The Affordable Health Care Act annual wellness visits: the effectiveness of a nurse-run clinic in promoting adherence to mammogram and colonoscopy recommendations.

OBJECTIVE: The aim of this study was to determine the effectiveness of the nurse-run annual wellness visit (AWV) in improving adherence to cancer screening recommendations for colonoscopies and/or mammograms. BACKGROUND: The Affordable Health Care Act provides Medicare beneficiaries access to AWVs. Nurse-run AWVs offer individualized education, reinforce health screening recommendations, and may enhance the patients' intent to complete the screenings. METHODS: A nonexperimental comparative study was conducted using data collected from chart audits comparing patients who only attended the AWV, patients who attended the AWV linked with a physician visit, and patients who have not attended an AWV. RESULTS: Patients who attended the AWV showed greater adherence to mammogram completion regardless of the link to the physician follow-up visit. Differences in adherence to colonoscopy recommendations were not significant, likely because of the low number of colonoscopies reported. CONCLUSIONS: Nurse-run AWV clinics are associated with adherence to mammograms and show promise of increasing colonoscopy compliance.

Estrogen Promotes Resistance to Bevacizumab in Murine Models of NSCLC.

INTRODUCTION: Subgroup analyses from clinical studies have suggested that among patients with metastatic NSCLC receiving chemotherapy, females may derive less benefit from the addition of the vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (BV) than males. This has raised the question of whether estrogen may affect the response to antiangiogenic therapy. METHODS: To address this, we investigated the effects of estrogen on tumor growth, angiogenesis, and the response to BV in human xenograft models of NSCLC. RESULTS: We observed that estrogen induced marked resistance to BV, which was accompanied by a 2.3-fold increase in tumor vascular pericyte coverage (p = 0.01) and an up-regulation of proangiogenic factors, VEGF and platelet-derived growth factor-BB. We also investigated the role of infiltrating myeloid cells, a population that has been associated with resistance to anti-VEGF therapies. We observed that estrogen induced a greater than twofold increase (p = 0.001) in the recruitment of tumor-infiltrating myeloid cells and concomitant increases in the myeloid recruitment factors, G-CSF and CXCL1. Blockade of the estrogen receptor pathway using fulvestrant resensitized tumors to VEGF targeting as evidenced by reduced tumor vasculature and an increase in overall survival in our NSCLC xenograft models. CONCLUSIONS: Collectively, these data provide evidence that estrogen may promote resistance to VEGF-targeted therapies, potentially by enhancing pericyte coverage and myeloid recruitment, and suggest that estrogen receptor blockade merits further investigation as an approach to enhance the effects of antiangiogenic therapy.

Altered Regulation of HIF-1α in Naive- and Drug-Resistant EGFR-Mutant NSCLC: Implications for a Vascular Endothelial Growth Factor-Dependent Phenotype.

INTRODUCTION: The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. METHODS: We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models in vitro and in vivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. RESULTS: NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. CONCLUSIONS: In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor-naive and refractory settings.

A hypersensitive estrogen receptor alpha mutation that alters dynamic protein interactions.

Estrogen receptor alpha (ERalpha) is highly regulated through multiple mechanisms including cell signaling, posttranslational modifications, and protein-protein interactions. We have previously identified a K303R ERalpha mutation within the hinge region of ERalpha. This mutation results in an altered posttranslational regulation and increased in vitro growth in the presence of low estrogen concentrations. We sought to determine if cells expressing this mutant ERalpha would display hypersensitive tumor growth in in vivo athymic ovariectomized nude mice. MCF-7 cells, stably expressing the K303R ERalpha, formed tumors in nude mice faster than cells expressing wild-type ERalpha in the presence of low levels of estrogen. When estrogen was withdrawn, all tumors regressed but half of the K303R ERalpha-expressing tumors became estrogen-independent and regrew. We evaluated potential mechanisms for the observed hypersensitivity. The mutant ERalpha did not demonstrate increased estrogen binding affinity, but did exhibit increased interactions with members of the SRC family of coactivators. The mutant ERalpha demonstrated increased levels and occupancy time on the pS2 promoter. In the presence of the K303R ERalpha, the SRC-3 and p300 coactivators also displayed increased levels and time on the pS2 promoter. The K303R ERalpha has, in part, lost critical negative regulation by the F domain. Collectively, these data demonstrate an important role for the K303R ERalpha mutation in hormonal regulation of tumor growth and estrogen-regulated promoter dynamics in human breast cancer.

Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells.

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) alpha mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERalpha-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERalpha that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Estrogen receptor mutations in human disease.

