abstract Background. Biomarkers are needed to complement current clinicopathologic variables towards distinguishing prostate tumors that are likely to be metastatic from those that are indolent. We previously used a bioinformatic analytical approach to identify ZFP36, also known as Tristetraprolin (TTP), as a tumor suppressor in the NFκB pathway. The current study aims to evaluate the prognostic potential of tumor mRNA expression of ZFP36. Methods. Our primary analysis leveraged data from a case-control study nested in the Health Professionals Follow-up Study and Physicians’ Health Study. Gene expression for ZFP36 was quantified from archival surgical tumor tissue using Affymetrix Human Gene 1.0 ST microarrays. Cases (n=113) were men who died of prostate cancer or developed metastatic disease, and controls (n=291) were men who lived at least 8 years after diagnosis and remained metastasis free. A genetically validated PTEN immunohistochemistry (IHC) assay was performed on tissue microarrays (TMAs) for a subset of the men (n=257). Results. Mean ZFP36 expression was significantly lower in the cases compared to the controls (p&amp;lt;0.001), and the crude odds ratio (OR) for lethal disease comparing those with low ZFP36 expression (below the lower quartile) to those with high ZFP36 expression (greater than or equal to the lower quartile) was 3.52 (95% CI: 2.18-5.69; p&amp;lt;0.001). The association persisted with adjustment for Gleason category, age at diagnosis, and clinical stage, with an adjusted OR of 1.93 (95% CI: 1.07-3.49; p=0.03). When modeled as a continuous variable in a logistic regression for prognosis, ZFP36 improved the AUC of the clinical factor model (Gleason, age, and clinical stage) from 0.83 (95% CI: 0.79-0.88) to 0.85 (95% CI: 0.81-0.89; p=0.10 for improvement). Among the patients with available PTEN staining, independent effects of ZFP36 and PTEN loss were observed. In the logistic regression on lethal disease with both variables included, PTEN negativity conferred an OR of 2.10 (95% CI: 1.11-3.93; p=0.02) and low ZFP36 produced an OR of 2.34 (95% CI: 1.24-4.36; p=0.01). Taken together, we observed an OR of 4.90 (95% CI: 2.05-11.72) comparing patients with PTEN negativity and low ZFP36 to those with PTEN staining present and high ZFP36. Conclusions. Loss of the tumor suppressor ZFP36 is prognostic for metastatic or lethal prostate cancer. Combining information on ZFP36 with measurements from other mechanistic pathways, such as PTEN, may lead to highly accurate models. Citation Format: Travis Gerke, Daniela Börnigen, Himisha Beltran, Svitlana Tyekucheva, Curtis Huttenhower, Gwo-Shu Lee, Bruce Trock, Lorelei Mucci, Christopher Sweeney. Loss of the tumor suppressor zinc finger protein-36 and risk of lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4740. doi:10.1158/1538-7445.AM2017-4740

PTEN loss and p27 loss differ among morphologic patterns of prostate cancer, including cribriform

The presence and extent of cribriform pattern of prostate cancer portend recurrence and cancer death. The relative expressions within this morphology of the prognostically adverse loss of PTEN, and the downstream inactivation of cell cycle inhibitor p27/Kip1 had been uncertain. In this study, we examined 52 cases of cribriform cancer by immunohistochemistry for PTEN, p27, and CD44 variant (v)7/8, and a subset of 17 cases by chromogenic in situ hybridization (ISH) using probes for PTEN or CDKN1B (gene for p27). The fractions of epithelial pixels positive by immunohistochemistry and ISH were digitally assessed for benign acini, high-grade prostatic intraepithelial neoplasia, and 8 morphologic patterns of cancer. Immunostaining results demonstrated that (1) PTEN loss was significant for fused small acini, cribriform-central cells, small cribriform acini, and Gleason grade 5 cells in comparison with other acini; (2) p27 loss was significant only for cribriform-peripheral cells and borderline significant for fused small acini in comparison with benign acini; and (3) CD44v7/8 showed expression loss in cribriform-peripheral cells; other comparisons were not significant. ISH showed that cribriform cancer had significant PTEN loss normalized to benign acini (P<.02), whereas Gleason 3 cancer or fused small acini did not. With CDKN1B, the degree of signal loss among various cancer morphologies was insignificant. In conclusion, molecular disparities emerged between the fused small acini and cribriform patterns of Gleason 4 cancer. PTEN or p27 loss as prognostic factors demands distinct assessment in the varieties of Gleason 4 cancer, and in the biphenotypic peripheral versus central populations in cribriform structures.

