A Longitudinal Comparison of Pharmacy Documentation Platforms Using the Technology Acceptance Model: Experiences With Opioid Risk Screening.

Background: Pharmacy practice continues to expand in scope, and technology platforms to assist with meeting the standards for documentation of billable services are needed. The ONE Program (Opioid and Naloxone Education) is an initiative centered on the community pharmacy focused on opioid risk screening for patients receiving opioid prescriptions. Objective: Opioid risk screening results and pharmacist interventions were documented using first REDCap and later the DocStation platforms. This study compared pharmacy staff experience with these 2 platforms. Methods: A survey using the Technology Acceptance Model (TAM) was designed to compare usability, ease of use, social influence, and facilitating conditions. Results: Analyses using descriptive statistics and open-ended responses showed similar results for each platform; however, pharmacy staff indicated that REDCap required less time when entering information, whereas the DocStation platform offered elevated pharmacy practice service opportunities, management support, and available informational technology support services. Conclusion: Health care technology continues to advance in meeting the needs of expanded service provision through pharmacy. This longitudinal study shows the value of the TAM framework in identifying efficiencies and deficiencies of health care technology systems.

Factors Likely to Affect the Uptake of Genomic Approaches to Cancer Screening in Primary Care: A Scoping Review.

Genomic tests are being developed for use in cancer screening. As most screening is offered in primary care settings, primary care provider and patient perceptions of such tests are likely to affect uptake. We conducted a scoping review to synthesize information on factors likely to affect patient and provider use of biospecimen collection and analysis for cancer screening, methods referred to as liquid biopsy or multi-cancer early detection (MCED) testing when used to detect multiple cancers. We ultimately identified 7 articles for review and analyzed them for major themes. None reported on primary care provider perspectives. Six articles focused on patient perceptions about testing for a single cancer (colorectal), and 1 reported on patient views related to testing for multiple cancers. Factors favoring this type of testing included its non-invasiveness, and the perceived safety, convenience, and effectiveness of testing. There is a dearth of information in the literature on primary care provider perceptions about liquid biopsy and MCED testing. The limited information on patient perceptions suggests that they are receptive to such tests. Research on primary care provider and patient test-related knowledge, attitudes, and behavior is needed to guide future implementation in primary care settings.

Genomic Characterization of Testicular Germ Cell Tumors Relapsing After Chemotherapy.

BACKGROUND: Although both seminomatous and nonseminomatous testicular germ cell tumors (TGCTs) have favorable outcomes with chemotherapy, a subset is chemorefractory, and novel therapeutic options are needed. OBJECTIVE: To molecularly characterize chemotherapy-refractory TGCTs. DESIGN, SETTING, AND PARTICIPANTS: Archival tissues from 107 chemotherapy-treated and relapsed TGCT patients (23 seminomas; 84 nonseminomas) underwent hybrid-capture-based genomic profiling to evaluate four classes of genomic alterations (GAs). Tumor mutational burden (TMB) and microsatellite instability (MSI) were also measured. INTERVENTION: Genomic profiling on tumor samples from chemotherapy-refractory TGCTs. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Descriptive analyses and differences between seminoma and nonseminoma subgroups were reported. RESULTS AND LIMITATIONS: The mean GA/tumor was 2.9 for seminomas and 4.0 for nonseminomas (p=0.04). KRAS alterations (mainly amplifications) were the most common GAs at the single-gene level (47.8% of seminomas and 51.2% of nonseminomas). RAS-RAF pathway (56.5% vs 52.3%) and cell-cycle pathway (52.2% vs 56.0%) were the most common GA classes in seminomas and nonseminomas, respectively. Receptor tyrosine kinase pathway and PI3K pathway GAs were more frequent in seminomas (p=0.02). Median TMB was 1.8 mutations/Mb for seminomas and 2.7 mutations/Mb for nonseminomas (p=0.098), and MSI-high status was found in one nonseminoma only (1.2%). A lack of clinical outcome correlation is a limitation of the present analyses. CONCLUSIONS: In chemotherapy-refractory TGCTs, trials with agents targeting the KRAS pathway may be pursued due to the high frequency of KRAS GAs. Overall, the GAs found in refractory seminomas and nonseminomas differ significantly. Considering the frequency of high TMB or MSI-high status, immunotherapy may benefit a small subset of nonseminomas. PATIENT SUMMARY: Testicular cancers that are resistant to or relapse after standard chemotherapy may harbor genomic alterations that are potentially druggable, particularly in the clinical trial setting, and genomic profiling can guide clinical research and disclose therapeutic opportunities for these patients.

