PGR Gene Expression Research Articles

A study of the prevalence of PGR gene expression with ER and PR Receptor markers positivity in female breast cancer cases in Al-Najaf province, Iraq

A study of the prevalence of PGR gene expression with ER and PR Receptor markers positivity in female breast cancer cases in Al-Najaf province, Iraq

Regulation of PGR gene expression in immortalized human endometrial stromal cells

Regulation of PGR gene expression in immortalized human endometrial stromal cells

Abstract PO1-14-08: Real-world clinical genomics study of HR+/HER2- metastatic breast cancers treated by CDK4/6i plus endocrine therapies revealed a drug resistant tumor segment characterized by ER independence

Abstract Background CDK4/6 inhibitors (CDK4/6i) plus endocrine therapies (ET) are the standard-of-care for hormone receptor–positive/human epidermal receptor 2–negative metastatic breast cancer (HR+/HER2− mBC). However, drug resistance remains a major unmet need. Investigations of drug resistance mechanisms has been hampered by a dearth of tumor molecular profiling data from the post-treatment setting. To address this challenge, we have conducted a real-world clinical genomics study to better understand the molecular mechanism of CDK4/6i resistance as well as to stratify patients based on integrated multi-omics profiles. Methods We retrospectively analyzed a multi-omics dataset of 400 HR+/HER2- mBC patients who had received CDK4/6i plus ET and developed progressive disease (PD) from the de-identified Tempus database. Pre-treatment and post-progression biopsies were taken  1 year prior to starting the CDK4/6i treatment or following PD respectively. Tempus xT next-generation sequencing (DNA-seq of 648 genes) and RNA sequencing assays were performed on 427 tumor FFPE samples, including 200 pre-treatment, 227 post-progression and 26 longitudinal pairs. Results The median age of the patients was 57 (54.9-57.4) and median progression free survival (PFS) is 379 (341-433) days. Two genes were found to harbor a significant increase in genomic alteration frequencies (GAF) after adjusting for FDR at post-progression vs. pre-treatment – ESR1 (41.9% vs. 15%, p=5.4e-10), RB1 (13.2% vs. 3%, p=8.5e-05). ESR1 and RB1 also harbored high frequencies of acquired genomic alterations among 26 paired samples at 34.6% and 11.5% respectively. TP53 mutation at baseline was significantly associated with shorter PFS at baseline (p=4.23e-05, HR=2.081) and TP53 GAF significantly increased after PD (37% vs. 28.5%, p=0.039). BRCA1/2 pathogenic mutations (p=1.63e-04, HR=3.066), APOBEC mutation signature S13 (p=0.0125, HR=1.55) and CCNE1 gene expression (p=0.024, HR=1.46) were significantly associated with shorter PFS. APOBEC signature (p=0.0035) and CCNE1 expression (p=1.33e-06) also significantly increased post-progression. Among the top molecular features associated with longer PFS were markers of estrogen signaling such as PGR gene expression (p=6.76e-04, HR=0.565) and the Hallmark estrogen response signature (p=0.021, HR=0.679). Applying a multi-omics pattern recognition algorithm, we identified a molecularly distinct cluster (IC1) characterized by down-regulation of estrogen signaling. IC1 is significantly associated with shorter PFS (p=3.72e-05, HR=0.22) and increased from 4% pre-treatment to 23% post-progression (p=7.3e-08). Further, IC1 is strongly enriched in markers previously implicated in CDK4/6i resistance including CCNE1 expression, RB1 mutation and MYC/E2F activation. We then developed machine learning models to predict gene-level dependency trained on cancer cell line expression and CRISPR-KO screen data. These models predicted decreased dependency on ESR1 and CDK4 and increased dependency on CDK2 in IC1, strengthening the association between ER independence and CDK4/6i resistance. Conclusions Our real-world clinical genomics study identified a comprehensive list of biomarkers associated with resistance to CDK4/6i plus ET and estimated patient prevalence for these markers in the post-treatment setting. Integrated and machine-learning analyses identified a subset of aggressive tumors with estrogen independence characteristics that are implicated in CDK4/6i resistance and suggested new therapeutic strategies. Citation Format: Zhengyan Kan, Ji Wen, Jennifer Webster, Vinicius Bonato, Whijae Roh, Xinmeng Jasmine Mu, Paul Rejto, Jadwiga Bienkowska. Real-world clinical genomics study of HR+/HER2- metastatic breast cancers treated by CDK4/6i plus endocrine therapies revealed a drug resistant tumor segment characterized by ER independence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-08.

Abstract PO4-16-02: Can high progesterone receptor (PgR) expression identify tumours with low-risk tumour gene expression scores?

Abstract Background Strong PgR expression predicts favorable outcomes for ER+ve HER2-ve breast cancer and has been proposed as a surrogate marker to distinguish between IHC-defined luminal A and luminal B subtypes. It is therefore possible that strong PgR expression may be able to predict tumor gene expression test results and independently identify tumors that are unlikely to be chemotherapy sensitive. PgR expression is traditionally determined by immunohistochemistry (IHC). Several validated RNA-based PGR expression tests have been developed that may outperform IHC. Methods We compared Oncotype DX RS with Oncotype DX reported PGR in 4 independent datasets which included 407 cases from the OPTIMA prelim trial. We further analyzed 251 OPTIMA prelim cases which had additional tumor gene expression data. All gene expression assays were performed by the test vendor, including PGR gene expression determined using the Mammatyper assay. PgR IHC was determined in a single laboratory on triplicate tissue micro-arrays using quantitative image analysis including a 10% manual quality control check. We analyzed PGR expression using cutoffs that correspond to approximately 20% staining; the standard Oncotype DX PGR assay is reported as positive if the score is >5.4, corresponding to approximately 1% staining by IHC. We used Spearman’s rank correlation coefficient to compare PGR data. Results The four Oncotype DX datasets consistently demonstrated that high Oncotype PGR expression was associated with a low RS (table). Combining the 3 validation data sets consisting of 633 cases, 70.9% had high PGR expression of which 92.7% had a an Oncotype RS ≤25. Approximately 50% of cases with low Oncotype PGR expression had an Oncotype RS >25. Mammatyper and Oncotype PGR were highly correlated (Rs = 0.9258, P< 0.001) in the OPTIMA prelim dataset (n=251), with only 8.4% of tumors having discordant high/low Mammatyper and Oncotype PGR scores. 93.2% of 176 Mammatyper high PGR expression cases had an RS ≤25. The Mammatyper PGR and PgR IHC correlation was weaker (Rs=0.763, P< 0.001); 87.2% of 211 cases with >20% staining had RS ≤25. PgR IHC staining had a bimodal distribution and there was little effect on the prediction of low RS score up to a 67% cut-off. Mammatyper and Oncotype PGR scores both appear to have a normal distribution. We took advantage of this to perform an exploratory analysis using a higher Mammatyper PGR cutoff. We were able to show superior prediction of a low RS (96.8%) but with a reduced proportion (50.2%) of high PGR score tumors. High PGR gene expression was weakly associated with low (≤60) Prosigna ROR_PT score and MammaPrint low risk (72.2% and 65.9% respectively) and with Prosigna and MammaPrint luminal A subtype (both 64.8%). Conclusion High progesterone receptor gene expression measured using locally performed RNA-based assays may allow the reliable prediction of Oncotype DX low-risk tumours. This analysis provides additional information for the clinical utility of PGR measurement. Additional data will be presented on the optimal PGR cutoff. OPTIMA prelim is registered as ISRCTN42400492 and funded by the UK NIHR Health Technology Assessment Programme, award number 10/34/01. Views expressed are those of the authors and not those of the HTA Programme, NIHR, NHS or the Department of Health. Table. Oncotype DX RS and PGR in 4 datasets Distribution of RS according to %cases with high or low PGR Citation Format: Robert Stein, Ralph Wirtz, Andrea Marshall, Jane Bayani, Sebastian Eidt, Claudia Schumacher, Hans-Peter Sinn, Andreas Schneeweiss, Andreas Makris, Iain Macpherson, Luke Hughes-Davies, Tammy Piper, Monika Sobol, Georgina Dotchin, Helen Higgins, Sarah Pinder, Abeer Shaaban, Janet Dunn, John MS Bartlett. Can high progesterone receptor (PgR) expression identify tumours with low-risk tumour gene expression scores? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-02.

CAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization.This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR.Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL.In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

Prospective longitudinal multi-omics study of palbociclib resistance in hormone receptor+/HER2- metastatic breast cancer.

1013 Background: The development of CDK4/6 inhibitors represents a significant advance in the treatment of metastatic breast cancer (MBC). To better understand the impact of treatment and drug resistance at the molecular level, we performed multi-omics profiling of matched pre-treatment, on-treatment, and post-progression tumor biopsies from patients treated with palbociclib combined with endocrine blockades (AIs and fulvestrant). The purpose of the study was to identify biomarkers of palbociclib resistance as well as to assess molecular changes during treatment, and those appearing at disease progression. Methods: Patients with Hormone Receptor positive (HR+)/HER2- MBC treated with palbociclib in combination with endocrine therapies were prospectively enrolled from July 2017 to June 2020. Of the 89 patients enrolled, we obtained tumor biopsies and matched blood samples, taken at pre-treatment, on-treatment (6 weeks or 12 weeks) and progressive disease (PD) from 71 patients (first line: 55, second line or later: 16 pts.) who had agreed to informed consent form. Tumor biopsies were profiled using whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-Seq). Results: Median follow-up duration was 20 (3-48) months and median age of the patients was 45 (range 30-71) years. The median progression free survival (PFS) of 71 patients was 15 (0.7-39.8) months. The Luminal B subtype is associated with shorter PFS compared to Luminal A (8.6 m vs 19.3 m, p=0.03, HR=1.96). The Luminal B subtype along with multiple cell cycle regulatory genes such as CCNE1 (8.3 m, p=0.015, HR=2.07), CCNE2 (8.5 m, p=8.5e-3, HR=2.2), CDK2 (8.45 m, p=0.05, HR=1.79) are associated with shorter PFS while estrogen response signatures (20.9 m, p=4.6e-3, HR=0.43) and PGR gene expression (20.2 m, p=6.2e-2, HR=0.56) are associated with more favorable prognosis. A Cox-regression multivariate model (p=5.17e-5, C-Index=0.72) was developed and revealed that PFS is independently associated with BRCA1/2 (HR=1.44; CI=[0.49, 4.29]), TP53 mutation statuses (HR=3.58; CI=[1.58, 8.13]), the HRD mutation signature (HR=1.40; CI=[1.09, 1.81]) and the proliferative index (HR=1.53; CI=[1.00, 2.34]), an expression signature of cell proliferation. Tumors classified as Luminal A at baseline frequently switched into Luminal B or Her2-enriched subtypes at PD, along with up-regulation of cell cycle markers and proliferation signatures. Further, tumor mutation burden (TMB) and HRD index, a DNA-based measure of genomic instability, significantly increased from baseline to PD in patients with TP53 and BRCA1/2 wild-type tumors. Conclusions: Our longitudinal multi-omics study identified prognostic biomarkers as well as post-treatment enrichment of HRD related genomic scars and frequent switching into molecular subtypes with aggressive and estrogen independence characteristics. Clinical trial information: NCT03401359 .

Abstract PS10-02: A good prognosis of endocrine-dependent tumors among residual invasive cancer after anti-HER2 therapy: CALGB 40601 (Alliance) and validation studies

Abstract Background: Patients with HER2+ breast cancer who have residual disease after neoadjuvant anti-HER2 plus chemotherapy have a high risk of recurrence and benefit from adjuvant trastuzumab emtansine (T-DM1). We hypothesize that endocrine-responsive residual tumors after neoadjuvant treatments may have good outcomes among patients receiving only adjuvant endocrine therapy plus trastuzumab. Using paired pre- and post-treatment samples from CALGB 40601 and other neoadjuvant cohorts that did not include adjuvant T-DM1, we investigated survival by pretreatment and residual disease ESR1 and PgR gene expression in CALGB 40601, and by estrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry (IHC) in the other cohorts. We considered endocrine-responsive tumors those with ER and/or PR expression by gene expression or IHC. Methods: CALGB 40601 is a randomized neoadjuvant trial of single vs. dual HER2-targeting (trastuzumab and/or lapatinib added to paclitaxel). We only included those patients who had not suffered from disease progression or death during their preoperative treatments. In total, 77 patients with paired pretreatment and residual disease tumors were profiled by mRNA sequencing and studied. Cutoffs for ESR1 and PgR mRNA expression mimicking clinical positivity were obtained using 265 pretreatment CALGB 40601 tumors (and 1,045 TCGA samples for ESR1). We also examined ER and PR IHC in paired tumors from 202 patients treated at 4 different collaborating institutions; all had residual disease after neoadjuvant HER2 targeting plus chemotherapy. We considered ER- or PR-positive as ≥10% positively staining cells. The primary endpoint was EFS, defined as the time from randomization to event in CALGB 40601 and from the first systemic therapy to event in the 4-institution validation cohort. Results: In 77 patients from CALGB 40601 with paired (pretreatment/residual disease) specimens, 38 (49.3%) had ESR1+/ESR1+ tumors. The EFS was superior in the ESR1+/ESR1+ (n=38) group than in the remaining others (the log-rank test, p=0.011). The 5- and 7-year EFS rates for the ESR1+/ESR1+ (n=38) were 92.1% and 89.2%, whereas the rates were uniformly < 70% in the others. In particular, the 5-year EFS rate among 11 patients with ESR1+/ESR1- tumors was 61.4%. This remained significant in multivariable analysis with clinical stage and treatment arm; the hazard ratio (HR) for EFS in ESR1+/ESR1+ versus all others was 0.29 (95% CI, 0.09-0.90). In ESR1+/ESR1+ tumors, 5-year EFS rates were high for those whose residual disease and also being PgR+ (n=32) or PAM50 LumA or Normal-like (n=34) (93.8% and 97.1%, respectively). In the institutional validation cohort, pretreatment /residual disease ER(+)/ER(+) tumors (n=113) had superior 3-year EFS versus all others (p=0.010). At a median follow-up of 35.9 months, the 3-year EFS rates for ER(+)/ER(+) and all other groups were 96.6% and 82.6%, respectively. This remained strongly significant in multivariable analysis with clinical stage; the HR for EFS in ER(+)/ER(+) versus all other groups was 0.28 (95% CI, 0.09-0.88). Among 46 who also had PR+ in the residual disease, the 3-year EFS was 100.0%. Conclusions: HER2+ patients with ER+ pretreatment and ER+ residual disease after neoadjuvant chemotherapy + HER2-targeting have a very good survival outcome despite not receiving additional anti-HER2 targeting with T-DM1. This may provide a simple mechanism to better tailor therapy within residual disease patients using serial ER measurements. Citation Format: Sung Gwe Ahn, Aranzazu Fernandez-Martinez, Mei-Yin C. Polley, Soong June Bae, Seho Park, Núria Chic, Fara Brasó-Maristany, Benedetta Conte, Valentina Guarneri, Maria Vittoria Dieci, Gaia Griguolo, PierFranco Conte, Joon Jeong, Aleix Prat, Lisa A. Carey, Charles M Perou. A good prognosis of endocrine-dependent tumors among residual invasive cancer after anti-HER2 therapy: CALGB 40601 (Alliance) and validation studies [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 PS10-02.

O16 Phyto-oestrogenic property of mimosine: A phenylalanine alkaloid

Hormone replacement therapy (HRT) has been generally used in postmenopausal symptoms in women caused by a lack of endogenous oestrogen. However, due to the increased concern around the potential associated risks of breast and endometrial cancer there is an increased demand for natural oestrogenic agents with lesser side effects. Mimosine is a phenylalanine class of alkaloid. It has been reported that, another phenylalanine alkaloid capsaicine and its analogues possess oestrogenic activity. Therefore, this study was designed to investigate the oestrogen like activity of mimosine by using in vitro assays with the ER positive MCF-7 cell line and in silico models. Cell viability by cellular count and MTT cell proliferation assays were performed using the oestrogen-dependent MCF-7 breast cancer cells. The regulation of oestrogen marker TFF1 and PGR gene expression were also studied. The binding ability of mimosine to ER α was predicted by in silico docking method. The results indicated that mimosine caused significant cell proliferation ( P P P P α binding site as compared to estradiol. The results indicate the oestrogenicity of mimosine for the first time and suggest that, it can be further studied and developed as an alternative to HRT for postmenopausal women.

Postcoital administration of RU486 induces a hormonally under-stimulated rat endometrium

RU486 is a partial progesterone and estrogen receptor antagonist, functioning to actively silence progesterone receptor gene-associated transcription. For this reason, it has been used as both a contraceptive and an abortive agent. In the present study, cellular and gene specific effects of RU486 were investigated in a rat model of early pregnancy, including key phases of the window of receptivity and early implantation. As these stages are hormonally regulated by progesterone and estrogens, the focus here was to elucidate the mechanism of action of a single dose of RU486, used as a postcoital contraceptive, to successfully prevent implantation of a viable blastocyst. Immunofluorescent techniques were used to examine the change in protein levels of PR in RU486-treated endometria at days 4.5, 5.5 and 6.5 of pregnancy. Changes in the Pgr gene expression level as a consequence of RU486 administration was evaluated using quantitative real-time reverse transcription polymerase chain reaction. The progesterone receptor gene and protein expression was ubiquitously decreased throughout pregnancy as a direct consequence of RU486 administration. The overall effects of postcoital RU486 administration during early pregnancy indicate highly effective inhibition of progesterone and estrogen effects on the endometrium, mediated by their receptors. More specifically, the expression and localization of the progesterone receptor mirrors that described in ovariectomized animal models, suggesting a hormonally under-stimulated endometrium. Clearly from the present study, the precise priming of the endometrium by progesterone, in preparation for blastocyst implantation, is severely impaired by RU486, thus predisposing the uterus to pregnancy failure.

26P - Gene Expression of ESR1, PGR and HER2 in Lymph Node Homogenates and Concordance with IHC in Primary Breast Tumours

Aim: To determine whether extracts from the molecular assessment of sentinel lymph node (LN) status may be used (i) to assess ER, PR or HER2 status and (ii) as a resource for assessing other molecular markers in axillary LNs. Background: One-step nucleic acid amplification (OSNA) is used for rapid detection of sentinel LN metastases. Based on the RNA level of cytokeratin 19 (KRT19) an epithelial marker, in LN extracts, the LN is classified as negative, ‘+’ positive or ‘+ +’ positive. Measurement of molecular markers in these extracts could be clinically useful when insufficient invasive tissue is present in the primary to characterize these markers and may have research applications. Patients and methods: 800 consecutive LN homogenates remaining after OSNA tests yielded 39 OSNA ++ cases. Additional HER2 + (IHC 3+) and ER -ve cases were identified from 400 further LN homogenates to give a final set of 48 OSNA ‘+ +’ cases (7 HER2 +, 7 ER -ve and 9 PR -ve). 23 OSNA -ve cases were used as controls. RNA was extracted from the homogenates and gene expression of KRT19, ESR1, HER2 and PGR was assessed by QPCR. IHC data was available from routine analysis of the primary tumours. Results: As expected, KRT19 expression levels in OSNA ++ samples were much higher (mean 1840-fold) than in OSNA -ve samples. Expression levels of ESR1, HER2 and PGR were 30-40-fold higher in OSNA ++ samples compared to OSNA -ve samples. 6 of the 7 IHC HER2 + cases had the highest HER2 gene expression levels. When HER2 expression levels were normalized to KRT19 level (to account for the amount of tumour present in each LN homogenate) there was complete concordance between LN RNA and primary tumour IHC. The 7 ER -ve cases were among the 9 lowest ESR1 gene expression levels; normalization to KRT19 did not improve the association. The 9 PR -ve cases were among the 13 lowest PGR gene expression levels; normalization to KRT19 lost any concordance for PR. Conclusion: These novel data indicate that LN extracts remaining after OSNA may have clinical utility for the measurement of HER2 and possibly ER and PR status. The data also suggest that RNA extracts from LNs are a resource for understanding the molecular characteristics of those cells with the greatest potential for metastatic spread. Disclosure: All authors have declared no conflicts of interest.

DNA methylation patterns of steroid receptor genes ESR1, ESR2 and PGR in deep endometriosis compromising the rectum

Endometriosis is characterized by the presence of endometrial-like tissue located outside the uterine cavity. Recent evidence suggests that endometriosis may be an epigenetic disease, as well as an estrogen-dependent disease. Based on the unique steroid hormone receptor expression profile observed in endometriotic lesions as compared to eutopic endometrium, the present study aimed to gain further insight into the DNA methylation patterns of alternative promoters of the steroid receptor genes ESR1, ESR2 and PGR in intestinal deep endometriosis, one of the most aggressive forms of endometriosis. The DNA methylation patterns were evaluated by methylation-specific polymerase chain reaction (MS-PCR) after bisulfite modification in 44 endometriotic tissues as well as in 7 matched eutopic endometrium. No differences in the DNA methylation were observed for the ESR1 and ESR2 genes. Methylation of the PGR gene was observed in 39% (17 out of 44) and 19% (7 out of 37) of the cases in the promoter regions B (PGRB) and A (PGRA), respectively. Both PGR promoter regions were methylated in 3 cases. PGRB methylated alleles were detected exclusively in the endometriotic lesions when compared to the eutopic endometrium obtained from the same patient. The effect of DNA methylation in inhibiting the PGR gene expression was corroborated by immuno-staining for PgR protein in a subset of tissue samples. The present study demonstrated that epigenetic changes occur in both promoter regions of the PGR gene in intestinal endometriosis. Since eutopic and ectopic tissues do not respond sufficiently to progesterone in women with endometriosis, further study is necessary to evaluate the effect of epigenetic alterations in progesterone-resistance in this enigmatic disease.

RELATIONSHIP OF GENE EXPRESSION AND SEX HORMONE RECEPTORS IN PRIMARY BREAST CARCINOMA AND METASTASES FROM THE SAME PATIENT

Using microarray results and published reports, 32 genes were selected whose expression appears related to clinical outcome of breast cancer. Candidate gene expression and estrogen (ER) and progestin receptor (PR), determined by ligand binding or EIA, were evaluated to ascertain their relationship during lymph node invasion. RNA was isolated from tissue sections of de‐identified frozen carcinoma biopsies. cDNA was prepared with Superscript RT III reagents (Invitrogen) and qPCR reactions were performed using Power Sybr Green PCR Master Mix (Applied Biosystems). Although levels of ER and PR protein varied between the primary and the metastasis, no primary lesion that was ER− or PR−exhibited a positive ER/PR result in the metastasis. Only 7 patients with either ER+ or PR+ primaries exhibited loss of receptor expression in the metastasis. ESR1 and PGR gene expression correlated highly with protein levels of ER and PR. Expression of 6 of 32 genes (TBC1D9, TRIM29, SCUBE2, IL6ST, SLC39A6, LRBA) was discordant in at least half of the primary lesion‐lymph node pairs, while expression of 6 genes (TRIM29, SCUBE2, SLC39A6, TCEAL1, PFKP, XBP1) was discordant in at least half of the primary‐distant metastasis pairs. These results address the molecular basis for progression of a primary lesion to a metastatic phenotype. Supported in part by grants from Phi Beta Psi Charity Trust and a CTSP Grant from the Commonwealth of Kentucky.

Establishment of a standardized gene-expression analysis system using formalin-fixed, paraffin-embedded, breast cancer specimens

It has recently being emphasized that gene-expression profiles are important clinical decision-making tools, and as such must be standardized across hospital laboratories in the same way as pathological investigations. In this study our objective was to independently establish a standardized gene-expression assay system using routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues. To verify gene expression by quantitative real-time polymerase chain reaction, the most stably expressed reference genes were explored using 30 matched FFPE and fresh frozen (FF) tissues. FFPE specimens from 290 female breast cancer patients were used for further RNA extraction; ESR1 and PGR were measured using 203 matched FFPE and FF specimens and normalized to these reference genes. RNA extracted from FFPE specimens was highly degraded, but almost the same selection of genes was identified-TAF, PUM1, and ACTB, and, for FFPE specimens only, FKBP15. Eventually 88.6% of all the FFPE samples were identified as quantitatively and qualitatively adequate for downstream analysis. The results revealed good correlation and excellent concordance with ERα and PgR protein expression evaluated by immunohistochemistry. Moreover, the distribution of ESR1 and PGR gene expression values was quite reasonable, reflecting differences between the transcriptional mechanisms of the respective genes. We successfully confirmed that our gene-expression analysis system provides good quality control for larger scale assays; it may therefore be suitable for development, in the near future, of a multiple gene assay as a routine clinical judgment tool.

