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Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

The table provides a step by step guide to calculate methylation in each gene followed by cumulative methylation using the algorithm derived in this paper.

Figure shows the perfomance of LBx-BCM in the training set samples. A histogram shows cumulative methylation for each sample, and a box plot shows significnt difference of methylation in serum of metastatic breast cancer patients compared to normal individuals (Mann Whitney p= 0.002)

Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 hours. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75–300 DNA copies) spiked into plasma (coefficient of variation, CV = 7.1%–10.9%) and serum (CV = 19.1%–36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples [MBC N = 40, controls N = 26; Spearman r = 0.891; 95% confidence interval (CI) = 0.825–0.933, P < 0.0001]. LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836–0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817–0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer.Significance:We technically validated an automated, cartridge-based, liquid biopsy prototype assay, to quantitatively measure breast cancer methylation in serum or plasma of patients with MBC, that demonstrated high sensitivity and specificity.

Study Schema

Purpose:We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we evaluated the clinical utility of a novel liquid biopsy-breast cancer methylation (LBx-BCM) prototype assay using the GeneXpert cartridge system for early assessment of disease progression in MBC.Experimental Design:The 9-marker LBx-BCM prototype assay was evaluated in TBCRC 005, a prospective biomarker study, using plasma collected at baseline, week 4, and week 8 from 144 patients with MBC.Results:At week 4, patients with MBC with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months vs. 6.60 months, P = 0.001) and OS (14.52 months vs. 22.44 months, P = 0.005) compared with those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR, 1.90; 95% CI, 1.20–3.01; P = 0.006). Change in CM from baseline to week 4 (OR, 4.60; 95% CI, 1.77–11.93; P = 0.002) and high levels of CM at week 4 (OR, 2.78; 95% CI, 1.29–5.99; P = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment.Conclusions:The automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression a month after initiating treatment in women with MBC undergoing routine care. The next step is to validate its clinical utility for specific treatments.

Association between week 4 CM and disease status at first restaging after adjusting for CEA.

Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 hours. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75–300 DNA copies) spiked into plasma (coefficient of variation, CV = 7.1%–10.9%) and serum (CV = 19.1%–36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P < 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples [MBC N = 40, controls N = 26; Spearman r = 0.891; 95% confidence interval (CI) = 0.825–0.933, P < 0.0001]. LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836–0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817–0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer. Significance: We technically validated an automated, cartridge-based, liquid biopsy prototype assay, to quantitatively measure breast cancer methylation in serum or plasma of patients with MBC, that demonstrated high sensitivity and specificity.

Purpose: We previously demonstrated that high levels of circulating methylated DNA are associated with subsequent disease progression in women with metastatic breast cancer (MBC). In this study, we validated the clinical utility of a Liquid Biopsy-Breast Cancer Methylation (LBx-BCM) prototype assay using the GeneXpert® cartridge system for early assessment of disease progression in MBC. Study Design: The 9-marker, LBx-BCM prototype assay was evaluated in TBCRC-005, a prospective biomarker study, using plasma collected at baseline, week 4 and week 8 from 144 MBC patients. Results: At week 4 MBC patients with high cumulative methylation (CM) had a significantly shorter median PFS (2.88 months v 6.60 months, p = 0.001) and OS (14.52 months v 22.44 months, p=0.005) compared to those with low CM. In a multivariable model, high versus low CM was also associated with shorter PFS (HR = 1.90, 95%CI 1.20-3.01; p =0.006). Change in CM from baseline to week 4 (OR = 4.60, 95%CI 1.77, 11.93; p = 0.002) and high levels of CM at week 4 (OR = 2.78, 95%CI 1.29, 5.99; p = 0.009) were associated with progressive disease at the time of first restaging. A robust risk model (AUC = 0.733) based on week 4 circulating CM levels was developed to predict disease progression as early as 3 months after initiating a new treatment. Conclusion: The easy to perform and automated LBx-BCM prototype assay is a promising clinical tool for detecting disease progression early after initiating treatment in women with MBC. Further validation in larger clinical datasets is needed. Citation Format: Kala Visvanathan, Leslie Cope, Mary Jo Fackler, Michael Considine, Lisa Carey, Andres Forero-Torres, James N. Ingle, Nancy U. Lin, Rita Nanda, Anna Maria Storniolo, Suzana Tulac, Natalie C. Wu, Sudhakar Marla, Neesha R. Venkatesan, Antonio C. Wolff, Saraswati Sukumar. Evaluation of the Automated Liquid Biopsy-Breast Cancer Methylation (LBx-BCM) Cartridge Assay for Predicting Early Disease Progression and Survival: TBCRC-005 Prospective Trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-09.