Clinical Activity of Nivolumab for Human Papilloma Virus-Related Juvenile-Onset Recurrent Respiratory Papillomatosis.

BACKGROUND: Juvenile-onset recurrent respiratory papillomatosis (JO-RRP) is a human papilloma virus-mediated progressive benign neoplasm that affects children and young adults. Primary management consists of regular surgical debulking to maintain airway patency and vocal function. Like condyloma acuminata, JO-RRP is associated with immune dysregulation, and T cells isolated from papillomas express an anergic phenotype. Therefore, we hypothesized that programmed death protein 1 axis inhibition could stabilize tumor growth. MATERIALS AND METHODS: We treated two patients with refractory JO-RRP using nivolumab, with the primary objective of assessing clinical activity. We explored baseline papilloma features using immunohistochemistry and comprehensive genomic profiling. RESULTS: Both patients experienced symptomatic improvement, and interval laryngoscopies revealed a reduction in papillomatosis burden. One patient has not required subsequent surgical debridement for almost 2 years. On pathologic examination of pretreatment papillomas from both cases, infiltrating T cells were evident in the papilloma stroma, and papilloma programmed death ligand 1 expression was absent. Papilloma mutational load ranged between three and six mutations per megabase for each case. From on-treatment biopsy tissue, a higher amount of intraepithelial T cells and programmed death ligand 1 expression were detected in the papilloma. CONCLUSION: Nivolumab appears to have promising activity in JO-RRP, and further clinical investigation with more patients in clinical trials is warranted. IMPLICATIONS FOR PRACTICE: To the authors' knowledge, this article is the first report describing clinical activity with a programed cell death-1 (PD-1) inhibitor to treat a rare but detrimental type of respiratory tract epithelial neoplasm that afflicts young adults. Two patients were treated, and tumor features, such as mutational load, were examined with the intent to stimulate future hypotheses for translational research. The safety and activity of PD-1 inhibitors in this population still need to be corroborated in clinical trials and should not yet be adopted into clinical practice.

Hybrid Capture-Based Genomic Profiling of Circulating Tumor DNA from Patients with Advanced Non-Small Cell Lung Cancer.

INTRODUCTION: Genomic profiling informs selection of matched targeted therapies as part of routine clinical care in NSCLC. Tissue biopsy is the criterion standard; however, genomic profiling of blood-derived circulating tumor DNA (ctDNA) has emerged as a minimally invasive alternative. METHODS: Hybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 1552 patients with NSCLC. RESULTS: Evidence of ctDNA was detected in 80% of samples, and in 86% of these cases, at least one reportable genomic alteration (GA) was detected. Frequently altered genes were tumor protein p53 gene (TP53) (59%), EGFR (25%), and KRAS (17%). Comparative analysis with a tissue genomic database (N = 21,500) showed similar frequencies of GAs per gene, although KRAS mutation and EGFR T790M were more frequent in tissue and ctDNA, respectively (both p < 0.0001), likely reflecting the use of liquid versus tissue biopsy after relapse during targeted therapy. In temporally matched ctDNA and tissue samples from 33 patients with evidence of ctDNA in their blood, 64% of GAs detected in tissue were also detected in ctDNA, including 78% of short variants (58 of 74) and 100% of rearrangements (four of four), but only 16% of amplifications (four of 25). CONCLUSIONS: Genomic profiling of ctDNA detected clinically relevant GAs in a significant subset of NSCLC cases. Most alterations detected in matched tissue were also detected in ctDNA. These results suggest the utility of ctDNA testing in advanced NSCLC as a complementary approach to tissue testing. Blood-based ctDNA testing may be particularly useful at the time of progression during targeted therapy.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

A prolonged response to platinum-based therapy in a patient with metastatic urothelial carcinoma harboring a single rearranged and truncated NF2 gene.

Tumor genome sequencing has become an invaluable resource in determining targets for new therapies. In this report, we describe the case of a patient with metastatic urothelial carcinoma with sarcomatoid features. Sarcomatoid differentiation is a rare histologic subtype that confers a more aggressive course. The first-line treatment for patients with urothelial carcinoma is platinum-based chemotherapy. Next generation tumor sequencing performed using the FoundationOne assay revealed loss of one NF2 allele and an unbalanced der(22)t(10;22)(p11.22;q12.2) chromosomal rearrangement involving the other NF2 allele, resulting in truncation and predicted loss of function. Fluorescence in situ hybridization (FISH) analysis confirmed the presence of one NF2 signal. NF2 mutations have been found in a variety of cancers and result in activation of the mTOR pathway. As such, the use of mTOR inhibitors, such as everolimus are thought to be particularly effective in the case of NF2 loss. Our patient had a dramatic response to first-line chemotherapy, but unfortunately experienced subsequent progression of his cancer and could not tolerate everolimus. Although our patient's tumor demonstrated unique acquired genetic features including both loss of heterozygosity and truncation of the NF2 locus, he still achieved a meaningful response to platinum-based chemotherapy.

