Validation of the 21-gene test as a predictor of clinical response to neoadjuvant hormonal therapy for ER+, HER2-negative breast cancer: the TransNEOS study.

PURPOSE: The Recurrence Score test is validated to predict benefit of adjuvant chemotherapy. TransNEOS, a translational study of New Primary Endocrine-therapy Origination Study (NEOS), evaluated whether Recurrence Score results can predict clinical response to neoadjuvant letrozole. METHODS: NEOS is a phase 3 clinical trial of hormonal therapy ± adjuvant chemotherapy for postmenopausal patients with ER+, HER2-negative, clinically node-negative breast cancer, after six months of neoadjuvant letrozole and breast surgery. TransNEOS patients had tumors ≥ 2 cm and archived core-biopsy samples taken before neoadjuvant letrozole and subsequently sent for Recurrence Score testing. The primary endpoint was to evaluate clinical (complete or partial) response to neoadjuvant letrozole for RS < 18 versus RS ≥ 31. Secondary endpoints included evaluation of clinical response and rate of breast-conserving surgery (BCS) by continuous Recurrence Score result, ESR1 and PGR single-gene scores, and ER gene-group score. RESULTS: Of 295 TransNEOS patients (median age 63 years; median tumor size 25 mm; 66% grade 1), 53.2% had RS < 18, 28.5% had RS18-30, and 18.3% had RS ≥ 31. Clinical response rates were 54% (RS < 18), 42% (RS18-30), and 22% (RS ≥ 31). A higher proportion of patients with RS < 18 had clinical responses (p < 0.001 vs. RS ≥ 31). In multivariable analyses, continuous Recurrence Score result (p < 0.001), ESR1 score (p = 0.049), PGR score (p < 0.001), and ER gene-group score (p < 0.001) were associated with clinical response. Recurrence Score group was significantly associated with rate of BCS after neoadjuvant treatment (RS < 18 vs. RS ≥ 31, p = 0.010). CONCLUSION: The Recurrence Score test is validated to predict clinical response to neoadjuvant letrozole in postmenopausal patients with ER+, HER2-negative, clinically node-negative breast cancer.

The 21-gene Recurrence Score® assay predicts distant recurrence in lymph node-positive, hormone receptor-positive, breast cancer patients treated with adjuvant sequential epirubicin- and docetaxel-based or epirubicin-based chemotherapy (PACS-01 trial).

BACKGROUND: The 21-gene Recurrence Score (RS) result predicts outcome and chemotherapy benefit in node-negative and node-positive (N+), estrogen receptor-positive (ER+) patients treated with endocrine therapy. The purpose of this study was to evaluate the prognostic impact of RS results in N+, hormone receptor-positive (HR+) patients treated with adjuvant chemotherapy (6 cycles of FEC100 vs. 3 cycles of FEC100 followed by 3 cycles of docetaxel 100 mg/m2) plus endocrine therapy (ET) in the PACS-01 trial (J Clin Oncol 2006;24:5664-5671). METHODS: The current study included 530 HR+/N+ patients from the PACS-01 parent trial for whom specimens were available. The primary objective was to evaluate the relationship between the RS result and distant recurrence (DR). RESULTS: There were 209 (39.4%) patients with low RS (< 18), 159 (30%) with intermediate RS (18-30) and 162 (30.6%) with high RS (≥ 31). The continuous RS result was associated with DR (hazard ratio = 4.14; 95% confidence interval: 2.67-6.43; p <  0.001), adjusting for treatment. In multivariable analysis, the RS result remained a significant predictor of DR (p <  0.001) after adjustment for number of positive nodes, tumor size, tumor grade, Ki-67 (immunohistochemical status), and chemotherapy regimen. There was no statistically significant interaction between RS result and treatment in predicting DR (p = 0.79). CONCLUSIONS: After adjustment for clinical covariates, the 21-gene RS result is a significant prognostic factor in N+/HR+ patients receiving adjuvant chemoendocrine therapy. TRIAL REGISTRATION: Not applicable.

21-Gene Recurrence Score for prognosis and prediction of taxane benefit after adjuvant chemotherapy plus endocrine therapy: results from NSABP B-28/NRG Oncology.