As early as the 1800s, the actions of estrogen have been implicated in the development and progression of breast cancer. The estrogen receptor (ER) was identified in the late 1950s and purified a few years later. However, it was not until the 1980s that the first ER was molecularly cloned, and in the mid 1990s, a second ER was cloned. These two related receptors are now called ERalpha and ERbeta, respectively. Since their discovery, much research has focused on identifying alterations within the coding sequence of these receptors in clinical samples. As a result, a large number of naturally occurring splice variants of both ERalpha and ERbeta have been identified in normal epithelium and diseased or cancerous tissues. In contrast, only a few point mutations have been identified in human patient samples from a variety of disease states, including breast cancer, endometrial cancer, and psychiatric diseases. To elucidate the mechanism of action for these variant isoforms or mutant receptors, experimental mutagenesis has been used to analyze the function of distinct amino acid residues in the ERs. This review will focus on ERalpha and ERbeta alterations in breast cancer.

Association between the estrogen receptor alpha A908G mutation and outcomes in invasive breast cancer.

PURPOSE: Estrogen receptor alpha (ERalpha) predicts the natural history of breast cancer without intervening therapy. Here, we have optimized the detection of a somatic mutation, an A908G transition of ERalpha, and examined its association with clinical and biological features of invasive breast cancer. EXPERIMENTAL DESIGN: We compared two methods of sequencing to detect the A908G ERalpha mutation. We then used primer extension sequencing with genomic DNA isolated from invasive breast tumors to determine whether the mutation was associated with clinical outcome in 267 axillary node-negative and axillary node-positive breast tumors. The presence of the mutation and clinical variables were analyzed for association with recurrence-free survival and overall survival by Cox proportional hazards regression models. RESULTS: We determined that dye-labeled terminator sequencing was not adequate for detection of the A908G ERalpha mutation. The mutation was detected at a high frequency (50%) in invasive breast tumors using primer extension sequencing, and was found to be associated with clinical measures of poor outcome, including larger tumor size and axillary lymph node positivity. Although the mutation was associated with recurrence-free survival in univariate analysis, it was not an independent predictor of outcomes in multivariate analysis. CONCLUSIONS: Consistent with our previous finding of this somatic ERalpha mutation in breast ductal hyperplasias, we now present evidence that the A908G mutation is present in invasive breast tumors using an optimized sequencing method. We find that the mutation is significantly associated with aggressive biological tumor features, and with an unfavorable prognosis, but was not an independent prognostic marker in untreated patients.

Activation of Src by c-Met overexpression mediates metastatic properties of colorectal carcinoma cells.

Overexpression and/or activation of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF), and the protein tyrosine kinase, Src, have been implicated in the progression and metastasis of human colorectal carcinoma (CRC). Previously, through ribozyme-mediated c-Met downregulation, we demonstrated a causal role for c-Met in CRC tumorigenesis and the production of liver metastases from the highly metastatic CRC cell line, KM20. Analysis of signaling intermediates downstream of c-Met demonstrated a specific reduction of Src activity with minimal effect on Erk1/2 and Akt following c-Met downregulation. As Src has been shown to upregulate Vascular Endothelial Growth Factor (VEGF), we sought to determine if the reduced tumor size and incidence could be due to reduced vessel formation in the c-Met downregulated clones. Here we show that tumors produced from c-Met downregulated cells have significantly reduced vessel density in tumors, correlating with a reduction in VEGF production. Additionally, the Src selective inhibitor, PP2, significantly reduced basal VEGF production in KM20 parental cells, and this effect could be rescued by HGF treatment. PP2 reduced basal proliferation, migration, and anchorage-independent growth. Furthermore, PP2 treatment demonstrated a dose-dependent decrease in HGF induced migration. In contrast, PP2 treatment only had a minor effect on HGF induced anchorage-independent growth. Collectively, these data demonstrate a significant role for c-Met-mediated Src activation in processes involved in CRC tumorigenesis and liver metastasis.

Estrogen receptors in resistance to hormone therapy.

Estrogen and its receptors alpha and beta (ERalpha and ERbeta) play a major role in tumor progression and approximately two-thirds of breast cancers express these functional receptors. Thus, the ER is a major target for current and developing therapies. Although most ER-positive tumors initially respond to hormonal therapies such as tamoxifen, many tumors will eventually become resistant to tamoxifen induced growth inhibition. This chapter will discuss molecular mechanisms that contribute to hormonal resistance of current therapies including ERalpha mutations, the roles of proliferation and apoptosis in tumor homeostasis and receptor coregulator proteins. Additionally, the role of nonclassical ERalpha signaling through growth factor receptors and the subsequent downstream-initiated signaling, and the role of the progesterone receptors will be discussed.

Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor-positive human breast cancer cells.