Hybrid Capture-Based Genomic Profiling of Circulating Tumor DNA from Patients with Advanced Cancers of the Gastrointestinal Tract or Anus.

Purpose: Genomic profiling of tumor biopsies from advanced gastrointestinal and anal cancers is increasingly used to inform treatment. In some cases, tissue biopsy can be prohibitive, and we sought to investigate whether analysis of blood-derived circulating tumor DNA (ctDNA) may provide a minimally invasive alternative.Experimental Design: Hybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 417 patients with gastrointestinal carcinomas to assess the presence of genomic alterations (GA) and compare with matched tissue samples.Results: Evidence of ctDNA was detected in 344 of 417 samples (82%), and of these, ≥1 reportable GA was detected in 89% (306/344) of samples. Frequently altered genes were TP53 (72%), KRAS (35%), PIK3CA (14%), BRAF (8%), and EGFR (7%). In temporally matched ctDNA and tissue samples available from 25 patients, 86% of alterations detected in tissue were also detected in ctDNA, including 95% of short variants, but only 50% of amplifications. Conversely, 63% of alterations detected in ctDNA were also detected in matched tissue. Examples demonstrating clinical utility are presented.Conclusions: Genomic profiling of ctDNA detected potentially clinically relevant GAs in a significant subset of patients with gastrointestinal carcinomas. In these tumor types, most alterations detected in matched tissue were also detected in ctDNA, and with the exception of amplifications, ctDNA sequencing routinely detected additional alterations not found in matched tissue, consistent with tumor heterogeneity. These results suggest feasibility and utility of ctDNA testing in advanced gastrointestinal cancers as a complementary approach to tissue testing, and further investigation is warranted. Clin Cancer Res; 24(8); 1881-90. ©2018 AACR.

Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation.

PURPOSE: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. EXPERIMENTAL DESIGN: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. RESULTS: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. CONCLUSIONS: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 1-11. ©2016 AACR.

Automated measurement of estrogen receptor in breast cancer: a comparison of fluorescent and chromogenic methods of measurement.

Whereas FDA-approved methods of assessment of estrogen receptor (ER) are 'fit for purpose', they represent a 30-year-old technology. New quantitative methods, both chromogenic and fluorescent, have been developed and studies have shown that these methods increase the accuracy of assessment of ER. Here, we compare three methods of ER detection and assessment on two retrospective tissue microarray (TMA) cohorts of breast cancer patients: estimates of percent nuclei positive by pathologists and by Aperio's nuclear algorithm (standard chromogenic immunostaining), and immunofluorescence as quantified with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). Reproducibility was excellent (R(2)>0.95) between users for both automated analysis methods, and the Aperio and QIF scoring results were also highly correlated, despite the different detection systems. The subjective readings show lower levels of reproducibility and a discontinuous, bimodal distribution of scores not seen by either mechanized method. Kaplan-Meier analysis of 10-year disease-free survival was significant for each method (Pathologist, P=0.0019; Aperio, P=0.0053, AQUA, P=0.0026); however, there were discrepancies in patient classification in 19 out of 233 cases analyzed. Out of these, 11 were visually positive by both chromogenic and fluorescent detection. In 10 cases, the Aperio nuclear algorithm labeled the nuclei as negative; in 1 case, the AQUA score was just under the cutoff for positivity (determined by an Index TMA). In contrast, 8 out of 19 discrepant cases had clear nuclear positivity by fluorescence that was unable to be visualized by chromogenic detection, perhaps because of low positivity masked by the hematoxylin counterstain. These results demonstrate that automated systems enable objective, precise quantification of ER. Furthermore, immunofluorescence detection offers the additional advantage of a signal that cannot be masked by a counterstaining agent. These data support the usage of automated methods for measurement of this and other biomarkers that may be used in companion diagnostic tests.

Quantitative analysis of estrogen receptor expression shows SP1 antibody is more sensitive than 1D5.