Expression characteristics of PTEN and NDRG1 in colorectal carcinoma and their prognostic value

Objective: To study the expression status and clinical significance of PTEN and NDRG1 in colorectal carcinoma. Methods: Tissue samples of 91 colorectal cancers, 30 colorectal adenomas and 21 colorectal normal mucosa tissues were collected. Postoperative specimens were examined by immunohistochemistry for PTEN and NDRG1 expression. The expression of PTEN and NDRG1 was correlated with clinicopathological feature. Results: The expression of PTEN and NDRG1 in the studied cases was detected in 55.0%(50/91) and 76.9%(70/91), respectively. Their expression was significantly different from that of colorectal adenomas and normal colorectal mucosa tissues(P<0.05). Decreased expression of PTEN and over expression of NDRG1 were significantly related to the lymph node metastasis (P<0.05). The expression of PTEN was negatively related to that of NDRG1 in colorectal carcinoma(rs'=-0.251, P=0.016). The patients with negative expression of PTEN showed a lower disease free survival and overall survival(P<0.05). Conclusions: Loss of expression of PTEN protein may be an important molecular marker in predicting the occurrence and PTEN may be useful as a prognostic marker of colorectal carcinoma. NDRG1 plays a role in the development of colorectal carcinoma, although not a prognostic indicator.The ancillary study with combined detection of PTEN and NDRG1 may be useful in difficult cases.

Abstract P5-06-07: PTEN immunohistochemistry is a predictor of mismatch repair status in breast cancer

Abstract Phosphatase and tensin homolog (PTEN), a potent downregulator of the PI3K-Akt pathway, has been shown to mediate the interaction between poly (ADP-ribose) polymerases (PARPs) and the mismatch repair (MMR) complex in endometrial and ovarian cancer. Drugs inhibiting PARPs (iPARPs) are currently considered promising therapeutic tools in a subset of PTEN-defective tumors. Regrettably, the frequency and significance of MMR alterations in breast cancer is debated, and their relationship with PTEN status has not been investigated in the breast. Furthermore, many of the studies on the DNA damage response and its therapeutic implications in breast cancer focus on inherited syndromes (e.g. Lynch syndrome and hereditary breast-ovarian cancer syndrome). Aims: We sought to explore the interplay between PTEN and the MMR system and to define whether PTEN immunohistochemistry (IHC) is a predictor of MMR proficiency in non-familial breast cancers. Methods: 373 cases of non-familial breast cancers, including a representative number of no special (n=295) and special types (n=78), carefully characterized from clinical and pathological standpoints, were reviewed and used to construct 14 tissue microarrays (TMAs). For each case, a mean of 4.5 tumor tissue cores (range 3 to 6 cores) was sampled, incorporating distinct topographic areas of the tumor, as well as matched non-neoplastic breast tissue. Taken together, 1876 spots were generated. Each TMA was subjected to IHC for PTEN and the DNA MMR proteins MLH1, MSH2, MSH6 and PMS2. In order to minimize human-related biases, each stained slide was digitalized and two pathologists blindly analyzed each tumor spot using a dedicated software able to segment and randomize TMA cores. The pattern of expression was therefore annotated manually on a digital database using a specific add-on module to reconstruct the original topography. Results: According to clinicopathologic surrogate definition of intrinsic subtypes, PTEN protein loss or heterogeneous expression was more frequent in estrogen receptor negative cancers. Furthermore, 100% of the MMR-proficient luminal B-like (HER2+) and triple-negative breast cancers displayed strong and diffuse homogeneous PTEN expression, while PTEN-positive status identified MMR-proficient luminal A-like and luminal B (HER2-) like tumors with accuracy rates of 89.3% and 92.7%, respectively (p=0.001, Fisher's exact test). Conclusions: The present study is the first to investigate PTEN protein loss in a large set of non-familial breast carcinomas based on their DNA MMR status by IHC. Here, we demonstrated that PTEN strong and homogeneous expression by IHC is able to capture the vast majority of MMR-proficient non-familial breast cancers. Our findings broaden the understanding of the biology underpinning these tumors, suggesting that PTEN is likely play a role in the development of MMR alterations. Given that PTEN-defective breast cancers have the propensity to develop additional somatic alterations in the MMR system, our results suggest that IHC for PTEN and MMR proteins may be emplyed as an ancillary study to define new subclasses of sporadic breast cancers potentially eligible for iPARPs therapies. Citation Format: Fusco N, Gambini D, Runza L, Lopez G, Ercoli G, Despini L, Bosari S. PTEN immunohistochemistry is a predictor of mismatch repair status in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-07.

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