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1.  Loss of CD73-mediated actin polymerization promotes endometrial tumor progression 
Ecto-5′-nucleotidase (CD73) is central to the generation of extracellular adenosine. Previous studies have highlighted a detrimental role for extracellular adenosine in cancer, as it dampens T cell–mediated immune responses. Here, we determined that, in contrast to other cancers, CD73 is markedly downregulated in poorly differentiated and advanced-stage endometrial carcinoma compared with levels in normal endometrium and low-grade tumors. In murine models, CD73 deficiency led to a loss of endometrial epithelial barrier function, and pharmacological CD73 inhibition increased in vitro migration and invasion of endometrial carcinoma cells. Given that CD73-generated adenosine is central to regulating tissue protection and physiology in normal tissues, we hypothesized that CD73-generated adenosine in endometrial carcinoma induces an innate reflex to protect epithelial integrity. CD73 associated with cell-cell contacts, filopodia, and membrane zippers, indicative of involvement in cell-cell adhesion and actin polymerization–dependent processes. We determined that CD73-generated adenosine induces cortical actin polymerization via adenosine A1 receptor (A1R) induction of a Rho GTPase CDC42–dependent conformational change of the actin-related proteins 2 and 3 (ARP2/3) actin polymerization complex member N-WASP. Cortical F-actin elevation increased membrane E-cadherin, β-catenin, and Na+K+ ATPase. Together, these findings reveal that CD73-generated adenosine promotes epithelial integrity and suggest why loss of CD73 in endometrial cancer allows for tumor progression. Moreover, our data indicate that the role of CD73 in cancer is more complex than previously described.
doi:10.1172/JCI79380
PMCID: PMC4701552  PMID: 26642367
2.  Identifying aggressive forms of endometrioid-type endometrial cancer: new insights into molecular subtyping 
Summary
Clinical heterogeneity represents a great challenge for cancer therapeutics. Molecular classification of patients into different subtypes based on genetic or epigenetic characteristics has the potential to revolutionize the clinical care and mechanistic understanding of a wide spectrum of cancers, including endometrial carcinoma, the most common gynecological cancer affecting women.
doi:10.1586/14737140.2015.992420
PMCID: PMC4638381  PMID: 25494844
Molecular subtyping; Endometrial carcinoma; CTNNB1 mutation; Wnt signaling pathway; Cancer therapeutics
3.  Mig-6 suppresses endometrial cancer associated with Pten deficiency and ERK activation 
Cancer research  2014;74(24):7371-7382.
PTEN mutations are the most common genetic alterations in endometrial cancer. Loss of PTEN and subsequent AKT activation stimulate ERα-dependent pathways that play an important role in endometrial tumorigenesis. The major pathologic phenomenon of endometrial cancer is the loss of ovarian steroid hormone control over uterine epithelial cell proliferation and apoptosis. However, the precise mechanism of PTEN/AKT signaling in endometrial cancer remains poorly understood. The progesterone signaling mediator MIG-6 suppresses estrogen signaling and it has been implicated previously as a tumor suppressor in endometrial cancer. In this study, we show that MIG-6 also acts as a tumor suppressor in endometrial cancers associated with PTEN deficiency. Transgenic mice where Mig-6 was overexpressed in PR-expressing cells exhibited a relative reduction in uterine tumorigenesis caused by Pten deficiency. ERK1/2 was phosphorylated in uterine tumors and administration of an ERK1/2 inhibitor suppressed cancer progression in PRcre/+Ptenf/f mice. In clinical specimens of endometrial cancer, MIG-6 expression correlated inversely with ERK1/2 phosphorylation during progression. Taken together, our findings suggest that Mig-6 regulates ERK1/2 phosphorylation and that it is crucial for progression of PTEN-mutant endometrial cancers, providing a mechanistic rationale for the evaluation of ERK1/2 inhibitors as a therapeutic treatment in human endometrial cancer.
doi:10.1158/0008-5472.CAN-14-0794
PMCID: PMC4268053  PMID: 25377472
4.  Clinically Significant Endometrial Cancer Risk Following a Diagnosis of Complex Atypical Hyperplasia 
Gynecologic oncology  2014;135(3):451-454.