Preoperative staging of suspicious axillary lymph nodes (ALNs) allows patients to be triaged to ALN dissection or to sentinel lymph node biopsy (SLNB). Ultrasound-guided fine needle aspiration (FNA) and cytology of ALN is moderately sensitive but its clinical utility relies heavily on the cytologist’s experience. We proposed that the 5-h automated GeneXpert system-based prototype breast cancer detection assay (BCDA) that quantitatively measures DNA methylation in ten tumor-specific gene markers could provide a facile, accurate test for detecting cancer in FNA of enlarged lymph nodes. We validated the assay in ALN-FNA samples from a prospective study of patients (N = 230) undergoing SLNB. In a blinded analysis of 218 evaluable LN-FNAs from 108 malignant and 110 benign LNs by histology, BCDA displayed a sensitivity of 90.7% and specificity of 99.1%, achieving an area under the ROC curve, AUC of 0.958 (95% CI: 0.928–0.989; P N = 72, benign, N = 53 by cytology) collected in the outpatient setting prior to neoadjuvant chemotherapy (NAC). Using the ROC-threshold determined in the prospective study, compared to cytology, BCDA achieved a sensitivity of 94.4% and a specificity of 92.5% with a ROC-AUC = 0.977 (95% CI: 0.953–1.000; P

Purpose:An unmet need in low-resource countries is an automated breast cancer detection assay to prioritize women who should undergo core breast biopsy and pathologic review. Therefore, we sought to identify and validate a panel of methylated DNA markers to discriminate between cancer and benign breast lesions using cells obtained by fine-needle aspiration (FNA).Experimental Design: Two case–control studies were conducted comparing cancer and benign breast tissue identified from clinical repositories in the United States, China, and South Africa for marker selection/training (N = 226) and testing (N = 246). Twenty-five methylated markers were assayed by Quantitative Multiplex-Methylation-Specific PCR (QM-MSP) to select and test a cancer-specific panel. Next, a pilot study was conducted on archival FNAs (49 benign, 24 invasive) from women with mammographically suspicious lesions using a newly developed, 5-hour, quantitative, automated cartridge system. We calculated sensitivity, specificity, and area under the receiver-operating characteristic curve (AUC) compared with histopathology for the marker panel.Results:In the discovery cohort, 10 of 25 markers were selected that were highly methylated in breast cancer compared with benign tissues by QM-MSP. In the independent test cohort, this panel yielded an AUC of 0.937 (95% CI = 0.900–0.970). In the FNA pilot, we achieved an AUC of 0.960 (95% CI = 0.883–1.0) using the automated cartridge system.Conclusions:We developed and piloted a fast and accurate methylation marker–based automated cartridge system to detect breast cancer in FNA samples. This quick ancillary test has the potential to prioritize cancer over benign tissues for expedited pathologic evaluation in poorly resourced countries.

BACKGROUND: Previously, we demonstrated the clinical validity of cMethDNA, a circulating methylated tumor DNA (ctDNA) assay, in serum samples from TBCRC 005 (J Clin Oncol, 2017; 35:751-758) to predict progression free- and overall-survival, and to monitor therapeutic response in patients with stage IV breast cancer. Here, Johns Hopkins (JH) and Cepheid partnered to develop an automated GeneXpert (GX) cartridge-based system to provide quantitative measures of DNA methylation within 5 hours.METHODS: With a goal of discriminating stage IV breast cancer from healthy and benign breast disease with high sensitivity and specificity, we evaluated breast cancer-specific DNA methylation markers (selected through comprehensive methylome analysis) in STRECK tube plasma of 46 patients with metastatic breast cancer enrolled in Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE II trial), 17 benign breast disease and 9 healthy normal controls (J0888 repository). Blood from IMAGE II participants was collected upon disease progression. A newly designed GX Breast Cancer Monitoring Assay for research use only (RUO*) first converted unmethylated CpG sites in ctDNA from 1 ml plasma with bisulfite. The sample was then split into two methylation detection cartridges, which quantitated DNA methylation of 9 markers along with an ACTB reference. Cumulative methylation (CM) of the 9-gene panel was calculated using a novel algorithm. Performance was assessed based on Receiver Operating Characteristic (ROC) curves and Mann-Whitney analyses.RESULTS: The GX Breast Cancer Monitoring Assay (RUO)* showed that the 9-gene panel was significantly more methylated in cancer compared to normal/benign plasma samples (median for cancer: 428.0 CM units versus for benign: 0.0 CM units; P< 0.0001), and revealed a sensitivity of 85% and specificity of 92%, using a cumulative methylation threshold of 35.5 units based on ROC area under the curve (AUC) = 0.909 (95% CI 0.836 – 0.982, P Citation Format: Mary Jo Fackler, Suzana Tulac, Neesha Venkatesan, Adam J. Aslam, Leslie M. Cope, Jennifer Lehman, Rita Denbow, Jeffrey Reynolds, Morgan Buckley, Bradley M. Downs, Kala Visvanathan, Christopher B. Umbricht, Antonio C. Wolff, Vered Stearns, Edwin W. Lai, Saraswati Sukumar. An automated DNA methylation assay for monitoring treatment response in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-03.