It has long been appreciated that estrogenic signaling contributes to breast cancer progression. c-Src is also required for a number of processes involved in tumor progression and metastasis. We have previously identified the K303R mutant estrogen receptor alpha (ERalpha) that confers hypersensitivity to low levels of estrogen. Because ERalpha and c-Src have been shown to interact in a number of different systems, we wanted to evaluate the role of c-Src kinase in estrogen-stimulated growth and survival of ERalpha-positive breast cancer cells. MCF-7 cells stably expressing the mutant receptor showed increased c-Src kinase activity and c-Src tyrosine phosphorylation when compared with wild-type ERalpha-expressing cells. A c-Src inhibitor, AZD0530, was used to analyze the biological effects of pharmacologically inhibiting c-Src kinase activity. MCF-7 cells showed an anchorage-dependent growth IC50 of 0.47 micromol/L, which was increased 4-fold in the presence of estrogen. In contrast, cells stably expressing the mutant ERalpha had an elevated IC50 that was only increased 1.4-fold by estrogen stimulation. The c-Src inhibitor effectively inhibited the anchorage-independent growth of both of these cells, and estrogen was able to reverse these effects. When cells were treated with suboptimal concentrations of c-Src inhibitor and tamoxifen, synergistic inhibition was observed, suggesting a cooperative interaction between c-Src and ERalpha. These data clearly show an important role for ERalpha and estrogen signaling in c-Src-mediated breast cancer cell growth and survival. Here, we show that c-Src inhibition is blocked by estrogen signaling; thus, the therapeutic use of c-Src inhibitors may require inhibition of ERalpha in estrogen-dependent breast cancer.

The guanine nucleotide exchange factor Tiam1 increases colon carcinoma growth at metastatic sites in an orthotopic nude mouse model.

Alterations in migration and adhesion are critical to invasion and metastasis. To examine signaling pathways important for colon tumor metastasis, cells of increased migratory potential from the low migratory SW480 human colorectal carcinoma parental cell line were biologically selected by serial migration through modified Boyden chambers. Several sublines were obtained with statistically significantly increased migration relative to the parental cell line. One highly migratory population was single-cell cloned and characterized. The migratory clones exhibit a four- to five-fold increase in protein and mRNA expression of T-lymphoma invasion and metastasis gene 1 (Tiam1), a guanine nucleotide exchange factor. To determine directly the role of Tiam1 in the migration of these migratory sublines, the parental SW480 cell line was transfected with a plasmid encoding the Tiam1 protein, and single cell clones were established. Ectopic expression of Tiam1 in these clones led to morphologic changes identical to biologically selected clones and increased migration. Finally, the implantation of clones that overexpress Tiam1 into the cecum of athymic mice resulted in tumor growth in the spleen, liver, and lung, whereas parental cells do not form tumors by this route of injection. These results demonstrate that overexpression of Tiam1 contributes to the metastatic phenotype of colon cancer cells.

Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cells.

Overexpression of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. To examine the role of c-Met on in vitro and in vivo growth of human colon tumor cell lines, stable subclones of the high metastatic human colorectal carcinoma cell line, KM20, isolated from a Dukes' D patient, with reduced c-Met expression were obtained after transfection with a c-Met-specific targeting ribozyme. The subclones were only modestly reduced in c-Met expression because of c-Met playing an important role in cellular survival. However, a 60-90% reduction in basal c-Met autophosphorylation and kinase activity were observed. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates and soft-agar colony-forming abilities. The in vivo growth of these cells was examined at both the ectopic SQ site and the orthotopic site of metastatic growth, the liver. SQ growth was delayed significantly in the c-Met down-regulated clones compared with controls, with tumors growing on loss of the ribozyme construct. In contrast, tumor incidence was significantly reduced when the c-Met down-regulated cells were grown in the orthotopic liver site. Thus, c-Met activation may be important in metastatic growth of colon tumor cells in the liver. Collectively these data demonstrate that a small reduction in c-Met protein levels leads to profound biological effects, and potential c-Met inhibitors may be of therapeutic value in treatment of colon cancer.

Reduced c-Met expression by an adenovirus expressing a c-Met ribozyme inhibits tumorigenic growth and lymph node metastases of PC3-LN4 prostate tumor cells in an orthotopic nude mouse model.

PURPOSE: The expression of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, frequently increases during prostate tumor progression. However, whether reduced c-Met expression inhibits tumor growth and metastasis has not been ascertained. EXPERIMENTAL DESIGN: c-Met expression was reduced by infection of an adenovirus expressing a c-Met ribozyme into the highly metastatic human prostate cancer cell line PC3-LN4. In vitro, effects on c-Met, Akt, and extracellular signal-regulated kinase 1/2 expression and phosphorylation, Src expression and activity, and vascular endothelial growth factor expression were determined, as were effects on cell migration and invasion. Prostate tumor formation and metastasis to regional lymph nodes in nude mice were examined after both ex vivo and in vivo infection of cells. RESULTS: Infection of PC3-LN4 cells with the Ad-c-Met-expressing ribozyme decreased steady-state c-Met levels, decreased Src kinase activity, decreased vascular endothelial growth factor expression, and decreased migration and invasion versus the pU1 (control) virus. Significant inhibition of tumorigenicity (histologically confirmed tumors in only 1 of 10 mice) and consequent lymph node metastasis were observed upon ex vivo infection of Ad-c-Met. Similarly, gene therapy experiments led to complete inhibition of tumor growth in 7 of 8 mice. CONCLUSIONS: Reduction in c-Met expression substantially inhibits both tumor growth and lymph node metastasis of PC3-LN4 cells in orthotopic nude mouse models. Therefore, targeting the c-Met signaling pathways may be important in controlling tumor growth and metastasis in human prostate cancers.

