Mohammed S. Orloff

Lifetime Cancer Risks in Individuals with Germline PTEN Mutations

Purpose: Age-adjusted cancer incidence and age-related penetrance studies have helped guide cancer risk assessment and management. PTEN hamartoma tumor syndrome (PHTS) is a term encompassing subsets of several clinical syndromes with germline mutations in the PTEN tumor suppressor gene. We conducted the first prospective study to clarify corresponding cancer risks to shed biologic insights on human germline PTEN mutations, and to better inform current surveillance recommendations on the basis of expert opinion. Experimental Design: A series of 3,399 individuals meeting relaxed International Cowden Consortium PHTS criteria were prospectively recruited; 368 individuals were found to have deleterious germline PTEN mutations. Age-adjusted standardized incidence ratio (SIR) calculations and genotype–phenotype analyses were carried out. Results: Elevated SIRs were found for carcinomas of the breast [25.4, 95% confidence interval (CI), 19.8–32.0], thyroid (51.1, 38.1–67.1), endometrium (42.9, 28.1–62.8), colorectum (10.3, 5.6–17.4), kidney (30.6, 17.8–49.4), and melanoma (8.5, 4.1–15.6). Estimated lifetime risks were, respectively, 85.2% (95% CI, 71.4%–99.1%), 35.2% (19.7%–50.7%), 28.2% (17.1%–39.3%), 9.0% (3.8%–14.1%), 33.6% (10.4%–56.9%), and 6% (1.6%-9.4%). Promoter mutations were associated with breast cancer, whereas colorectal cancer was associated with nonsense mutations. Conclusion: Lifetime risks for a variety of cancers, now extending to colorectal cancer, kidney cancer, and melanoma, are increased in patients with PTEN mutations. The genotype–phenotype associations here may provide new insights on PTEN structure and function. We propose a comprehensive approach to surveillance of patients with PTEN mutations. Clin Cancer Res; 18(2); 400–7. ©2012 AACR.

A Clinical Scoring System for Selection of Patients for PTEN Mutation Testing Is Proposed on the Basis of a Prospective Study of 3042 Probands

Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome are allelic, defined by germline PTEN mutations, and collectively referred to as PTEN hamartoma tumor syndrome. To date, there are no existing criteria based on large prospective patient cohorts to select patients for PTEN mutation testing. To address these issues, we conducted a multicenter prospective study in which 3042 probands satisfying relaxed CS clinical criteria were accrued. PTEN mutation scanning, including promoter and large deletion analysis, was performed for all subjects. Pathogenic mutations were identified in 290 individuals (9.5%). To evaluate clinical phenotype and PTEN genotype against protein expression, we performed immunoblotting (PTEN, P-AKT1, P-MAPK1/2) for a patient subset (n = 423). In order to obtain an individualized estimation of pretest probability of germline PTEN mutation, we developed an optimized clinical practice model to identify adult and pediatric patients. For adults, a semiquantitative score-the Cleveland Clinic (CC) score-resulted in a well-calibrated estimation of pretest probability of PTEN status. Overall, decreased PTEN protein expression correlated with PTEN mutation status; decreasing PTEN protein expression correlated with increasing CC score (p < 0.001), but not with the National Comprehensive Cancer Network (NCCN) criteria (p = 0.11). For pediatric patients, we identified highly sensitive criteria to guide PTEN mutation testing, with phenotypic features distinct from the adult setting. Our model improved sensitivity and positive predictive value for germline PTEN mutation relative to the NCCN 2010 criteria in both cohorts. We present the first evidence-based clinical practice model to select patients for genetics referral and PTEN mutation testing, further supported biologically by protein correlation.

Frequent Gastrointestinal Polyps and Colorectal Adenocarcinomas in a Prospective Series of PTEN Mutation Carriers

BACKGROUND & AIMS Germline phosphatase and tensin homolog (PTEN) mutations cause Cowden syndrome (CS), associated with breast and thyroid cancers. Case reports found 35%-85% of CS patients had gastrointestinal (GI) hamartomas. The association of benign and malignant GI neoplasias with CS remains debatable. Our goal is to describe the GI phenotype in a prospective series of PTEN mutation carriers. METHODS Patients who met relaxed International Cowden Consortium criteria (N = 2548) or with 5 or more GI polyps, 1 or more of which was hyperplastic or hamartomatous (N = 397), were prospectively recruited. Germline PTEN mutation/deletion analysis was performed. Of the 2945 patients, 127 (123 of 2548 and 4 of 397, respectively) patients having clear pathogenic PTEN mutations were eligible for this study. Esophagogastroduodenoscopy, colonoscopy, and pathology reports were reviewed. The Fisher 2-tailed exact test, unpaired t tests, and age- and sex-adjusted standardized incidence ratio were calculated. RESULTS Of 127 PTEN mutation carriers, 69 underwent 1 or more endoscopies with 64 (93%) having polyps. Of the 64, half had hyperplastic polyps. There were one to innumerable polyps in the colorectum, ileum, duodenum, stomach, and/or esophagus, with 24 subjects having both upper and lower GI polyps. Nine (13%) subjects had colorectal cancer, all younger than the age of 50. The adjusted standardized incidence ratio was 224.1 (95% confidence interval, 109.3-411.3; P < .0001). CONCLUSIONS PTEN-associated CS should be considered a mixed polyp syndrome, with hyperplastic polyps most prevalent, with a risk of early onset colorectal cancer. Routine colonoscopy should be considered in PTEN-associated CS, especially in the context of hyperplastic and/or adenomatous polyps.