Background: Axillary lymph node metastasis is associated with a high risk of breast cancer recurrence and is the single most powerful predictor of patient prognosis. Various imaging tests alone or in combination with preoperative fine needle aspiration (FNA) cytology are used to evaluate suspicious lymph nodes (LNs). However, these methods have low sensitivity (70%) and their utility varies depending on the local surgical practice and experience of the cytologists. In previous work, we have demonstrated higher than 90% sensitivity/specificity of a panel of ten hypermethylated biomarkers in FNA to distinguish between cancer and benign breast lesions. Analysis of methylated genes in FNA of suspicious LNs may improve sensitivity of cancer detection and assist in staging disease more accurately. Methods: We used Quantitative Multiplex Methylation-Specific PCR (QM-MSP) to determine the utility of the 10-gene panel for detecting tumor cells in LN FNAs. Two studies were performed to test the accuracy of a newly developed automated, cartridge-based GeneXpert® Breast Cancer Detection Assay (RUO*). First, we conducted a prospective case-control study in China in breast cancer patients with palpable LNs undergoing sentinel lymph node biopsy. FNA specimens were collected intraoperatively from a single palpable LN and assayed. Histopathology of the same LN (88 metastatic and 104 benign), was used as the gold standard for comparison. Next, using the automated assay, a pilot validation study was conducted on archival ultrasound-guided FNAs (69 metastatic and 53 benign by cytopathology) collected preoperatively from enlarged axillary LNs in patients with and without breast cancer in the outpatient centers in China and U.S.A. We calculated sensitivity, specificity and area under the receiver operating characteristic curve (AUC) for the marker panel tested by the GeneXpert® assay compared to histopathology for the case control study, and cytopathology for the pilot validation study. We also compared its performance to the highly sensitive laboratory assay, QM-MSP. Results: In the case-control study in China, compared to histopathology, cytopathology of the palpable enlarged LNs achieved cancer detection sensitivity of 85% and specificity of 95% (ROC; AUC=0.898, 95% CI: 0.843-0.953). Using the GeneXpert® assay on the same samples, compared to histopathology, the methylated biomarker panel showed a sensitivity of 92.0% and a specificity of 98.1% based on receiver operating characteristic statistics (ROC; AUC = 0.960, 95% CI: 0.928-0.993). QM-MSP yielded a similar sensitivity of 91.8% and a specificity of 97.7% (ROC; AUC = 0.966, 95% CI: 0.934-0.998). In the pilot validation set, compared to cytopathology, the automated assay achieved a sensitivity of 94.2% and a specificity of 92.5% (ROC; AUC=0.976 (95%CI: 0.950-1.001). Conclusions: We have developed and piloted a 5-hour, automated GeneXpert® Breast Cancer Detection Assay (RUO*) on FNA to detect cancer in suspicious LNs. This assay performs with an accuracy equivalent to that of the highly sensitive but labor-intensive assay, QM-MSP. This test has the potential to determine axillary involvement with higher sensitivity than cytopathology within a very short time in the outpatient setting. A positive test preoperatively could provide guidance for decision-making regarding eligibility of patients for neoadjuvant treatment, post-mastectomy radiotherapy and breast reconstruction. Further evaluation of this automated assay in larger, blinded, prospective studies is warranted. Citation Format: Juanjuan Li, Bradley Downs, Leslie Cope, Xiuyun Zhang, Chuan-gui Song, Kejing Zhang, Yong Han, Mary Jo Fackler, Edwin Lai, Suzana Tulac, Neesha Venkatesan, Timothy Guzman, Chuang Chen, Jingping Yuan, Saraswati Sukumar. Automated molecular diagnosis of suspicious axillary lymph nodes in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-02-06.