Objectives
Because of the frequent detection of carcinoma in surgical specimens after hysterectomy for endometrial complex atypical hyperplasia (CAH), it has been suggested that patients with a preoperative diagnosis of CAH be referred to gynecologic oncology for potential lymphadenectomy. However, the risk of lymph node metastasis in such patients is unknown. We sought to determine the risk of endometrial cancer and to estimate the risk of lymphatic spread in women with a preoperative diagnosis of CAH.
Study Design
We retrospectively reviewed the medical records of 150 consecutive patients with a preoperative diagnosis of CAH who subsequently underwent hysterectomy. Clinical characteristics and pathologic information were abstracted. Risk of lymphatic spread was modeled using previously published criteria and nomograms.
Results
Fifty-five of the 150 patients (36.7%) had an incidental endometrial carcinoma at the time of hysterectomy. Among patients with a preoperative office biopsy compared to dilation and curettage, the rate of an incidental finding of cancer was 43.5% and 28.1%, respectively (p=0.054). Of patients with cancer, 1 (1.8%) had a grade 3 endometrial carcinoma, 4 (7.3%) had lymphovascular space involvement, and 6 (10.9%) had deep (>50%) myometrial invasion. For the 10 patients who underwent lymphadenectomy, one (10%) had lymph node metastases. Based on multiple models, the estimated risk of lymph node spread was 1.6-2.1% for all women with a preoperative diagnosis of CAH and 4.4-6.8% for the 55 women with endometrial cancer.
Conclusions
Given the high rates of underlying endometrial cancer and the potential need for lymphadenectomy, care for patients with a preoperative diagnosis of CAH desiring definitive management with hysterectomy should be referred to a gynecologic oncologist.
doi:10.1016/j.ygyno.2014.10.008
PMCID: PMC4268403  PMID: 25316176
5.  Long non-coding RNA profiling links subgroup classification of endometrioid endometrial carcinomas with trithorax and polycomb complex aberrations 
Oncotarget  2015;6(37):39865-39876.
Background
Integrative analysis of endometrioid endometrial carcinoma (EEC) using multiple platforms has distinguished four molecular subgroups. However, the landscape of expressed long non-coding RNAs (lncRNA) and their role in charting EEC subgroups and determining clinical aggressiveness remain largely unknown.
Materials and Methods
We performed integrative analysis of lncRNAs in EEC using The Cancer Genome Atlas (TCGA) molecular RNAseq profiles of 191 primary tumors for which genomic data were also available. We established lncRNA subgroup classification, correlated it with chromatin modifying gene expression, and described correlations between our lncRNA classification and clinico-genomic tumor features.
Results
Using stringent criteria, we identified 1,931 expressed lncRNAs and predicted potential drivers through integrative analysis. Unsupervised clustering of lncRNA expression revealed three robust categories: basal-like, luminal-like and CTNNB1-enriched subgroups. Basal-like subgroup was enriched for aggressive tumors with higher pathological grade (p < 0.0001), TNM stage (p = 0.01), and somatic mutations in trithorax-group genes (MLL, MLL2 and MLL3); and it overexpressed polycomb genes EZH2 and CBX2. In contrast to the luminal-like subgroup, progesterone (PGR) and estrogen receptor (ESR1) genes were highly down-regulated in the EEC basal-like subgroup. Consistent with its enrichment for CTNNB1 mutations (69%), lncRNA profile of the CTNNB1-enriched EEC subgroup was highly similar to that of the CTNNB1-enriched liver cancer subgroup.
Conclusions
Our results reveal the utility of systematic characterization of clinically annotated EEC in three clinically relevant subgroups. They also highlight the convergence of aberrations in polycomb- and trithorax-group genes in aggressive basal EEC subtypes, providing a rationale for further investigation of epigenetic therapy in this setting.
PMCID: PMC4741866  PMID: 26431491
endometrioid endometrial carcinoma; long non-coding RNA; RNA-Seq; expression profiling; polycomb complex
6.  Complete loss of PTEN protein expression correlates with shorter time to brain metastasis and survival in stage IIIB/C melanoma patients with BRAFV600 mutations 
PURPOSE
Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAFV600 mutations. The prevalence, pathological features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event.
EXPERIMENTAL DESIGN
Archival tissue from lymphadenectomy specimens among patients (n=136) with stage IIIB or IIIC melanoma were assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry (IHC) for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined.
RESULTS
The prevalence of BRAFV600 mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathological substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone. Analysis of PTEN in mutationally-defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAFV600 mutations.