BACKGROUND: The 21-gene recurrence score (RS) predicts outcome and benefit from adjuvant chemotherapy benefit in breast cancer patients treated with adjuvant endocrine therapy. In the NSABP B-28 study, we evaluated the 21-gene RS for its prognostic impact and its ability to predict benefit from paclitaxel (P) in node-positive, estrogen receptor-positive (ER+) breast cancer patients treated with adjuvant chemotherapy plus tamoxifen. METHODS: The B-28 trial compared doxorubicin/cyclophosphamide (AC) with AC followed by P in 3060 patients. Tamoxifen for 5 years was also given to patients > 50 years and those < 50 years with ER+ and/or progesterone receptor-positive (PR+) tumors. The present study includes 1065 ER-positive, tamoxifen-treated patients with RS assessment. Median follow-up time was 11.2 years. RESULTS: In univariate analyses, RS was a significant predictor of outcome. In multivariate analyses, RS remained a significant independent predictor of outcome beyond clinico-pathologic factors, age, and type of surgery (p < 0.001). In the study population (n = 1065), the disease-free survival (DFS) hazard ratio (HR) with adding P to AC was 0.87 (95% CI 0.72-1.05; p = 0.14). RS was not a significant predictor of P benefit: for DFS, HRs for adding P to AC in RS low, intermediate, and high subgroups were 1.01 (95% CI 0.69-1.47; p = 0.99), 0.84 (95% CI 0.62-1.14; p = 0.26), and 0.81 (95% CI 0.60-1.10; p = 0.21), respectively (interaction p = 0.64). Similar findings were observed for the other study endpoints. CONCLUSIONS: RS maintains significant prognostic impact in ER-positive, node-positive patients treated with adjuvant chemotherapy plus tamoxifen. However, RS did not significantly predict benefit from adding paclitaxel to AC chemotherapy. (Trial Registration: PDQ: NSABP-B-28).

21-Gene Recurrence Score and Locoregional Recurrence in Node-Positive/ER-Positive Breast Cancer Treated With Chemo-Endocrine Therapy.

Background: The 21-gene recurrence score (RS) predicts risk of locoregional recurrence (LRR) in node-negative, estrogen receptor (ER)-positive breast cancer. We evaluated the association between RS and LRR in node-positive, ER-positive patients treated with adjuvant chemotherapy plus tamoxifen in National Surgical Adjuvant Breast and Bowel Project B-28. Methods: B-28 compared doxorubicin/cyclophosphamide (AC X 4) with AC X 4 followed by paclitaxel X 4. Tamoxifen was given to patients age 50 years or older and those younger than age 50 years with ER-positive and/or progesterone receptor-positive tumors. Lumpectomy patients received breast radiotherapy. Mastectomy patients received no radiotherapy. The present study includes 1065 ER-positive, tamoxifen-treated patients with RS assessment. Cumulative incidence functions and subdistribution hazard regression models were used for LRR to account for competing risks including distant recurrence, second primary cancers, and death from other causes. Median follow-up was 11.2 years. All statistical tests were one-sided. Results: There were 80 LRRs (7.5%) as first events (68% local/32% regional). RS was low: 36.2%; intermediate: 34.2%; and high: 29.6%. RS was a statistically significant predictor of LRR in univariate analyses (10-year cumulative incidence of LRR = 3.3%, 7.2%, and 12.2% for low, intermediate, and high RS, respectively, P < .001). In multivariable regression analysis, RS remained an independent predictor of LRR (hazard ratio [HR] = 2.59, 95% confidence interval [CI] = 1.28 to 5.26, for a 50-point difference, P = .008) along with pathologic nodal status (HR = 1.91, 95% CI = 1.20 to 3.03, for four or more vs one to three positive nodes, P = .006) and tumor size (HR = 1.28, 95% CI = 1.05 to 1.55, for a 1 cm difference, P = .02). Conclusions: RS statistically significantly predicts risk of LRR in node-positive, ER-positive breast cancer patients after adjuvant chemotherapy plus tamoxifen. These findings can help in the selection of appropriate candidates for comprehensive radiotherapy.

Multigene Expression Assay and Benefit of Radiotherapy After Breast Conservation in Ductal Carcinoma in Situ.