Introduction: Breast cancer incidence is rapidly increasing globally with 1.7 million new cases and 600,000 deaths due to the disease annually. Death due to breast cancer is four to six times more frequent in the developing world compared to the USA due to lack of early detection, definitive diagnosis, and limited access to treatment. Ultrasound imaging for early detection and ultrasound guided fine needle aspiration (FNA) of suspicious lesions followed by an automated, cartridge-based analysis of methylated genes could triage malignancies for faster pathology assessment and treatment, thus enabling better use of scarce resources. Experimental Procedures: To identify a panel of methylated DNA markers to enable sensitive and specific detection of malignant breast lesions, we screened 24 methylated genes (known to be frequently methylated in malignant breast tissue and unmethylated in normal breast tissue) by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) analysis. Formalin-fixed paraffin-embedded (FFPE) sections of biopsies of both benign and malignant breast lesions from the USA, China and South Africa were analyzed. Samples were divided into Training and Test sets. The Training set consisted of 206 tissues [66 invasive ductal carcinoma (IDC), 30 ductal carcinoma in situ (DCIS), 99 benign breast disease (BBD) and 11 normal breast (NB)]. The gene panel selected in the Training set was examined in an independent Test set of tissues (n=204) [65 IDC, 29 DCIS, 99 BBD and 11 NB]. Further, we optimized the technical performance of an automated, prototype breast cancer detection cartridge system for quantitative assessment of gene methylation and tested it in pilot study using FNA samples from breast cancers. Results: Analysis of the tissues in the Training set (n=206) led to the selection of a panel of 10 genes highly methylated in malignant lesions with little or no methylation in benign lesions. For the 10-gene panel to achieve a sensitivity greater than 90%, a laboratory cutoff of 14.5 cumulative methylation (CM) units (out of a possible 10,000 units) was set. In the Training set, the 10-gene panel achieved a sensitivity of 90% and a specificity of 85% with receiver operating characteristic (ROC) statistics: ROC, AUC= 0.947. In a blinded Test set of tissue samples (n=204), with a laboratory cutoff of 14.5 CM units, the 10-gene panel achieved a sensitivity of 87% and a specificity of 89%, with ROC statistics: p Conclusions: QM-MSP of breast lesions led to the selection of a panel of 10 genes methylated for detection of breast cancer. We have validated the technical performance of an automated, prototype cartridge system. The study reveals the potential of methylation markers to provide fast, accurate and automated cancer detection at a low cost in developing regions globally. Research Use Only. Not for diagnostic tests. A sponsored research agreement from Cepheid to Dr. Sukumar's Lab at Johns Hopkins University. Citation Format: Bradley M. Downs, Mary Jo Fackler, Claudia Mercado-Rodriguez, Ashley Cimino-Mathews, Chuang Chen, Jing-Ping Yuan, Eunice van den Berg, Leslie M. Cope, Susan C. Harvey, Syed Z. Ali, Suzana Tulac, Kriszten J. Kocmond, Edwin W. Lai, Brian Rhees, Mike Bates, Saraswati Sukumar. An automated breast cancer detection assay for screening in the developing world [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-220.

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.

Lefty/Ebaf polypeptides, novel members of the TGF-beta superfamily, are involved in endometrial differentiation and embryo implantation. Recently, we showed that, during undisturbed estrous cycle, lefty is present in mouse uterine horn primarily in a precursor form. Here, we show that decidual differentiation of endometrial stroma leads to increased lefty (approximately 3.1- to 3.6-fold in vivo and 5- to 8-fold in vitro) and processing of its precursor primarily to its long form. This event occurs on d 5 of pregnancy, and is paralleled by proprotein convertase (PC)5/6 up-regulation (approximately 6-fold increase for PC5A and 3-fold increase for PC5B) in decidualized uterine horn, independent of embryo implantation. Among the known convertases, only PC5/6A processes lefty to its long form. Taken together, the findings show that decidualized differentiation of stroma, which is a prerequisite for embryo implantation, leads to processing of lefty by PC5/6A.

Problem: Changes in the immune environment in the endometrium are believed to be important for successful implantation and maintenance of pregnancy. We have previously investigated global gene profiling in human endometrium during the window of implantation by oligonucleotide microarray technology, and analysis of these data underscore the regulation of a group of immune-related genes. The present study was therefore conducted to examine the pattern of expression and regulation of these genes including decay accelerating factor (DAF), indoleamine 2,3 dioxygenase (IDO), interleukin-15 (IL-15), IL-15 receptor alpha subunit (IL-15Rα), interferon regulatory factor-1 (IRF-1), lymphotactin (Lpn), natural killer-associated transcript 2 (NKAT2) and NKG5 in secretory and proliferative human endometrium. Method of Study: Endometrial biopsies were obtained from normally cycling women in the late proliferative and mid-secretory phase of the menstrual cycle. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to determine the expression and regulation of these genes in secretory and proliferative human endometrium. Cellular localization of NKG5, Lpn and IDO by in situ hybridization in secretory-phase endometrium was also examined. Results: Semi-quantitative RT-PCR and Northern blot results demonstrate that there is a coordinated upregulation of this group of genes during the window of implantation. Conclusions: We demonstrate the upregulation of immune-related genes IL-15Rα, Lpn and NKG5 in secretory versus proliferative human endometrium. We also demonstrate a similar upregulation in secretory endometrium of other immune-related genes, viz, DAF, IDO, IL-15, IRF-1 and NKAT2. The functions of these genes include stimulation of proliferation of uterine natural killer (uNK) cells, inhibition of cytolytic activity of uNK cells, inhibition of cell growth of T cells and other pathogens and inhibition of the classical complement pathway. Upregulation of these immune-related genes in the window of implantation suggests their role during the process of implantation and in immune tolerance of the implanting conceptus.

Interaction between the endocrine and the immune systems has been suggested by observations of sexual dimorphism of the immune response, differential susceptibility to autoimmunity between the sexes, changes in autoimmune disease activity during the menstrual cycle and in pregnancy and in vitro studies of hormonal influence on cytokine production.We hypothesized that if there is hormonal regulation of the immune response, this would be manifest in peripheral blood leukocytes (PBLs) at different phases of the menstrual cycle. In this study, we describe gene profiling of PBLs from the follicular and luteal phases of the menstrual cycle. We observe important differences in immune gene expression, with significant down-regulation of the Th1 immune response in the luteal phase. A significant number of interferon (IFN)-related genes are amongst the downregulated genes. These results support significant hormonal regulation of the immune system and may have therapeutic implications in diseases of autoimmunity in women.

Decidualization of endometrial stromal cells is a prerequisite for human implantation and occurs in vivo in response to progesterone and involves activation of the protein kinase A (PKA) pathway. The objective of this study was to determine the molecular signatures and patterns of gene expression during stimulation of this pathway with an analog of cAMP. Endometrial stromal cells from two subjects were treated with or without 8-Br-cAMP (1 mM) for 0, 2, 12, 24, 36, and 48 h and were processed for microarray analysis, screening for 12,686 genes and ESTs. Most abundantly upregulated genes included neuropeptides, immune genes, IGF family members, cell cycle regulators, extracellular matrix proteases, cholesterol trafficking, cell growth and differentiation, hormone signaling, and signal transduction. Most abundantly downregulated genes included activator of NF-κB, actin/tropomyosin/calmodulin binding protein, cyclin B, IGFBP-5, α1 type XVI collagen, lipocortin III, l-kynurenine hydrolase, frizzle-related protein, and cyclin E2. RT-PCR validated upregulation of IGFBP-1, preprosomatostatin, and IL-11, and Northern analysis validated their kinetic upregulation. RT-PCR confirmed downregulation of IGFBP-5, cyclin B, and TIL-4. K-means analysis revealed four major patterns of up- and downregulated genes, and genes within each ontological group were categorized into these four kinetic patterns. Within each ontological group different patterns of temporal gene expression were observed, indicating that even genes within one functional category are regulated differently during activation of the PKA pathway in human endometrial stromal cells. Overall, the data demonstrate kinetic reprogramming of genes within specific functional groups and changes in genes associated with nucleic acid binding, cell proliferation, decreased G protein signaling, increased STAT pathway signaling, structural proteins, cellular differentiation, and secretory processes. These changes are consistent with cAMP modulating early events (0–6 h) primarily involving cell cycle regulation, subsequent events (12–24 h) involving cellular differentiation (including changes in morphology and secretory phenotype), and late events (24–48 h) mediating more specialized function, including immune modulators, in the human endometrial stromal cell.

Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. Herein, we have investigated global gene expression during the window of implantation (peak E2 and progesterone levels) in well characterized human endometrial biopsies timed to the LH surge, compared with the late proliferative phase (peak E2 level) of the menstrual cycle. Tissues were processed for poly(A(+)) RNA and hybridization of chemically fragmented, biotinylated cRNAs on high density oligonucleotide microarrays, screening for 12,686 genes and expressed sequence tags. After data normalization, mean values were obtained for gene readouts and fold ratios were derived comparing genes up- and down-regulated in the window of implantation vs. the late proliferative phase. Nonparametric testing revealed 156 significantly (P0.05) up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport [apolipoprotein (Apo)E being the most induced gene, 100-fold], prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins [glycodelin, mammaglobin, Dickkopf-1 (Dkk-1, a Wnt inhibitor)], IGF binding protein (IGFBP), and TGF-beta superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (gamma aminobutyric acid A receptor pi subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport [e.g. Clostridia Perfringens Enterotoxin (CPE) 1 receptor (CPE1-R) and K(+) ion channel], among others. Down-regulated genes included intestinal trefoil factor (ITF) [the most repressed gene (50-fold)], matrilysin, members of the G protein-coupled receptor signaling pathway, frizzled-related protein (FrpHE, a Wnt antagonist), transcription factors, TGF-beta signaling pathway members, immune modulators (major histocompatibility complex class II subunits), and other cellular functions. Validation of select genes was conducted by Northern analysis and RT-PCR using RNA from endometrial biopsies obtained in the proliferative phase and the implantation window and by RT-PCR using RNA from cultured endometrial epithelial and stromal cells. These approaches confirmed up-regulation of genes corresponding to IGFBP-1, glycodelin, CPE1-R, Dkk-1, mammaglobin, and ApoD and down-regulation for PR membrane component 1, FrpHE, matrilysin, and ITF, as with the microarray data. Cultured endometrial epithelial cells were found to express mRNAs for glycodelin, CPE-1R, Dkk-1, the gamma aminobutyric acid A receptor pi subunit, mammaglobin, matrilysin, ITF and PR membrane component 1. The expression of IGFBP-1, CPE1-R, Dkk-1, and ApoD mRNAs increased upon decidualization of stromal cells in vitro with progesterone after E2 priming, whereas FrpHE decreased, consistent with the microarray results. Overall, the data demonstrate numerous genes and gene families not heretofore recognized in human endometrium or associated with the implantation process. Reassuringly, several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were verified, and the striking regulation of select secretory proteins, water and ion channels, signaling molecules, and immune modulators underscores the important roles of these systems in endometrial development and endometrial-embryonic interactions. In addition, the current study validates using high density oligonucleotide microarray technology to investigate global changes in gene expression in human endometrium.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRα, mPRβ, and mPRγ have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRα and mPRβ but not mPRγ, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRα and mPRβ proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRα mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [3H]progesterone binding to T- and Jurkat cell membranes (Kd 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (Gi), suggesting that mPRs are coupled to Gi in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE→ESE, ESE→MSE, and MSE→LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Changes in the immune environment in the endometrium are believed to be important for successful implantation and maintenance of pregnancy. We have previously investigated global gene profiling in human endometrium during the window of implantation by oligonucleotide microarray technology, and analysis of these data underscore the regulation of a group of immune-related genes. The present study was therefore conducted to examine the pattern of expression and regulation of these genes including decay accelerating factor (DAF), indoleamine 2,3 dioxygenase (IDO), interleukin-15 (IL-15), IL-15 receptor alpha subunit (IL-15Ralpha), interferon regulatory factor-1 (IRF-1), lymphotactin (Lpn), natural killer-associated transcript 2 (NKAT2) and NKG5 in secretory and proliferative human endometrium.Endometrial biopsies were obtained from normally cycling women in the late proliferative and mid-secretory phase of the menstrual cycle. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to determine the expression and regulation of these genes in secretory and proliferative human endometrium. Cellular localization of NKG5, Lpn and IDO by in situ hybridization in secretory-phase endometrium was also examined.Semi-quantitative RT-PCR and Northern blot results demonstrate that there is a coordinated upregulation of this group of genes during the window of implantation.We demonstrate the upregulation of immune-related genes IL-15Ralpha, Lpn and NKG5 in secretory versus proliferative human endometrium. We also demonstrate a similar upregulation in secretory endometrium of other immune-related genes, viz, DAF, IDO, IL-15, IRF-1 and NKAT2. The functions of these genes include stimulation of proliferation of uterine natural killer (uNK) cells, inhibition of cytolytic activity of uNK cells, inhibition of cell growth of T cells and other pathogens and inhibition of the classical complement pathway. Upregulation of these immune-related genes in the window of implantation suggests their role during the process of implantation and in immune tolerance of the implanting conceptus.

Silencing of a target mRNA by small interfering RNA (siRNA) has emerged as a new and powerful tool to study gene function, and post-transcriptional gene silencing can now be accomplished with 21-23 nucleotide RNA that mediate sequence-specific mRNA degradation. In the current study we employed lamin A/C siRNA to silence lamin A/C expression in cultured human endometrial stromal cells and investigated downstream cellular markers for proof of concept. Human endometrial stromal cells from three subjects were transfected with lamin A/C siRNA or non-silencing fluorescein-labelled siRNA, and flow cytometric analysis revealed 95-98% transfection efficiency after 6 h of treatment. RT-PCR and quantitative RT-PCR were used to measure mRNA degradation of lamin A/C, and 75-88% silencing was observed 48 h post-transfection. Western blotting and immunocytochemistry confirmed corresponding decrease in lamin A/C protein within 48 h of gene silencing. The downstream effect of lamin A/C silencing was investigated by immunocytochemical analysis of the cellular localization of the protein, emerin, an important component of the nuclear lamina and known to be regulated by lamin expression. Marked displacement of emerin from the nuclear lamina to the cytoplasm was observed when lamin A/C was silenced in human endometrial stromal cells, confirming functional silencing of lamin A/C resulting in a nuclear lamina assembly defect. Silencing target mRNA by siRNA in human endometrial stromal cells can be more broadly applied to investigate the function and regulation of other genes in this cell type, and the methodology and data presented herein strongly support the more widespread use of this powerful tool in endometrial biology research.

Molecular mechanisms underlying fetal growth restriction due to placental insufficiency and in utero hypoxia are not well understood. In the current study, time-dependent (3 h-11 days) changes in fetal tissue gene expression in a rat model of in utero hypoxia compared with normoxic controls were investigated as an initial approach to understand molecular events underlying fetal development in response to hypoxia. Under hypoxic conditions, litter size was reduced and IGFBP-1 was up-regulated in maternal serum and in fetal liver and heart. Tissue-specific, distinct regulatory patterns of gene expression were observed under acute vs. chronic hypoxic conditions. Induction of glycolytic enzymes was an early event in response to hypoxia during organ development; consistently, tissue-specific induction of calcium homeostasis-related genes and suppression of growth-related genes were observed, suggesting mechanisms underlying hypoxia-related fetal growth restriction. Furthermore, induction of inflammation-related genes in placentas exposed to long-term hypoxia (11 days) suggests a mechanism for placental dysfunction and impaired pregnancy outcome accompanying in utero hypoxia.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

The pharmaceutical industry is in the midst of an information overload triggered by the sequencing of the human genome (Lander et al. 2001; Venter et al. 2001). The challenge now is to elucidate the function of the encoded gene products and to determine their possible involvement in disease. Approaches that measure protein binding (two-hybrid analysis) or changes in expression (microarrays) across the entire genome have been initiated to connect sets of genes functionally. A prime example is the use of microarrays to monitor genome-wide differences in transcription between normal and diseased tissue (DeRisi et al. 1996; Schena et al. 1998; Zweiger 1999; Diehn et al. 2000). Already, enormous strides have been made using such expression profiling to associate unique transcriptional patterns with stages of development in certain cancers (Golub et al. 1999; Perou et al. 1999, 2000; Sgroi et al. 1999; Alizadeh et al. 2000; Bittner et al. 2000; Ross et al. 2000). In addition to serving as diagnostic markers for disease, the unique gene patterns identified might also provide drug development targets for therapeutic intervention by pharmaceutical compounds. However, of the hundreds of up- or down-regulated genes observed, only a small percentage might actually play a functional role in the disease being studied. One way to simplify the problem is to sift through the genes whose expression is altered and identify those genes that might encode activities that are similar to those that have been important historically in drug development. Over the years, researchers have shown that the malfunction of certain classes of proteins, for example, kinases, proteases, phosphodiesterases, phosphatases, and G protein coupled receptors (GPCRs), occurs in a variety of diseases. These findings are not surprising given that these proteins play key roles in signaling pathways that act to coordinate internal cellular functions with the external environment (Cohen 1999; Kowaluk and Jarvis 2000; Pawson and Nash 2000; Stein and Waterfield 2000). Often these proteins belong to large gene families whose members share significant sequence identity, although many members have no established biological function. That is, even if they possess domains suggesting a given biochemical activity, their substrate(s) and role in cellular physiology are unknown. How then does one begin to build specific biochemical assays for hundreds of genes with no clearly defined substrate? Additionally, many proteins will be intractable for various other reasons, such as that they are unstable in vitro or require membrane localization for activity. Moreover, the sheer number of genes indicates the need for an approach encompassing an increase in scale and parallel processing. In this respect, cell-based assays have an inherent advantage over biochemical assays in that they eliminate the time investment required to gain enough knowledge about each protein to prepare a purified target or to modify the target for activation. More importantly, in cell-based assays, proteins are examined in a cellular context that simulates more closely the normal physiological state (Hertzberg 1993; Silverman et al. 1998). There has been widespread recognition that cell-based assays designed in model organisms such as the yeast Saccharomyces cerevisiae provide greater ease of genetic manipulation and can be screened rapidly at a low cost (Kirsch 1993; Silverman et al. 1998; Munder and Hinnen 1999). Although the yeast cell wall has been thought to limit permeability of small molecules during screening, there is now evidence to indicate that compound efflux, as opposed to permeability, is the culprit, and this can be controlled by deletion of the major ABC transporters in yeast (Broach and Thorner 1996; Kolaczkowski et al. 1998; Kaur and Bachhawat 1999). Yeast cell-based assays have been designed in which mammalian proteins with yeast homologs, such as GPCRs, or enzymes such as Topoisomerase II, are made to function in yeast (van Hille and Hill 1998). These types of assays require that the mammalian gene product couple to yeast pathways in such a way as to provide a functional readout that can be used for screening. This approach has been used traditionally to isolate genes from higher organisms by functional complementation of yeast mutations (Becker et al. 1991; Tugendreich et al. 1994; Jonassen et al. 1998). A requirement for this application is that the biochemical activity affected by the yeast mutation has a counterpart in a mammalian pathway (Becker et al. 1991; Jonassen et al. 1998). On the other hand, the presence of a yeast counterpart is not absolutely necessary for a foreign protein to be active in yeast. For instance, although apoptosis is a process generally associated with multicellular organisms, new mammalian genes that play roles in this pathway have been discovered through the use of genetic approaches in yeast (Shaham et al. 1998; Greenhalf et al. 1999). For example, the growth arrest and cell death induced by the mammalian apoptosis effector Bax is overcome when the known apoptosis inhibitors, Bcl-2 and Bcl-x (L), are coexpressed in yeast (Sato et al. 1994; Greenhalf et al. 1996). The reversal of the growth interference phenotype was used to isolate mammalian genes that were shown later to inhibit induction of apoptosis in mammalian cells (Xu and Reed 1998; Greenhalf et al. 1999). These studies confirm the potential use of yeast growth interference assays to study (and to even identify) new genes involved in key cellular pathways in disease, even if it happens that those pathways are not found in yeast. We have developed a high-throughput process in yeast that enables us to assay many heterologous genes for which no suitable assay can be applied easily. We describe here the initial phase of our process that establishes a phenotypic readout induced by expression of a heterologous gene. In later phases of assay development, we use genetic strategies to elaborate further on the phenotype or to broaden the response of yeast to a wider spectrum of heterologous cDNAs. The enhancement of the yeast phenotype is based on the synergistic or potentiating effect of such mutations. This type of “synthetic dosage lethality effect” has been described for overexpression of yeast proteins (Kroll et al. 1996). We describe here an example of such an interaction for the mammalian MAP kinase, p38. The assay development process reported here is depicted in Figure ​Figure11 and encompasses the following suite of methods: (1) a rapid, uniform, and parallel cloning method for the transfer of heterologous cDNAs into yeast expression vectors; (2) a quick method to convert high- or low-copy episomes into linear, integratable vectors; (3) an automated batch processing procedure for analyzing the data from highly sensitive liquid growth assays; (4) a random mutagenesis scheme for validation of the yeast phenotype and for structure/activity analysis of proteins that are characterized poorly or have no known active site; and (5) analysis of the selected mutants and small molecule inhibitors in mammalian secondary assays. The process of profiling cDNAs in a rapid, parallel, and systematic manner eliminates the costly need for a separate assay development strategy for each cDNA and therefore shortens the time between “gene and screen”. These kinds of rapidly constructed assays (which in principle could be derived from any set of heterologous genes, including those from plants or pathogens) allow identification of small molecules as potential leads for drug development and as tools to better understand gene function. Figure 1 Steps for parallel profiling of mammalian and other heterologous cDNAs in yeast cell-based assays. Heterologous cDNAs from cDNA collections are transferred in parallel to yeast expression vectors and transformed into yeast strains optimized for drug screening. ...