Germline Mutations and Variants in the Succinate Dehydrogenase Genes in Cowden and Cowden-like Syndromes

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

Germline PIK3CA and AKT1 Mutations in Cowden and Cowden-like Syndromes

Cowden syndrome (CS) is a difficult-to-recognize multiple hamartoma syndrome with high risks of breast, thyroid, and other cancers. Germline mutations in PTEN on 10q23 were found to cause 85% of CS when accrued from tertiary academic centers, but prospective accrual from the community over the last 12 years has revealed a 25% PTEN mutation frequency. PTEN is the phosphatase that has been implicated in a heritable cancer syndrome and subsequently in multiple sporadic cancers and developmental processes. PTEN antagonizes the AKT1/PI3K signaling pathway and has roles in cell cycle, migration, cell polarity, and apoptosis. We report that 8 of 91 (8.8%) unrelated CS individuals without germline PTEN mutations carried 10 germline PIK3CA mutations (7 missense, 1 nonsense, and 2 indels) and 2 (2.2%) AKT1 mutations. These mutations result in significantly increased P-Thr308-AKT and increased cellular PIP3. Our observations suggest that PIK3CA and AKT1 are CS susceptibility genes.

Genetic and phenotypic heterogeneity in the PTEN hamartoma tumour syndrome

Germline PTEN (Phosphatase and TENsin homologue deleted on chromosome TEN) mutations predispose to phenotypically diverse disorders that share several overlapping clinical features: Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome, Proteus syndrome and Proteus-like syndrome, collectively classified as PTEN hamartoma tumour syndrome (PHTS). The meticulous acquisition and documentation of PHTS phenotypic data at different levels and the profiling of the plethora of genetic changes in PTEN and other genes within the same or related pathways are important in resolving the challenge of discriminating heritable cancers from sporadic PHTS-mimicking clinical features. The characterization of PTEN and PTEN-related pathways from a multidisciplinary perspective underscores the importance of incorporating data from different -omics, which is crucial for the advancement of personalized medicine.

Subtypes of familial breast tumours revealed by expression and copy number profiling

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism–comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.

Resveratrol enhances the anti-tumor activity of the mTOR inhibitor rapamycin in multiple breast cancer cell lines mainly by suppressing rapamycin-induced AKT signaling

The anti-tumor activity of rapamycin is compromised by the feedback-loop-relevant hyperactive PI3K and ERK-MAPK pathway signaling. In breast cancer cells treated with rapamycin, we observed a moderate increase of AKT phosphorylation (P-AKT) in a rapamycin resistant cell line, MDA-MB-231, as well as a slight increase of P-AKT in a rapamycin sensitive cell line, MCF-7. We found that resveratrol, a natural phytoalexin, suppressed the phosphorylation and activation of the PI3K/AKT pathway in all the three breast cancer cell lines that we tested. It also had a weak inhibitory effect on the activation of the mTOR/p70S6K pathway in two cell lines expressing wildtype PTEN, MCF-7 and MDA-MB-231. The combined use of resveratrol and rapamycin resulted in modest additive inhibitory effects on the growth of breast cancer cells, mainly through suppressing rapamycin-induced AKT activation. We, therefore, reveal a novel combination whereby resveratrol potentiates the growth inhibitory effect of rapamycin, with the added benefit of preventing eventual resistance to rapamycin, likely by suppressing AKT signaling. We also present data herein that PTEN is an important contributor to resveratrols growth suppressive effects and its potentiation of rapamycin in this therapeutic scenario, as resveratrols suppression of rapamycin-mediated induction of P-AKT is both PTEN-dependent and -independent. Thus, the resveratrol-rapamycin combination may have therapeutic value in treating breast cancer and perhaps other processes where mTOR is activated.

Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines

The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Mammalian Target of Rapamycin (mTOR) regulates cellular proliferation and tumor growth in urothelial carcinoma

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.