Background: Most women with ductal carcinoma in situ (DCIS) will receive breast-conserving surgery (BCS) and radiation (RT). RT can be omitted for women at low risk of local recurrence (LR). The Oncotype DX DCIS score (DS) predicts LR risk after BCS alone. This study assesses the impact of RT and DS on LR risk. Methods: Population-based cohort analysis of individuals with DCIS treated by BCS ± RT from 1994-2003. Treatment and outcomes were determined by linkage and chart review. We used a propensity score-adjusted multivariable model to examine associations between DS and LR and evaluate the impact of RT. All statistical tests were two-sided. Results: The cohort includes 571 individuals treated by BCS alone, 689 cases treated with BCS + RT. Median follow-up was 9.4 years. On multivariable analysis, factors associated with LR include RT, age at diagnosis, tumor size, and multifocality. Adjusting for these factors, the DS risk group was statistically significantly associated with LR risk (hazard ratio high/intermediate = 1.75, 95% confidence interval = 1.28 to 2.41, P < .001). Women with a low-risk DS treated by BCS alone had an LR risk of 10.6% at 10 years and a small benefit from RT, while those with a high DS had a higher risk of LR (25.4%) after BCS alone and greater benefit from RT. A subgroup of patients with favorable clinicopathological features had a high-risk DS; these patients had a higher than expected risk of LR after BCS alone and a greater benefit with RT. Conclusions: The DS molecular assay improves risk stratification and estimates of RT benefit in individuals with DCIS treated with breast-conserving therapy.

Gene expression in normal-appearing tissue adjacent to prostate cancers are predictive of clinical outcome: evidence for a biologically meaningful field effect.

PURPOSE: We evaluated gene expression in histologically normal-appearing tissue (NT) adjacent to prostate tumor in radical prostatectomy specimens, assessing for biological significance based on prediction of clinical recurrence (cR - metastatic disease or local recurrence). RESULTS: A total of 410 evaluable patients had paired tumor and NT. Forty-six genes, representing diverse biological pathways (androgen signaling, stromal response, stress response, cellular organization, proliferation, cell adhesion, and chromatin remodeling) were associated with cR in NT (FDR < 20%), of which 39 concordantly predicted cR in tumor (FDR < 20%). Overall GPS and its stromal response and androgen-signaling gene group components also significantly predicted time to cR in NT (RM-corrected HR/20 units = 1.25; 95% CI: 1.01-1.56; P = 0.024). EXPERIMENTAL DESIGN: Expression of 732 genes was measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) separately in tumor and adjacent NT specimens from 127 patients with and 374 without cR following radical prostatectomy for T1/T2 prostate cancer. A 17-gene expression signature (Genomic Prostate Score [GPS]), previously validated to predict aggressive prostate cancer when measured in tumor tissue, was also assessed using pre-specified genes and algorithms. Analysis used Cox proportional hazards models, Storey's false discovery rate (FDR) control, and regression to the mean (RM) correction. CONCLUSIONS: Gene expression profiles, including GPS, from NT adjacent to tumor can predict prostate cancer outcome. These findings suggest that there is a biologically significant field effect in primary prostate cancer that is a marker for aggressive disease.

A Prospective Comparison of the 21-Gene Recurrence Score and the PAM50-Based Prosigna in Estrogen Receptor-Positive Early-Stage Breast Cancer.

INTRODUCTION: The 21-gene Recurrence Score(®) assay (Oncotype DX(®), Genomic Health, Inc.) is a validated predictor of recurrence risk/chemotherapy benefit in patients with estrogen receptor-positive (ER+) early-stage breast cancer treated with endocrine therapy. The Prosigna(®) assay (NanoString Technologies Inc.) is a validated prognosticator in postmenopausal patients with low-risk ER+ early-stage breast cancer treated with endocrine therapy. The assays were analytically/clinically developed and validated differently. This study focused on comparing recurrence risk estimates as determined by these assays and is the first blinded comparison of these assays on matched patient samples. METHODS: Sequential breast cancer specimens from postmenopausal, node-negative, ER+ patients treated at the Marin General Hospital were analyzed: first by the 21-gene assay then by the Prosigna assay in an independent lab blinded to the Recurrence Score results. RESULTS: The final analysis included 52 patients. Correlation between the Recurrence Score and the Prosigna assay results was poor (r = 0.08). Agreement between risk classifications based on these assays was 54%; 4/7 of patients classified as high risk by the Prosigna assay had low Recurrence Score results. Two tumors with high Recurrence Score results had low ER expression (close to positivity threshold); both of which had a low/intermediate Prosigna assay result. The Prosigna assay classified 73.1% and 23.1% of samples as luminal A and luminal B, respectively. A range of Recurrence Score results was observed within the subtypes; 83% of luminal B samples had a low Recurrence Score result. CONCLUSION: Consistent with prior comparisons between the 21-gene and other genomic assays, our study demonstrated substantial differences in the way patients are risk stratified, suggesting that the different assays are not interchangeable. FUNDING: Genomic Health, Inc.

