Michael W. Daniels

Continuous infusion of low-dose unfractionated heparin after aneurysmal subarachnoid hemorrhage: a preliminary study of cognitive outcomes

OBJECTIVECognitive dysfunction occurs in up to 70% of aneurysmal subarachnoid hemorrhage (aSAH) survivors. Low-dose intravenous heparin (LDIVH) infusion using the Maryland protocol was recently shown to reduce clinical vasospasm and vasospasm-related infarction. In this study, the Montreal Cognitive Assessment (MoCA) was used to evaluate cognitive changes in aSAH patients treated with the Maryland LDIVH protocol compared with controls.METHODSA retrospective analysis of all patients treated for aSAH between July 2009 and April 2014 was conducted. Beginning in 2012, aSAH patients were treated with LDIVH in the postprocedural period. The MoCA was administered to all aSAH survivors prospectively during routine follow-up visits, at least 3 months after aSAH, by trained staff blinded to treatment status. Mean MoCA scores were compared between groups, and regression analyses were performed for relevant factors.RESULTSNo significant differences in baseline characteristics were observed between groups. The mean MoCA score for the LDIVH group (n = 25) was 26.4 compared with 22.7 in controls (n = 22) (p = 0.013). Serious cognitive impairment (MoCA ≤ 20) was observed in 32% of controls compared with 0% in the LDIVH group (p = 0.008). Linear regression analysis demonstrated that only LDIVH was associated with a positive influence on MoCA scores (β = 3.68, p =0.019), whereas anterior communicating artery aneurysms and fevers were negatively associated with MoCA scores. Multivariable linear regression analysis resulted in all 3 factors maintaining significance. There were no treatment complications.CONCLUSIONSThis preliminary study suggests that the Maryland LDIVH protocol may improve cognitive outcomes in aSAH patients. A randomized controlled trial is needed to determine the safety and potential benefit of unfractionated heparin in aSAH patients.

Expression of Genes for Methylxanthine Pathway-Associated Enzymes Accompanied by Sex Steroid Receptor Status Impacts Breast Carcinoma Progression

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.

Abstract 669: Quantification of estrogen (ER) and progestin receptor (PR) as well as HER2/neu proteins and gene expression improves discrimination of clinical behavior by triple positive breast carcinomas

IHC analyses of ER, PR and HER2 proteins are used as clinical biomarkers for breast cancer management. However, IHC provides semi-quantitative results sometimes complicated by variation in methods and interpretation. Our goal was to ascertain clinical use of these three analytes to predict breast cancer recurrence when analyses for gene expression and protein products were quantified. Procedures: We examined ER and PR protein in 1059 de-identified carcinoma biopsies previously quantified by radio-ligand binding and/or enzyme immunoassay (EIA) using FDA approved reagents and HER2 oncoprotein determined by EIA to assess relationships between biomarker profiles and disease-free (DFS) and overall survival (OS) for 123 patients. Our comprehensive, IRB-approved Database also contained de-identified Microarray results obtained for ~ 22,000 genes from LCM-procured breast carcinoma cells of 247 primary breast biopsies. ESR1, PGR and ERBB2 gene expression levels were validated previously in 278 cancers by qPCR. Results: Generalized Pair Plots of the three protein biomarkers indicated a relationship only between ER and PR supporting evidence that ER complexed with estrogenic ligands promotes synthesis of PR. Of the 8 possible combinations of the 3 protein biomarkers, only breast cancers exhibiting ER+PR+HER2+ (triple positive breast cancer, TPBCa) exhibited increased OS, whereas those with triple negative breast cancers (TNBCa) had decreased OS. TPBCa was exhibited by 32.5 % of specimens compared to TNBCa, which represented 7.3% of biopsies when biomarker proteins were quantified. Number of TPBCa was less when expression of the 3 genes (ESR1+/PGR+/ERBB2+) was measured by microarray or by qPCR while those of TNBCa increased. Furthermore, when TPBCa were identified by either microarray or by qPCR measurements, collectively increased ESR1, PGR and ERBB2 expression was associated with increased PFS and OS of patients. In contrast, biopsies that were ESR1-/PGR-/ERBB+ (14.1%) correlated with poor prognosis and overall survival using qPCR data, while those exhibiting ESR1-PGR+ERBB2- (14.1%) profiles by microarray results exhibited decreased PFS and OS. Influence of patient menopausal status on biomarker profiles was examined. Conclusions: Quantitative measurements of ER, PR and HER2 proteins as well as microarray and qPCR assessment of ESR1, PGR and ERBB2 gene expression were correlated with prediction of clinical outcomes of breast carcinomas exhibiting Triple Positive Breast Cancers (TPBCa). Results reinforce importance of assessing levels of the three biomarkers in a quantified fashion to enhance their use in breast cancer management and prediction of risk of recurrence. Citation Format: Zohair R. Hameed, Michael W. Daniels, D. Alan Kerr, James L. Wittliff. Quantification of estrogen (ER) and progestin receptor (PR) as well as HER2/neu proteins and gene expression improves discrimination of clinical behavior by triple positive breast carcinomas [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 669. doi:10.1158/1538-7445.AM2017-669

Abstract P3-05-11: Clinical outcomes of estrogen-receptor negative breast carcinoma are associated with protein hormone-cognate receptor gene expression

Background: Since certain protein hormones appear to be associated with progression of breast cancer cells in vitro , we employed a global approach by evaluating relationships between genes for these hormones and their cognate receptors in human breast cancer biopsies as independent predictors of risk of recurrence. Our goal is to derive clinically relevant molecular signatures correlating various ER/PR subtypes. Methods: Microarray analyses of LCM-procured carcinoma cells from 247 de-identified biopsies determined expression of genes for 61 protein hormones (the ligands) and 81 cognate receptors. Total RNA was extracted, purified and amplified from cells to determine expression of 22,000 genes. Univariable and multivariable Cox regressions were determined using expression levels of each hormone/receptor gene. Kaplan-Meier plots were constructed with an adjusted p-value Results: Expression levels of 7 genes for protein hormones and 10 receptor genes were significant for OS at an adjusted p-value of Conclusion: We revealed many breast carcinomas synthesize mRNA species for various protein hormones and their cognate receptors by determining gene expression directly on pure populations of these cells procured by LCM. We report a novel ten-gene ER-/PR- signature containing four genes in common with that of a six-gene ER- only signature that predicts breast cancer recurrence. Collectively, results of mRNA expression suggest that often breast carcinomas exhibit substantial elements of endocrine autonomy for regulating progression, warranting investigation of protein products of gene candidates in isolated populations of breast carcinoma cells. Citation Format: Daniels MW, Wittliff JL, Brock GN. Clinical outcomes of estrogen-receptor negative breast carcinoma are associated with protein hormone-cognate receptor gene expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-11.

Abstract P6-03-14: Expression of genes for aromatase inhibitor targets to discriminate invasive lobular from invasive ductal carcinomas of the breast using LCM-procured cells to complement endocrine biomarkers