CONCLUSIONS
Loss of PTEN protein expression correlates significantly with decreased OS and time to MBM in stage IIIB/C melanoma patients with BRAFV600 mutations. The findings add to evidence supporting a significant role for PTEN loss and the PI3K-AKT pathway in melanoma.
doi:10.1158/1078-0432.CCR-14-1027
PMCID: PMC4216767  PMID: 25165098
melanoma; BRAF; NRAS; PTEN; brain metastasis
7.  Expression of Estrogen-Induced Genes and Estrogen Receptor Beta in Pancreatic Neuroendocrine Tumors: Implications for Targeted Therapy 
Pancreas  2014;43(7):996-1002.
Objective
The indolent nature and expression of progesterone receptor (PR), a well-known estrogen-induced gene, in a subset of pancreatic neuroendocrine tumors (PanNETs), raise the possibility of hormonal regulation in these tumors.
Methods
Immunohistochemical expression of estrogen receptors (ER) α and β and mRNA expression of estrogen-induced genes (PR, EIG121, IGF-1, IGF-1R, sFRP1, and sFRP4) by qRT-PCR were examined in 131 WHO G1-G2 PanNETs and correlated expression with clinicopathologic features.
Results
39 PanNETs (30%) showed high positive ERβ staining and 87 cases (66%) had low positive ERβ staining; only 5 cases (4%) had no nuclear staining. PanNETs with small size (P=.02), low WHO grade (P=.02) and low AJCC stage (P=.006) more frequently showed high positive ERβ staining. Among estrogen-induced genes studied, PanNETs had significantly higher expression of PR, EIG121, IGF-1, sFRP1, and sFRP4 compared to normal pancreas, independent of age or gender. High positive ERβ staining was associated with increased expression of PR (P < .001) and EIG121 (P=.02).
Conclusions
Our study showed that PanNETs with favorable prognostic features have higher ERβ expression, which is associated with upregulated PR and EIG121 mRNA expression. Estrogen regulation in PanNETs could potentially help in risk stratification and provide a rational target for novel treatment strategies.
doi:10.1097/MPA.0000000000000203
PMCID: PMC4628823  PMID: 25058880
Pancreatic neuroendocrine tumor; ERβ; estrogen-induced genes
8.  Naturally occurring neomorphic PIK3R1 mutations activate the MAPK pathway dictating therapeutic response to MAPK pathway inhibitors 
Cancer cell  2014;26(4):479-494.
Summary
PIK3R1 (p85α regulatory subunit of PI3K) is frequently mutated across cancer lineages. Herein, we demonstrate that the most common recurrent PIK3R1 mutation PIK3R1R348* and a nearby mutation PIK3R1L370fs, in contrast to wild-type and mutations in other regions of PIK3R1, confers an unexpected sensitivity to MEK and JNK inhibitors in vitro and in vivo. Consistent with the response to inhibitors, PIK3R1R348* and PIK3R1L370fs unexpectedly increase JNK and ERK phosphorylation. Surprisingly, p85α R348* and L370fs localize to the nucleus where the mutants provide a scaffold for multiple JNK pathway components facilitating nuclear JNK pathway activation. Our findings uncover an unexpected neomorphic role for PIK3R1R348* and neighboring truncation mutations in cellular signaling providing a rationale for therapeutic targeting of these mutant tumors.
doi:10.1016/j.ccell.2014.08.017
PMCID: PMC4198486  PMID: 25284480
9.  Role of microsatellite instability-low as a diagnostic biomarker of Lynch syndrome in colorectal cancer 
Cancer genetics  2014;207(0):495-502.
Lynch syndrome is the most common Mendelian disorder predisposing to hereditary colorectal cancer. Carriers of MSH6 mutations constitute less than 10% of total cases and present with a weaker clinical phenotype, including low levels of microsatellite instability (MSI-L) in colorectal tumors. The frequency of MSH6 mutation carriers among patients presenting with MSI-L colorectal cancer has yet to be determined, as has the appropriate genetic work-up in this context. We have reviewed here the clinicopathological characteristics, immunohistochemistry and genetic testing results for 71 patients at a single institution diagnosed with MSI-L colorectal cancers. Of 71 patients with MSI-L tumors, 21 underwent genetic testing for MSH6 mutations, three of them presented with loss of staining of MSH6 and only one carried a pathogenic germline MSH6 mutation in exon 4 (c.2677_2678delCT; p.Leu893Alafs*6). This latter patient had a significant family history and had a rectal primary that showed instability only in mononucleotide markers. In this cohort of MSI-L patients, we detected no notable clinicopathological and molecular characteristics that would help to distinguish a group most likely to harbor germline MSH6 mutations. Therefore, we conclude that the prevalence of MSH6 mutations among subjects with MSI-L tumors is very low. MSI analysis combined with immunohistochemistry of mismatch repair proteins adequately detects potential MSH6 carriers among MSI-L colorectal cancers.