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

INTRODUCTION: The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. METHODS: N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence. RESULTS: Median follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. CONCLUSIONS: Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. TRIAL REGISTRATION: Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

A population-based validation study of the DCIS Score predicting recurrence risk in individuals treated by breast-conserving surgery alone.

Validated biomarkers are needed to improve risk assessment and treatment decision-making for women with ductal carcinoma in situ (DCIS) of the breast. The Oncotype DX DCIS Score (DS) was shown to predict the risk of local recurrence (LR) in individuals with low-risk DCIS treated by breast-conserving surgery (BCS) alone. Our objective was to confirm these results in a larger population-based cohort of individuals. We used an established population-based cohort of individuals diagnosed with DCIS treated with BCS alone from 1994 to 2003 with validation of treatment and outcomes. Central pathology assessment excluded cases with invasive cancer, DCIS < 2 mm or positive margins. Cox model was used to determine the relationship between independent covariates, the DS (hazard ratio (HR)/50 Cp units (U)) and LR. Tumor blocks were collected for 828 patients. Final evaluable population includes 718 cases, of whom 571 had negative margins. Median follow-up was 9.6 years. 100 cases developed LR following BCS alone (DCIS, N = 44; invasive, N = 57). In the primary pre-specified analysis, the DS was associated with any LR (DCIS or invasive) in ER+ patients (HR 2.26; P < 0.001) and in all patients regardless of ER status (HR 2.15; P < 0.001). DCIS Score provided independent information on LR risk beyond clinical and pathologic variables including size, age, grade, necrosis, multifocality, and subtype (adjusted HR 1.68; P = 0.02). DCIS was associated with invasive LR (HR 1.78; P = 0.04) and DCIS LR (HR 2.43; P = 0.005). The DCIS Score independently predicts and quantifies individualized recurrence risk in a population of patients with pure DCIS treated by BCS alone.

A 17-gene assay to predict prostate cancer aggressiveness in the context of Gleason grade heterogeneity, tumor multifocality, and biopsy undersampling.

BACKGROUND: Prostate tumor heterogeneity and biopsy undersampling pose challenges to accurate, individualized risk assessment for men with localized disease. OBJECTIVE: To identify and validate a biopsy-based gene expression signature that predicts clinical recurrence, prostate cancer (PCa) death, and adverse pathology. DESIGN, SETTING, AND PARTICIPANTS: Gene expression was quantified by reverse transcription-polymerase chain reaction for three studies-a discovery prostatectomy study (n=441), a biopsy study (n=167), and a prospectively designed, independent clinical validation study (n=395)-testing retrospectively collected needle biopsies from contemporary (1997-2011) patients with low to intermediate clinical risk who were candidates for active surveillance (AS). OUTCOME MEASURES AND STATISTICAL ANALYSIS: The main outcome measures defining aggressive PCa were clinical recurrence, PCa death, and adverse pathology at prostatectomy. Cox proportional hazards regression models were used to evaluate the association between gene expression and time to event end points. Results from the prostatectomy and biopsy studies were used to develop and lock a multigene-expression-based signature, called the Genomic Prostate Score (GPS); in the validation study, logistic regression was used to test the association between the GPS and pathologic stage and grade at prostatectomy. Decision-curve analysis and risk profiles were used together with clinical and pathologic characteristics to evaluate clinical utility. RESULTS AND LIMITATIONS: Of the 732 candidate genes analyzed, 288 (39%) were found to predict clinical recurrence despite heterogeneity and multifocality, and 198 (27%) were predictive of aggressive disease after adjustment for prostate-specific antigen, Gleason score, and clinical stage. Further analysis identified 17 genes representing multiple biological pathways that were combined into the GPS algorithm. In the validation study, GPS predicted high-grade (odds ratio [OR] per 20 GPS units: 2.3; 95% confidence interval [CI], 1.5-3.7; p<0.001) and high-stage (OR per 20 GPS units: 1.9; 95% CI, 1.3-3.0; p=0.003) at surgical pathology. GPS predicted high-grade and/or high-stage disease after controlling for established clinical factors (p<0.005) such as an OR of 2.1 (95% CI, 1.4-3.2) when adjusting for Cancer of the Prostate Risk Assessment score. A limitation of the validation study was the inclusion of men with low-volume intermediate-risk PCa (Gleason score 3+4), for whom some providers would not consider AS. CONCLUSIONS: Genes representing multiple biological pathways discriminate PCa aggressiveness in biopsy tissue despite tumor heterogeneity, multifocality, and limited sampling at time of biopsy. The biopsy-based 17-gene GPS improves prediction of the presence or absence of adverse pathology and may help men with PCa make more informed decisions between AS and immediate treatment. PATIENT SUMMARY: Prostate cancer (PCa) is often present in multiple locations within the prostate and has variable characteristics. We identified genes with expression associated with aggressive PCa to develop a biopsy-based, multigene signature, the Genomic Prostate Score (GPS). GPS was validated for its ability to predict men who have high-grade or high-stage PCa at diagnosis and may help men diagnosed with PCa decide between active surveillance and immediate definitive treatment.

Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies.

BACKGROUND: The Oncotype DX Prostate Cancer Assay is a multi-gene RT-PCR expression assay that was developed for use with fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies containing as little as 1 mm of prostate tumor in the greatest dimension. The assay measures expression of 12 cancer-related genes representing four biological pathways and 5 reference genes which are algorithmically combined to calculate the Genomic Prostate Score (GPS). This biopsy-based assay has been analytically and subsequently clinically validated as a predictor of aggressive prostate cancer. The aim of this study was to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria. RESULTS: The lowest quartile of RNA yields from prostate needle biopsies (six 5 µm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as 0.005 ng to 320 ng). Analytical accuracy was excellent with average biases at qPCR inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively. CONCLUSIONS: The Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. The assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer.

A multigene expression assay to predict local recurrence risk for ductal carcinoma in situ of the breast.

BACKGROUND: For women with ductal carcinoma in situ (DCIS) of the breast, the risk of developing an ipsilateral breast event (IBE; defined as local recurrence of DCIS or invasive carcinoma) after surgical excision without radiation is not well defined by clinical and pathologic characteristics. METHODS: The Oncotype DX breast cancer assay was performed for patients with DCIS treated with surgical excision without radiation in the Eastern Cooperative Oncology Group (ECOG) E5194 study. The association of the prospectively defined DCIS Score (calculated from seven cancer-related genes and five reference genes) with the risk of developing an IBE was analyzed using Cox regression. All statistical tests were two-sided. RESULTS: There were 327 patients with adequate tissue for analysis. The continuous DCIS Score was statistically significantly associated with the risk of developing an IBE (hazard ratio [HR] = 2.31, 95% confidence interval [CI] = 1.15 to 4.49; P = .02) when adjusted for tamoxifen use (prespecified primary analysis) and with invasive IBE (unadjusted HR = 3.68, 95% CI = 1.34 to 9.62; P = .01). For the prespecified DCIS risk groups of low, intermediate, and high, the 10-year risks of developing an IBE were 10.6%, 26.7%, and 25.9%, respectively, and for an invasive IBE, 3.7%, 12.3%, and 19.2%, respectively (both log rank P ≤ .006). In multivariable analyses, factors associated with IBE risk were DCIS Score, tumor size, and menopausal status (all P ≤ .02). CONCLUSIONS: The DCIS Score quantifies IBE risk and invasive IBE risk, complements traditional clinical and pathologic factors, and provides a new clinical tool to improve selecting individualized treatment for women with DCIS who meet the ECOG E5194 criteria.

Characterization and functionality of proliferative human Sertoli cells.

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2'-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy.

Discovery of ADDL--targeting small molecule drugs for Alzheimer's disease.

Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.

Agonist-induced calcium response in single human platelets assayed in a microfluidic device.

To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.

Metal coordination-based inhibitors of adenylyl cyclase: novel potent P-site antagonists.

The adenylyl cyclases (ACs) are a family of intracellular enzymes associated with signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the so-called purine binding site (P-site) of the enzyme followed by metal-mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known, and unique isoform combinations are expressed in a tissue-specific manner. The development of isoform-specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of unique signal transduction inhibitors. To develop novel AC inhibitors, we have chosen an approach to inhibitor design utilizing an adenine ring system joined to a metal-coordinating hydroxamic acid via various linkers. Previous work in our group has validated this approach and identified novel inhibitors that possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible acyclic linkers (Levy, D. E., et al. Bioorg. Med. Chem. Lett. 2002, 12, 3085-3088). Subsequent studies have focused on the introduction of conformational restrictions into the tether of the inhibitors with the goal of increasing potency (Levy, D. E., et al. Bioorg. Med. Chem. Lett. 2002, 12, 3089-3092). Building upon the favorable spatial positioning of the adenine and hydroxamate groups coupled with potentially favorable entropic factors, the unit joining the carbocycle to the hydroxamate was explored further and a stereochemical-based SAR was elucidated, leading to a new series of highly potent AC inhibitors.

Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding.

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme.