Consumption of methylxanthine alkaloids appears to induce activities by antagonizing adenosine receptors, implicated in breast cancer behavior in vitro. Our goal was to evaluate expression of genes for methylxanthine receptors and metabolizing enzymes to assess risk of breast carcinoma recurrence. Clinical outcomes, estrogen/progestin receptor results, and gene expression assays guided selection. RNA was isolated from laser capture microdissection-procured carcinoma cells for microarray using established protocols. Gene expression levels of eight methylxanthine receptors, eight metabolizing enzymes, and various phosphodiesterases were retrieved from microarray results. Univariable Cox regressions and Kaplan-Meier plots were determined for each gene with R software. Individually, lower expressions of PDE4A, CYP2A6, or CYP2E were related to decreased progression-free survival (PFS) and overall survival (OS). PDE1A over-expression predicted decreased PFS and OS. ADORA2B and RYR1 over-expressions predicted diminished OS. ER+ cancers exhibited lower ADORA1, ADORA2B, and RYR1 and elevated PDE4A, CYP2A6, and CYP2E expressions. Of PR+ carcinomas, diminished ADORA2B and RYR1 and elevated expressions of ADORA3, PDE4A, CYP2C8, and CYP2E were noted. Least absolute shrinkage and selection operator (LASSO) revealed that CYP2E, PDE1A, and PDE4A expressions collectively predicted PFS whereas ADORA1, CYP2E, PDE1A, PDE1B, and PDE4A expressions jointly predicted OS. Models were clinically significant when validated externally. LASSO also derived a six-gene model and five-gene model that predicted PFS of ER- or PR- carcinomas, respectively. Similarly, five-gene and four-gene models predicted OS in ER- or PR- carcinomas, respectively. Collectively, expression of genes involved in methylxanthine action and metabolism in single-cell types predicted clinical outcomes of breast carcinoma indicating promise for developing diagnostics and design of new therapeutics.

Background uPA, its receptor uPAR, and inhibitors PAI‐1 and PAI‐2 play key roles in membrane remodeling/invasion and in predicting response to chemotherapy. We identified novel relationships of these biomarkers with ER/PR that indicate clinical utility for assessing breast carcinoma outcomes. Methods Retrospective studies were performed with de‐identified results of (a) uPA, uPAR, and PAI‐1; (b) estrogen (ER) and progestin receptor (PR); and (c) clinical outcomes. Relative expression of 22 000 genes from microarray of RNA from LCM‐procured breast cancer cells was used with R Studio version 3.4.1. Results Primary ER/PR status was related to uPA, uPAR, or PAI‐1 levels. ER− or PR− cancers expressed elevated uPA, uPAR, and PAI2 mRNA compared to ER+ or PR+ cells. Inverse relationships between ER/PR protein and expression of uPA, uPAR, and PAI‐2 were observed, whereas HER2 status was unrelated. qPCR analyses showed RERG and NQO‐1 expressions were elevated in uPA− lesions, while CD34 and EDG‐1 were elevated in uPAR− cancers. ERBB4 was overexpressed in PAI‐1+ carcinomas. Cox regression analyses revealed relationships of ER/PR status and uPA system members with regard to clinical outcomes of breast cancer. Conclusions uPA, uPAR, PAI1, or PAI2 expression was increased in either ER− or PR− cancers similar to that of protein content in ER−/PR− carcinomas, suggesting sex hormones regulate the uPA system in breast cancer. Results revealed protein content of uPA system members was related to ER/PR status of primary lesions. Use of LCM‐procured carcinoma cells uncovered relationships between expression of known cancer−associated genes and protein content of uPA system members. Collectively, results indicate evaluation of ER and PR protein of primary breast cancers combined with analyses of uPA, uPAR, and PAI‐1 protein content improves assessment of clinical outcomes.

Introduction: The urokinase plasminogen activator (uPA), its receptor uPAR and serine protease inhibitors PAI-1 or PAI-2 play a key role in tissue membrane remodeling and invasion of basement membranes by induction of a fibrinolytic pathway. Earlier studies reported that uPA and PAI-1 protein levels assist in prediction of breast cancer response to chemotherapy. Our goal is to develop molecular signatures of candidate genes and identify novel relationships with these four protein biomarkers that demonstrate clinical utility for assessment of breast carcinoma outcomes. Methods: The retrospective study used de-identified tissue biopsies from primary breast cancers on which biomarker and clinical outcomes were stored in an IRB-approved Database. ELISA analyses of uPA, uPAR and PAI-1 performed using IMUBIND kits (American Diagnostica Inc.) used cutoff values previously reported. Estrogen (ER) and progestin receptor (PR) assays were performed either by EIA (Abbott Labs) or by radioligand binding (NEN/DuPont). Relative expression levels of 22,000 genes were determined by microarray using RNA extracted and amplified from Laser-Capture Microdissection (LCM) procured breast carcinoma cells. Univariable and multivariable Cox regression analyses, Kaplan-Meier plots, Violin and scatter grams were performed by R Studio version 3.4.1. External validation of gene subsets derived were performed with SurvExpress. Results: uPA and PAI-1 protein content of a carcinoma were predictive of overall survival (OS). Examination of biomarker gene expression by Violin plots revealed that either ER- or PR- breast cancers expressed elevated levels of UPA, UPAR and PAI2 compared to that of ER+ or PR+ carcinoma cells. Scatter grams suggested an inverse relationship between ER/PR protein levels and expression of UPA, UPAR and PAI2. Univariable Cox regression analyses indicated that PAI2 expression was associated with progression-free survival (PFS) and OS while UPA and PAI1 expression was only associated with OS with a p value < 0.3 (selected as the discovery limit). When carcinomas were sorted by biomarker levels, qPCR expression of RERG and NQO-1 were elevated in uPA- lesions while CD34 and EDG-1 were elevated in uPAR- cancers. However, ERBB4 expression was elevated in PAI-1+ carcinomas. Multivariate Cox regression, performed with backward conditional selection using microarray data with ER or PR status revealed clinically relevant genes for PFS and OS. SurvExpress, an online tool, validated gene subsets externally. Conclusions: Use of LCM-procured breast carcinoma cells in a nondestructive manner uncovered relationships between gene expression and either uPA, uPAR, PAI-1 or PAI-2 protein content of a lesion to reveal candidates for predicting clinical outcomes. Certain of these gene subsets appear related to patient prognosis when considered with ER/PR status of the carcinomas. Citation Format: Seth B. Sereff, Michael W. Daniels, James L. Wittliff. Expression of genes of the uPA system from LCM-procured breast carcinoma cells and their relationships with clinical outcomes [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 1610.

Consumption of methylxanthine alkaloids such as caffeine, theophylline and theobromine may induce breast pain. Caffeine appears to induce its biological activities by antagonizing adenosine receptors, which have been implicated in breast cancer cell behavior in vitro. Our goal is to evaluate expression of genes for methylxanthine receptors and metabolizing enzymes for assessing risk of breast cancer recurrence. Procedures: De-identified primary cancers previously collected, stored and analyzed under stringent conditions were employed to amass an IRB-approved, de-identified comprehensive Database. Patient-related properties (e.g., nodal status, clinical outcome) and results from estrogen (ER) and progestin receptor (PR) analyses and gene expression assays guided selection. To decode clinical utility of gene expression profiles, Laser Capture Microdissection (PixCell IIe™ Arcturus/Thermo Fisher) was used previously to non-destructively collect carcinoma cells. RNA was extracted, amplified and analyzed by microarray (~ 22,000 genes). Results: Gene expression levels of 8 methylxanthine receptors, 8 metabolizing enzymes and various phosphodiesterases were retrieved from microarray results of 247 breast biopsies. Univariable Cox regressions and Kaplan Meier plots were determined for each candidate gene with R software. Kaplan-Meier plots of PDE4A, CYP2A6 or CYP2E individually indicated lower expression was related to decreased progression free (PFS) and overall survival (OS) while PDE1A over-expression predicted decreased PFS and OS. ADORA2B and RYR1 over-expression was associated with lower OS. Gene expression was examined with ER or PR status without regard to clinical outcome. Breast cancers that were ER+ exhibited lower ADORA1, ADORA2B and RYR1 expression and elevated expression of PDE4A, CYP2A6 and CYP2E. PR+ carcinomas also exhibited decreased expression of ADORA2B and RYR1 expression. However, over-expression of ADORA3, PDE4A, CYP2C8 and CYP2E was observed in PR+ cancers. Analysis of variance (ANOVA) of carcinomas according to ER/PR status indicated ADORA2B expression was elevated in ER- cancers regardless of PR status. ANOVA also showed PDE4A was over expressed in ER+ carcinomas regardless of PR status. LASSO analysis revealed CYP2E, PDE1A and PDE4A expression taken jointly predicted PFS which was validated externally (Breast Cancer Meta-base) with SurvExpress. Conclusions: Collectively, results suggest expression of genes involved in methylxanthine action and metabolism may be used to predict breast cancer behavior. Significantly, we identified gene expression signatures in single cell types that were highly associated with clinical outcome suggesting promise for development of novel prognostic tests for breast carcinoma management and design of new therapeutics. Citation Format: Seth B. Sereff, Michael W. Daniels, James L. Wittliff. Novel relationships of expression of methylxanthine alkaloid receptor genes and risk of breast carcinoma recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1973. doi:10.1158/1538-7445.AM2017-1973