doi:10.1016/j.cancergen.2014.10.002
PMCID: PMC4278939  PMID: 25432668
Microsatellite instability-low; Lynch syndrome; colorectal cancer; MSH6; mononucleotide markers
10.  Combined colonoscopy and endometrial biopsy cancer screening results in women with Lynch syndrome 
Gynecologic oncology  2014;135(1):85-89.
Objective
Endometrial biopsy (EMBx) and colonoscopy performed under the same sedation is termed combined screening and has been shown to be feasible and to provide a less painful and more satisfactory experience for women with Lynch syndrome (LS). However, clinical results of these screening efforts have not been reported. The purpose of this study was to evaluate the long-term clinical outcomes and patient compliance with serial screenings over the last 10.5 years.
Methods
We retrospectively analyzed the data for 55 women with LS who underwent combined screening every 1–2 years between 2002 and 2013. Colonoscopy and endometrial biopsy were performed by a gastroenterologist and a gynecologist, with the patient under conscious sedation.
Results
Out of 111 screening visits in these 55 patients, endometrial biopsies detected one simple hyperplasia, three complex hyperplasia, and one endometrioid adenocarcinoma (FIGO Stage 1A). Seventy one colorectal polyps were removed in 29 patients, of which 29 were tubular adenomas. EMBx in our study detected endometrial cancer in 0.9% (1/111) of surveillance visits, and premalignant hyperplasia in 3.6% (4/111) of screening visits. No interval endometrial or colorectal cancers were detected.
Conclusions
Combined screening under sedation is feasible and less painful than EMBx alone. Our endometrial pathology detection rates were comparable to yearly screening studies. Our results indicate that screening of asymptomatic LS women with EMBx every 1–2 years, rather than annually, is effective in the early detection of (pre)cancerous lesions, leading to their prompt definitive management, and potential reduction in endometrial cancer.
doi:10.1016/j.ygyno.2014.08.017
PMCID: PMC4389779  PMID: 25149916
Combined screening; endometrial cancer screening; colonoscopy; Lynch syndrome; endometrial biopsy; hereditary nonpolyposis colorectal cancer (HNPCC)
11.  Clinical Significance of CTNNB1 Mutation and Wnt Pathway Activation in Endometrioid Endometrial Carcinoma 
Background
Endometrioid endometrial carcinoma (EEC) is the most common form of endometrial carcinoma. The heterogeneous clinical course of EEC is an obstacle to individualized patient care.
Methods
We performed an integrated analysis on the multiple-dimensional data types including whole-exome and RNA sequencing, RPPA profiling, and clinical data from 271 EEC cases in The Cancer Genome Atlas (TCGA) to identify molecular fingerprints that may account for this clinical heterogeneity. Significance analysis of microarray was used to identify marker genes of each subtype that were subject to pathway analysis. Association of molecular subtypes with clinical features and mutation data was analyzed with the Mann Whitney, Chi-square, Fisher’s exact, and Kruskal-Wallis tests. Survival analysis was evaluated with log-rank test. All statistical tests were two-sided.
Results
Four transcriptome subtypes with distinct clinicopathologic characteristics and mutation spectra were identified from the TCGA dataset and validated in an independent sample cohort of 184 EEC cases. Cluster II consisted of younger, obese patients with low-grade EEC but diminished survival. CTNNB1 exon 3 mutations were present in 87.0% (47/54) of Cluster II (P < .001) that exhibited a low overall mutation rate; this was statistically significantly associated with Wnt/β-catenin signaling activation (P < .001). High expression levels of CTNNB1 (P = .001), MYC (P = .01), and CCND1 (P = .01) were associated with poorer overall survival in low-grade EEC tumors.
Conclusions
CTNNB1 exon 3 mutations are likely a driver that characterize an aggressive subset of low-grade and low-stage EEC occurring in younger women.
doi:10.1093/jnci/dju245
PMCID: PMC4200060  PMID: 25214561
12.  Clinical next generation sequencing to identify actionable aberrations in a phase I program 
Oncotarget  2015;6(24):20099-20110.
Purpose
We determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer.
Methods
We reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA).
Results
Of the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified.
Conclusion
Multiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials.
PMCID: PMC4652990  PMID: 26015395
genomic sequencing; actionable genes
13.  Consequences of Universal MSI/IHC in Screening Endometrial Cancer Patients for Lynch Syndrome 
Gynecologic oncology  2014;134(2):319-325.
Objective
Determine factors impacting the uptake of genetic counseling and results of genetic testing following universal tumor testing for Lynch syndrome in patients with endometrial cancer.
Methods
The study population consisted of two unselected cohorts of endometrial cancer patients, 408 identified retrospectively and 206 identified prospectively. Immunohistochemistry for mismatch repair protein expression and/or microsatellite instability analysis was performed on these tumors. MLH1 methylation analysis was performed on tumors with loss of MLH1 protein. Tumor studies were considered suggestive of Lynch Syndrome if they showed immunohistochemical loss of MSH2, MSH6, or PMS2, loss of MLH1 without MLH1 promoter methylation, and/or microsatellite instability. Participants with suggestive tumor studies were contacted and offered genetic counseling and testing.
Results
In the retrospective cohort, 11% had tumor studies suggestive of Lynch syndrome. 42% were seen for genetic counseling. A germline mutation was detected in 40%, and one had a variant of uncertain significance. In the prospective cohort, 8.7% of patients had tumor testing suggestive of Lynch syndrome. 72% were seen for genetic counseling. Germline mutations were found in 40%, and one had a variant of uncertain significance. Common challenges included timing of re-contact, age, perceived lack of relevance, inability to travel, and limited insurance coverage.
Conclusions
There are several barriers to genetic counseling and testing follow up after universal tumor testing, and uninformative genetic test results present a management challenge. It is important to consider these limitations when implementing an approach to screening endometrial cancer patients for Lynch syndrome.
doi:10.1016/j.ygyno.2014.06.009
PMCID: PMC4125501  PMID: 24933100
14.  Beyond BRAFV600: clinical mutation panel testing by next-generation sequencing in advanced melanoma 
The management of melanoma has evolved due to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent event in tumors with BRAFV600 and NRAS mutations. Melanomas with BRAFNon-V600 mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.
doi:10.1038/jid.2014.366
PMCID: PMC4289407  PMID: 25148578
melanoma; sequencing; mutation; BRAF; NRAS; TP53
15.  Evaluation of Clinical Criteria for the Identification of Lynch Syndrome among Unselected Endometrial Cancer Patients 
Clinical criteria, primarily young age of cancer onset and family history of signature cancers, have been developed to identify individuals at elevated risk for Lynch Syndrome with the goals of early identification and cancer prevention. In 2007, the Society of Gynecologic Oncology (SGO) codified criteria for women presenting with gynecologic cancers. These criteria have not been validated in a population-based setting. For 412 unselected endometrial cancers, immunohistochemical expression of DNA mismatch repair proteins and MLH1 methylation were assessed to classify tumors as sporadic or probable Lynch Syndrome. In this cohort, 10.5% of patients were designated as probable Lynch Syndrome based on tumor testing. The sensitivity and specificity of SGO criteria to identify these same cases were 32.6% (95% CI 19.2–48.5) and 77% (95% CI 72.7–81.8), respectively. With the exception of tumor location in the lower uterine segment, multivariate analysis of clinical features, family history, and pathologic variables failed to identify significant differences between the sporadic and probable Lynch Syndrome groups. A simplified cost-effectiveness analysis demonstrated that SGO clinical criteria and universal tissue testing strategies had comparable costs per probable Lynch Syndrome patient identified. In conclusion, SGO criteria successfully identify probable Lynch Syndrome cases among women with endometrial cancer who are young or have significant family history of signature tumors. However, a larger proportion of probable Lynch Syndrome patients who are older and have less significant family history are not detected by this screening strategy. Universal tissue testing may be necessary to capture more individuals at risk for having Lynch Syndrome.
doi:10.1158/1940-6207.CAPR-13-0359
PMCID: PMC4090057  PMID: 24771847
Lynch Syndrome; Endometrial Cancer; Screening; Cost analysis
16.  Retrospective Review of MET Gene Mutations 
Oncoscience  2015;2(5):533-541.