Background: The BIG 1-98 trial and later the ABCSG 8 study reported that patients with invasive lobular carcinoma (ILC) exhibited better response to aromatase inhibitors (AIs) compared to those with invasive ductal carcinoma (IDC). Aromatase cytochrome P450 (CYP19) synthesizes estrogen from androgens and is the target of AI9s. CYP19 substrates are generated by upstream enzymes including estrone sulfatase (SULT1E1) and 3b-hydroxysteroid dehydrogenase type 1 (HSD3B1). Enzymes of the aromatase pathway have been reported to be expressed in intact tissue biopsies of breast cancer. To learn more about the pathogenic mechanisms that may underlie the survival benefit of ILCs treated with AIs, we analyzed expression levels of key enzymes related to the aromatase pathway in ILC and IDC. Unlike previous studies, we determined gene expression levels directly on pure populations of carcinoma cells procured by laser capture microdissection, eliminating the contribution of non-cancerous cells. Methods: Using an IRB-approved biorepository and database, total RNA was extracted from carcinoma cells of 247 de-identified biopsies to perform microarray analyses of 22,000 genes. Of the 247 samples, 16 were ILC, 13 were low grade IDC, 55 were intermediate grade IDC and 85 were high grade IDC, and 107 of these were hormone receptor positive. CYP19, HSD3B1 and SULT1E1 expression was directly detected in LCM-procured breast carcinoma cells of ILC and of IDC. Expression of other genes generally associated with the aromatase pathway, e.g., NADPH-cytochrome P450 reductase (POR), ATP-binding cassette gene (ABCG2), catechol-o-methyltransferase (COMT) and uridine-59-diphosphate glucuronosyltransferase (UGT1A3 & UGT1A9) as well as HSD17B2 were assessed with LCM-procured cells. Estrogen receptor (ER) and progesterone receptor (PR) proteins were quantified by radio-ligand binding and EIA, and gene expression was validated by qPCR. Results: Univariate Cox regression analyses indicated that ABCG2, HSD17B2and UGTA3 independently predicted disease free and/or overall survival of breast carcinomas. We found that CYP19 expression in carcinoma cells, as well as SULT1E1, COMT, POR, HSD17B2 and UTG1A3 expression, decreased as either ER or PR protein increased. HSD3B1 appeared to be over-expressed in ILC compared to IDC, however this difference did not approach statistical significance, likely due to the small sample size. No differences were seen in expression levels of CYP19 and SULT1E1 between ILC and IDC. Conclusions: An inverse relationship between CYP19 and ER and PR expression levels was observed and suggests that synthesis of estrogens by breast cancer cells in situ plays a significant role in defining tumor biology. Our results also indicate overexpression of HSD3B1 in ILC, although not statistically significant. This finding suggests that HSD3B1 may be a key contributor to the increased benefit of AI therapy seen in ILC. Collectively our results suggest a comprehensive study is warranted to ascertain the molecular basis for differences in expression of genes directing estrogen synthesis in situ in relationship to AI therapeutic responses of histologic subtypes of breast carcinomas. Citation Format: Sanders MAG, Daniels MW, Wittliff JL. Expression of genes for aromatase inhibitor targets to discriminate invasive lobular from invasive ductal carcinomas of the breast using LCM-procured cells to complement endocrine biomarkers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-14.

Abstract P3-04-06: Expression of genes for peptide/protein hormones and their receptors in breast carcinomas as biomarkers predicting risk of recurrence

Background: Several reports have suggested expression of certain peptide/protein hormones in breast cancer cells appear to be related to clinical behavior. We have taken a global approach by evaluating relationships between genes for these hormones and their cognate receptor proteins as independent predictors of risk of recurrence. Methods: Expression of genes for 55 peptide/protein hormones (the ligands) and 73 of their cognate receptor proteins were measured by microarray analyses of LCM-procured carcinoma cells from 247 breast carcinoma biopsies. Using an IRB-approved biorepository and comprehensive database, total RNA was extracted from carcinoma cells to determine expression levels of 22,000 genes. Univariate and multivariate Cox regressions with interaction were determined using expression values of each ligand and its cognate receptor with an interaction term. Results: When pairs of hormone ligand and cognate receptor genes were evaluated by multivariate Cox regression with interaction, the following sets of genes were identified that predicted risk of breast cancer recurrence (INS/IGF2R, HAMP/SLC40A1, POMC/MC4R, GH1/GHR and VIP/VIPR2) and OS (CORT/SSTR5, VIP/VIPR2 and GHRH/GHRHR, based on unadjusted p-value for interaction term Conclusion: As a result of determining gene expression directly on pure populations of breast carcinoma cells procured by LCM, we have demonstrated the many lesions synthesize mRNA species for a wide variety of peptide and protein hormones as well as for their cognate receptor proteins. Using clinical follow-up that extended as much as 12 years, univariate and multivariate Cox regression analyses with and without interaction models revealed a number of noteworthy candidates of hormone-receptor complexes that predicted risk of breast cancer recurrence as well as overall survival. Collectively, our results suggests that many breast carcinomas exhibit considerable endocrine autonomy for controlling progression, which warrants investigation of the protein products of the gene candidates in isolated populations of breast carcinoma cells. Citation Format: Wittliff JL, Daniels MW, Brock GN. Expression of genes for peptide/protein hormones and their receptors in breast carcinomas as biomarkers predicting risk of recurrence. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-06.