Activation of c-Met in colorectal carcinoma cells leads to constitutive association of tyrosine-phosphorylated beta-catenin.

Increased expression and/or activity of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, occurs commonly during colon tumor progression. To examine potential roles for c-Met in promoting metastasis, we compared the colon tumor cell line KM12C with low metastatic potential to the isogenic variants KM,12L4 and KM12SM with high metastatic potential. KM12C cells express c-Met with low levels of tyrosine phosphorylation in the absence of HGF. The high metastatic cells express a c-Met that is constitutively tyrosine phosphorylated, they have increased colony formation, and are minimally responsive to HGF relative to the parental cells. Tyrosine-phosphorylated beta-catenin was constitutively associated with c-Met in the more metastatic cells, but was inducible only after HGF addition in the less metastatic cells. Functions mediated by beta-catenin, including cell-cell adhesion and migration, and activation of the tcf (T-cell factor) family of transcription factors, were also elevated in the more metastatic KM12SM and L4 cells. Furthermore, analysis of the known tcf transcriptional target genes, cyclin D1, c-Myc, and uPAR, demonstrated increased expression in the high metastatic cells, correlating with the levels of tcf activity. Collectively, these results suggest that endogenous activation of c-Met in highly metastatic KM12SM CRC cells results in increased survival and growth under anchorage independent conditions, increased in vitro migration, and elevated levels of tcf target genes. Thus, beta-catenin association with activated c-Met may contribute to a more aggressive liver metastatic phenotype of these cells.

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor-resistant human lung adenocarcinoma.

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Dicer-mediated upregulation of BCRP confers tamoxifen resistance in human breast cancer cells.

PURPOSE: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. EXPERIMENTAL DESIGN: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. RESULTS: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. CONCLUSION: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity.

Expression of the K303R estrogen receptor-alpha breast cancer mutation induces resistance to an aromatase inhibitor via addiction to the PI3K/Akt kinase pathway.

Aromatase inhibitors (AI) are rapidly becoming the first choice for hormonal treatment of estrogen receptor-alpha (ERalpha)-positive breast cancer in postmenopausal women. However, de novo and acquired resistance frequently occurs. We have previously identified a lysine to arginine transition at residue 303 (K303R) in ERalpha in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to tamoxifen treatment. Thus, we questioned whether resistance to AIs could arise in breast cancer cells expressing the ERalpha mutation. As preclinical models to directly test this possibility, we generated K303R-overexpressing MCF-7 cells stably transfected with an aromatase expression vector. Cells were stimulated with the aromatase substrate, androstenedione, with or without the AI anastrozole (Ana). We found that Ana decreased androstenedione-stimulated growth of wild-type cells, whereas K303R-expressing cells were resistant to the inhibitory effect of Ana on growth. We propose that a mechanism of resistance involves an increased binding between the mutant receptor and the p85alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K), leading to increased PI3K activity and activation of protein kinase B/Akt survival pathways. Inhibition of the selective "addiction" to the PI3K/Akt pathway reversed AI resistance associated with expression of the mutant receptor. Our findings suggest that the K303R ERalpha mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and that targeting the PI3K/Akt pathway may be a useful strategy for treating patients with tumors resistant to hormone therapy.

Immunohistochemical expression of estrogen and progesterone receptors identifies a subset of NSCLCs and correlates with EGFR mutation.

PURPOSE: To determine the frequency of estrogen receptor alpha and beta and progesterone receptor protein immunohistochemical expression in a large set of non-small cell lung carcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports. EXPERIMENTAL DESIGN: Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors alpha and beta and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients' clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status. RESULTS: Estrogen receptors alpha and beta were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for alpha and 42-56% for beta; cytoplasm: <1-42% for alpha and 20-98% for beta) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor alpha nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor alpha expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor alpha expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor beta and progesterone receptor but not estrogen receptor alpha expressed in the normal epithelium adjacent to lung adenocarcinomas. CONCLUSIONS: Estrogen receptor alpha and beta expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor alpha expression correlates with EGFR mutations.