Characterization and Application of Oviductal Epithelial Cells In Vitro in Gallus domesticus1

Chicken oviductal epithelium produces large quantities of egg white protein in daily cycles. In this study, we cultured and characterized oviductal epithelial cells (OECs) from juvenile (10-wk-old) chickens and from actively laying (30-wk-old) hens. The juvenile OECs were maintained over passage 25 and were positive for toluidine blue, lectin-ConA, HPA, UEA-1, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR, whereas the adult OECs were cultured over passage 6 and were positive for toluidine blue, periodic acid-Schiff, lectin-ConA, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR. To investigate the optimal concentration of steroid hormones for inducing egg white protein genes in vitro, we examined the effects of estrogen, diethylstilbestrol, progesterone, and corticosterone on OECs. Results showed that oviduct-specific levels of avidin, ovalbumin, ovomucin, lysozyme, ESR1, and PGR gene expression were significantly elevated in steroid hormone-treated OECs compared with those of untreated cells (P < 0.05). Ovalbumin protein was also secreted into culture medium from hormone-treated OECs. In addition, to examine the application of OECs for avian transgenesis, we introduced human thrombopoietin (THPO)-expressing lentiviral vector controlled by a 3.5-kb ovalbumin promoter into cultured OECs, and THPO expression was significantly induced with diethylstilbestrol or progesterone in juvenile OECs (P < 0.05) and in adult OECs (P < 0.05). In conclusion, these data demonstrate the potential of cultured OECs as a model system for providing a better understanding of the regulation of gene expression and for the production of an avian transgenic bioreactor.

Lapatinib Restores Hormone Sensitivity with Differential Effects on Estrogen Receptor Signaling in Cell Models of Human Epidermal Growth Factor Receptor 2–Negative Breast Cancer with Acquired Endocrine Resistance

Acquired endocrine resistance in estrogen receptor (ER)alpha+/human epidermal growth factor receptor 2-negative (HER2-) breast cancer has been associated with modest adaptive increases in HER2, although exactly how aberrant HER2 signaling affects the ERalpha pathway is poorly understood. We investigated (a) whether the epidermal growth factor receptor/HER2 inhibitor lapatinib could restore endocrine responsiveness in cell models of acquired endocrine resistance with modest increases in HER2, and (b) the nature of ERalpha-HER2 cross-talk in this process. Combination growth studies, ERalpha transcription, immunoblot, and gene expression assays were conducted in two models of acquired resistance to (a) estrogen deprivation (long-term estrogen-deprived cells) and (b) tamoxifen (long-term tamoxifen-treated cells), and in hormone sensitive controls. Changes in ERalpha, PgR, and HER2 were assessed in samples from patients treated with tamoxifen. Both cell models of acquired endocrine resistance showed modest adaptive upregulation in HER2, and lapatinib restored endocrine sensitivity in both. The effect of lapatinib on ERalpha signaling varied markedly depending on the nature of the HER2/ERalpha cross-talk. In long-term estrogen-deprived cells characterized by enhanced ERalpha function, lapatinib suppressed ERalpha genomic activity (as measured by pERSer118, ERalpha transcriptional activity, and PGR gene expression). In contrast, in long-term tamoxifen-treated cells with reduced ERalpha activation, lapatinib reactivated ERalpha genomic function. Twenty percent of tamoxifen-resistant patients relapsed with modest increases in HER2 and either suppressed or enhanced ERalpha/PgR expression. Aberrant GFR signaling can augment or suppress ERalpha function. Regardless, interrupting the HER2/ERalpha cross-talk with lapatinib can restore endocrine sensitivity and should be investigated as a therapeutic strategy in combination with endocrine therapy in ERalpha+/HER2- patients with acquired endocrine resistance.