C-MET proto-oncogene is a tyrosine kinase situated on chromosome 7. C-MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) play a role in proliferation, differentiation and organ development. C-MET genetic aberrations are found associated with driving tumorigenesis. In this retrospective study, we reviewed molecular analysis data gathered from a cancer institute during a two-year period (2010-2012). Upon detection of tumors harboring c-MET mutations, we determined the status of the other mutations tested and evaluated c-MET expression by fluorescent in-situ hybridization (FISH). Our search resulted in identification of 134 c-MET mutations, 44% of which had mutations of at least one of the other genes tested. No c-MET expression aberrancy was detected in this subset by FISH. Survival amongst the patients with surgically resected metastatic colorectal cancers (CRC) was slightly better in those with only a c-MET mutation compared to those with no mutation detected, although the difference was not statistically significant. When c-MET inhibition becomes an integrated part of chemotherapy practice, our observed frequency of co-mutations will be an argument for utilizing c-MET targeted treatment in combination with other targeted drugs and therapeutic strategies. Larger studies can aid to further parse out c-MET prognostic and therapeutic significance.
PMCID: PMC4468339  PMID: 26097886
C-MET; co-mutations; FISH; targeted therapy
19.  PIK3CA, and PTEN Aberrations in Early-Phase Trials with PI3K/AKT/mTOR Inhibitors: Experience with 1,656 Patients at MD Anderson Cancer Center 
Cell reports  2014;6(2):377-387.
Despite a wealth of preclinical studies, it is unclear whether PIK3CA or PTEN gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CA or PTEN abnormalities, PIK3CA mutations were found in 9% (146/1,589), and PTEN loss and/or mutation in 13% (149/1,157). In multicovariable analysis, treatment with a PI3K/AKT/mTOR inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CA or PTEN aberration. The rate of stable disease (SD) ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CA mutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations.
doi:10.1016/j.celrep.2013.12.035
PMCID: PMC4409143  PMID: 24440717
PIK3CA; PTEN; KRAS; NRAS; BRAF; Cancer; Clinical trial
20.  Crosstalk between EphA2 and BRaf/CRaf is a Key Determinant of Response to Dasatinib 
Purpose
EphA2 is an attractive therapeutic target due to its diverse roles in cancer growth and progression. Dasatinib is a multi-kinase inhibitor that targets EphA2 and other kinases. However, reliable predictive markers and a better understanding of the mechanisms of response to this agent are needed.
Experimental design
The effects of dasatinib on human uterine cancer cell lines were examined using a series of in vitro experiments, including MTT, Western blot, and plasmid transfection. In vivo, an orthotopic mouse model of uterine cancer was utilized to identify the biological effects of dasatinib. Molecular markers for response prediction and the mechanisms relevant to response to dasatinib were identified by using RPPA, immunoprecipitation, and double immunofluorescence staining.
Results
We show that high levels of CAV-1, EphA2 phosphorylation at S897 and the status of PTEN are key determinants of dasatinib response in uterine carcinoma. A set of markers essential for dasatinib response was also identified and includes CRaf, pCRafS338, pMAPKT202/Y204 (MAPK pathway), pS6S240/244, p70S6kT389 (mTOR pathway) and pAKTS473. A novel mechanism for response was discovered whereby high expression level of CAV-1 at the plasma membrane disrupts the BRaf/CRaf heterodimer and thus inhibits the activation of MAPK pathway during dasatinib treatment.
Conclusions
Our in vitro and in vivo results provide a new understanding of EphA2 targeting by dasatinib and identify key predictors of therapeutic response. These findings have implications for ongoing dasatinib-based clinical trials.
doi:10.1158/1078-0432.CCR-13-2141
PMCID: PMC3975695  PMID: 24486585
Dasatinib; EphA2; Caveolin-1 (CAV-1); Uterine Cancer
21.  Potentiation of Colon Cancer Susceptibility in Mice by Colonic Epithelial PPAR-δ/β Overexpression 
Background
The nuclear receptor peroxisome proliferator-activated receptor-δ/β (PPAR-d) is upregulated in human colorectal cancers, but its role in colonic tumorigenesis remains controversial.
Methods
We generated a novel mouse model of intestinally targeted PPAR-d overexpression to simulate PPAR-d upregulation in human colon carcinogenesis. Colon-specific PPAR-d overexpression was confirmed by real-time reverse transcription polymerase chain reaction, immunoblotting, and activity assays. Mice with and without targeted PPAR-d overexpression were tested for azoxymethane (AOM)–induced colonic tumorigenesis. Mouse whole-genome transcriptome microarray analyses were performed to identify PPAR-d target genes to promote tumorigenesis. We used linear models to test for PPAR-d overexpression trend effects on tumor multiplicity. All statistical tests were two-sided.
Results
Targeted PPAR-d overexpression markedly increased colonic tumor incidence (from 0 of 10 wild-type [WT] littermate mice to 9 of 10 mice [P < .001] in 2 FVB/N background mouse lines [villin-PPAR-d-1 and villin-PPAR-d-2] at a 5-mg/kg AOM dose) and multiplicity (number of tumors per mouse per mg/kg dose of AOM increased from 0.47 [95% confidence interval [CI] = 0.22 to 0.72] for the WT littermates to 2.15 [95% CI = 1.90 to 2.40] [P < .001] for the villin-PPAR-d-1 mice and from 0.44 [95% CI = 0.09 to 0.79] for the WT littermates to 1.91 [95% CI = 1.57 to 2.25] [P < .001] for the villin-PPAR-d-2 mice). PPAR-d overexpression reversed resistance to AOM-induced colonic tumorigenesis in C57BL/6 mice. PPAR-d overexpression modulated expression of several novel PPAR-d target genes in normal-appearing colonic epithelial cells of mice with PPAR-d overexpression in a pattern that matched the changes in colonic tumors.
Conclusions
Our finding that PPAR-d upregulation profoundly enhances susceptibility to colonic tumorigenesis should impact the development of strategies of molecularly targeting PPAR-d in cancer and noncancerous diseases.
doi:10.1093/jnci/dju052
PMCID: PMC3982893  PMID: 24681603
22.  Clinicopathologic Features and Prognosis of Duodenal Adenocarcinoma and Comparison with Ampullary and Pancreatic Ductal Adenocarcinoma 
Human pathology  2013;44(12):2792-2798.
Due to the rarity of duodenal adenocarcinoma (DAC), the clinicopathologic features and prognostication data for DAC are limited. There are no published studies directly comparing the prognosis of DAC to ampullary adenocarcinoma (AA) and pancreatic ductal adenocarcinoma (PDA) after resection. In this study, we examined the clinicopathologic features of 68 patients with DAC, 92 patients with AA and 126 patients with PDA, who underwent resection. Patient clinicopathologic and survival information were extracted from medical records. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) with two-sided significance level of 0.05. Patients with DAC had higher American Joint Committee on Cancer (AJCC) stage than AA patients (P=0.001). Lymph node metastasis (P=0.013) and AJCC stage (P=0.02) correlated with overall survival in DAC patients. Patients with DAC or AA had lower frequencies of lymph node metastasis and positive margin and better survival than those with PDA (P<0.05). However, no differences in nodal metastasis, margin status or survival were observed between DAC patients and those with AA. Our study showed that lymph node metastasis and AJCC stage are important prognostic factors for overall survival in DAC patients. Patients with DAC had less frequent nodal metastasis and better prognosis than those with PDA. There was no significant difference in prognosis between DAC and AA.
doi:10.1016/j.humpath.2013.07.030
PMCID: PMC4378233  PMID: 24139211
Duodenal adenocarcinoma; pancreatic cancer; ampullary adenocarcinoma; survival; prognosis
23.  Utility of MLH1 Methylation Analysis in the Clinical Evaluation of Lynch Syndrome in Women with Endometrial Cancer 
Current pharmaceutical design  2014;20(11):1655-1663.
Clinical screening criteria, such as young age of endometrial cancer diagnosis and family history of signature cancers, have traditionally been used to identify women with Lynch Syndrome, which is caused by mutation of a DNA mismatch repair gene. Immunohistochemistry and microsatellite instability analysis have evolved as important screening tools to evaluate endometrial cancer patients for Lynch Syndrome. A complicating factor is that 15-20% of sporadic endometrial cancers have immunohistochemical loss of the DNA mismatch repair protein MLH1 and high levels of microsatellite instability due to methylation of MLH1. The PCR-based MLH1 methylation assay potentially resolves this issue, yet many clinical laboratories do not perform this assay. The objective of this study was to determine if clinical and pathologic features help to distinguish sporadic endometrial carcinomas with MLH1 loss secondary to MLH1 methylation from Lynch Syndrome-associated endometrial carcinomas with MLH1 loss and absence of MLH1 methylation. Of 337 endometrial carcinomas examined, 54 had immunohistochemical loss of MLH1. 40/54 had MLH1 methylation and were designated as sporadic, while 14/54 lacked MLH1 methylation and were designated as Lynch Syndrome. Diabetes and deep myometrial invasion were associated with Lynch Syndrome; no other clinical or pathological variable distinguished the 2 groups. Combining Society of Gynecologic Oncology screening criteria with these 2 features accurately captured all Lynch Syndrome cases, but with low specificity. In summary, no single clinical/pathologic feature or screening criteria tool accurately identified all Lynch Syndrome-associated endometrial carcinomas, highlighting the importance of the MLH1 methylation assay in the clinical evaluation of these patients.
PMCID: PMC3895501  PMID: 23888949
Lynch Syndrome; molecular diagnostics; MLH1 methylation; immunohistochemistry; endometrial cancer
24.  Sex hormone regulation of survivin gene expression 
The Journal of endocrinology  2010;207(2):237-243.
Survivin (BIRC5) is a cell survival gene that is overexpressed in endometrial cancer and has been implicated to have a physiological role in normal endometrial function. To determine whether survivin gene expression is regulated by reproductive steroid hormones in the human endometrium, RNA was prepared from normal cycling women in the proliferative and secretory phases of the menstrual cycle. RNA was also isolated from 21 endometrial biopsies from premenopausal women at baseline and following 3 months of treatment with depot medroxyprogesterone acetate. Finally, RNA was isolated from endometrial biopsies from ten healthy postmenopausal women participating in a clinical trial of estrogen replacement therapy at baseline and following 6 months of treatment with conjugated equine estrogen. Quantitative RT-PCR analysis was used to determine survivin, insulin-like growth factor binding protein 1 (IGFBP1), Ki67, and IGF1 gene expression levels. Survivin gene expression was highest in the proliferative phase of the menstrual cycle and showed a statistically significant 4-fold increase in expression following chronic treatment with estrogens; this was strongly correlated with increased Ki67, a marker of proliferation. Survivin gene expression decreased 4·6-fold following chronic progestin treatment in the human endometrium. These data suggest that survivin transcript is regulated by estrogens and progestins in the disease-free human endometrium. The data also suggest that survivin transcript may be used as a biomarker of estrogen and progestin treatment efficacy, but validation studies must be conducted to support this conclusion.
doi:10.1677/JOE-10-0128
PMCID: PMC4270120  PMID: 20798131
25.  Clinicodemographic factors influencing outcomes in patients with low-grade serous ovarian carcinoma 
Cancer  2011;117(16):3741-3749.
Background
Low-grade serous carcinoma (LGSC) of the ovary is a rare tumor that is distinct from its high-grade counterpart. Our aim was to estimate if patient demographic factors and clinical treatment histories affected survival in a population of women with this disease.
Methods
A review of patients with pathologically-confirmed LGSC of the ovary diagnosed between 1977- 2009 was performed. Abstracted data included medical and social histories, anthropometric measurements, and details about diagnosis, treatment, and follow-up. Statistical analyses included Fisher's exact test, Cox proportional hazards models, and the Kaplan-Meier method.
Results
The study sample included 194 patients with a median follow-up of 60.9 months (range 1-383). By multivariable analyses, smoking was negatively associated with both overall survival (OS) (HR 1.73; 95% CI [1.03-2.92], p=0.04) and progression-free survival (PFS) (HR 1.72; 95% CI [1.00-2.96], p=0.05). Median OS was shorter in current smokers than former/never smokers (48.0 vs. 79.9 months; p=.002). PFS was also predicted by year of diagnosis >1994 (HR1.74, p=0.01). Although not statistically significant, hormone consolidation appeared to be associated with better OS (HR 0.15, p=.06) and better PFS (HR 0.44; p=.07). A smaller proportion of patients treated with hormone consolidation experienced recurrence compared to those who did not receive hormone consolidation (66.7% vs. 87.6%, p=0.07).
Conclusion
Smoking is negatively associated with survival outcomes in women with LGSC of the ovary, while consolidation treatment with hormone antagonists demonstrated a protective associative trend with survival. Both lifestyle modification and innovative treatment plans should be considered in this group of patients.
doi:10.1002/cncr.25929
PMCID: PMC4254832  PMID: 21319148

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