Studies comparing rabbit monoclonal SP1 antibody with 1D5 for estrogen receptor (ER) immunohistochemical testing show conflicting results. Here we use a standardized quantitative immunofluorescent (QIF) ER assay to determine the level and significance of discordance between the antibodies. Both antibodies were assessed by QIF on our Index TMA of cell lines and case controls, followed by QIF and immunohistochemical analysis on 2 retrospective cohorts from Yale. On the Index TMA, SP1 displayed stronger signal-to-noise ratio compared with 1D5. On the patient cohorts, the range of discrepancy between the 2 antibodies was 8% to 16.9%, with the majority of discrepant cases being SP1 positive/1D5 negative. Kaplan-Meier analysis of the discrepant cases showed outcomes comparable to those of double-positive cases, suggesting that SP1 is more sensitive than 1D5. A series of cases with high levels of ER-ß shows that neither antibody cross-reacts, suggesting equivalent specificity. Future efforts are needed to determine whether response to endocrine therapies show superiority of either antibody as a companion diagnostic test.

Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen.

PURPOSE: Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein. METHODS: Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody. RESULTS: ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin. CONCLUSION: Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

Cytoplasmic estrogen receptor in breast cancer.

PURPOSE: In addition to genomic signaling, it is accepted that estrogen receptor-α (ERα) has nonnuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of nonnuclear ER in clinical specimens. EXPERIMENTAL DESIGN: A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, and 1D5) was validated by Western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, and 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. RESULTS: Four of the antibodies specifically recognized ER by Western and QIF analysis, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with Pearson correlations (r(2) values) ranging from 0.87 to 0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other four antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. CONCLUSIONS: Our data show ERα is present in the cytoplasm in a number of cases using multiple antibodies while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value.

Standardization of estrogen receptor measurement in breast cancer suggests false-negative results are a function of threshold intensity rather than percentage of positive cells.

PURPOSE: Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. METHODS: An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. RESULTS: Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/µg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. CONCLUSION: Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.

Antibody validation.

Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.

Analytic variability in immunohistochemistry biomarker studies.

BACKGROUND: Despite the widespread use of immunohistochemistry (IHC), there are no standardization guidelines that control for antibody probe variability. Here we describe the effect of variable antibody reagents in the assessment of cancer-related biomarkers by IHC. METHODS: Estrogen receptor (ER), epidermal growth factor receptor (EGFR) 1, and human epidermal growth factor receptor 3 (HER3) were evaluated by quantitative immunofluorescence. Correlations between ER clones 1D5, SP1, F10, and ER60c, and EGFR monoclonal 31G7, 2-18C9, H11, and 15F8, and polyclonal 2232 antibodies were assessed in 642 breast cancer patients. HER3 was measured by RTJ1, RTJ2, SGP1, M7297, RB-9211, and C-17 antibodies in 42 lung cancer patients. Survival analysis was done with the use of multiple cutoff points to reveal any prognostic classification. RESULTS: All ER antibodies were tightly correlated (Pearson's r(2) = 0.94-0.96; P < 0.0001) and western blotting confirmed their specificity in MCF-7 and BT474 cells. All EGFR antibodies but 2232 yielded specific results in western blotting; however, only 31G7 and 2-18C9 were strongly associated (Pearson's r(2) = 0.61; P < 0.0001). HER3 staining was nonspecific and nonreproducible. High EGFR-expressing patients had a worse prognosis when EGFR was measured with H11 or 31G7 (log rank P = 0.015 and P = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9, 15F8, or polyclonal 2232 antibodies. CONCLUSIONS: Antibody validation is a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. IMPACT: This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols.

Co-targeting the insulin-like growth factor I receptor enhances growth-inhibitory and pro-apoptotic effects of anti-estrogens in human breast cancer cell lines.

The insulin-like growth factor I receptor (IGF1R) interacts with estrogen receptor-alpha (ERalpha) and HER2. We examined the effect of combinations of IGF1R antagonists (alpha-IR3, AG1024) and anti-estrogens (4-hydroxy tamoxifen, fulvestrant) in two human ER+ breast cancer cell lines: BT474 (HER2 overexpressing, IGF1R low) and MCF7 (HER2 non-overexpressing, IGF1R high). In BT474 cells, growth was inhibited by anti-estrogens, but not by IGF1R antagonists; however, adding IGF1R inhibitors to anti-estrogens enhanced growth inhibition. In MCF7 cells, growth was inhibited by IGF1R and ER antagonists and more so by their combination. In both cell lines, no single agents could induce apoptosis, but combining IGF1R inhibitors with anti-estrogens induced dramatic levels of apoptosis. IGF1R antagonists enhanced the ability of the anti-estrogens to inhibit ER transcriptional activity in BT474 cells, but not in MCF7 cells. The drug combination synergistically inhibited ER and IGF1R activity. Such combinations may be useful therapy for breast cancer.