Martha E. Brown

Systemic Challenge with Endotoxin Stimulates Corticotropin-Releasing Hormone and Arginine Vasopressin Secretion into Hypophyseal Portal Blood: Coincidence with Gonadotropin-Releasing Hormone Suppression

We tested the hypothesis that systemic immune/inflammatory challenge (endotoxin) activates the neuroendocrine stress axis centrally by stimulating the secretion of CRH and arginine vasopressin (AVP) into hypophyseal portal blood. In addition, we examined the temporal association between this stimulation of the stress neuropeptides and the inhibition of pulsatile GnRH and LH secretion. Using alert, normally behaving ewes, hypophyseal portal and peripheral blood were sampled simultaneously at 10-min intervals for 14 h. Temperature was monitored remotely by telemetry at the same interval. Endotoxin (400 ng/kg, i.v. bolus) or saline as a control was injected after a 4-h baseline period. Portal blood was assayed for CRH, AVP, and GnRH, and peripheral blood was assayed for cortisol, progesterone, and LH. In controls, hypophyseal portal CRH and AVP remained just above or at assay sensitivity, and cortisol showed a regular rhythmic pattern unaffected by saline and typical of basal secretion. In contrast, endotoxin potently stimulated CRH and AVP secretion into portal blood, and cortisol and progesterone into peripheral blood. Both CRH and AVP generally rose and fell simultaneously, although the peak of the AVP response was approximately 10-fold greater than that of CRH. The AVP in portal blood was not due to recirculation of hormone secreted into the peripheral circulation by the posterior pituitary gland, because the AVP increase in peripheral blood was negligible relative to the marked increase in portal blood. The stimulation of CRH and AVP coincided with significant suppression of GnRH and LH pulsatile secretion in these same ewes and with the generation of fever. We conclude that endotoxin induces central activation of the neuroendocrine stress axis, stimulating both CRH and AVP release into the hypophyseal portal blood of conscious, normally behaving ewes. This response is temporally coupled to inhibition of pulsatile GnRH and LH release as well as with stimulation of adrenal cortisol and progesterone secretion and generation of fever.We tested the hypothesis that systemic immune/inflammatory challenge (endotoxin) activates the neuroendocrine stress axis centrally by stimulating the secretion of CRH and arginine vasopressin (AVP) into hypophyseal portal blood. In addition, we examined the temporal association between this stimulation of the stress neuropeptides and the inhibition of pulsatile GnRH and LH secretion. Using alert, normally behaving ewes, hypophyseal portal and peripheral blood were sampled simultaneously at 10-min intervals for 14 h. Temperature was monitored remotely by telemetry at the same interval. Endotoxin (400 ng/kg, iv bolus) or saline as a control was injected after a 4-h baseline period. Portal blood was assayed for CRH, AVP, and GnRH, and peripheral blood was assayed for cortisol, progesterone, and LH. In controls, hypophyseal portal CRH and AVP remained just above or at assay sensitivity, and cortisol showed a regular rhythmic pattern unaffected by saline and typical of basal secretion. In contrast, endotoxin ...

Prostaglandins mediate the endotoxin-induced suppression of pulsatile gonadotropin-releasing hormone and luteinizing hormone secretion in the ewe.

Five experiments were conducted to test the hypothesis that PGs mediate the endotoxin-induced inhibition of pulsatile GnRH and LH secretion in the ewe. Our approach was to test whether the PG synthesis inhibitor, flurbiprofen, could reverse the inhibitory effects of endotoxin on pulsatile LH and GnRH secretion in ovariectomized ewes. Exp 1–4 were cross-over experiments in which ewes received either flurbiprofen or vehicle 2 weeks apart. Jugular blood samples were taken for LH analysis throughout a 9-h experimental period. Depending on the specific purpose of the experiment, flurbiprofen or vehicle was administered after 3.5 h, followed by endotoxin, vehicle, or ovarian steroids (estradiol plus progesterone) at 4 h. In Exp 1, flurbiprofen reversed the endotoxin-induced suppression of mean serum LH concentrations and the elevation of body temperature. In Exp 2, flurbiprofen prevented the endotoxin-induced inhibition of pulsatile LH secretion and stimulation of fever, reduced the stimulation of plasma cortis...

Development of Circulating Tumor Cell-Endocrine Therapy Index in Patients with Hormone Receptor Positive Breast Cancer

Background: Endocrine therapy (ET) fails to induce a response in one half of patients with hormone receptor (HR)–positive metastatic breast cancer (MBC), and almost all will eventually become refractory to ET. Circulating tumor cells (CTC) are associated with worse prognosis in patients with MBC, but enumeration alone is insufficient to predict the absolute odds of benefit from any therapy, including ET. We developed a multiparameter CTC-Endocrine Therapy Index (CTC-ETI), which we hypothesize may predict resistance to ET in patients with HR-positive MBC. Methods: The CTC-ETI combines enumeration and CTC expression of four markers: estrogen receptor (ER), B-cell lymphoma 2 (BCL-2), Human Epidermal Growth Factor Receptor 2 (HER2), and Ki67. The CellSearch System and reagents were used to capture CTC and measure protein expression by immunofluorescent staining on CTC. Results: The feasibility of determining CTC-ETI was initially established in vitro and then in a prospective single-institution pilot study in patients with MBC. CTC-ETI was successfully determined in 44 of 50 (88%) patients. Eighteen (41%), 9 (20%), and 17 (39%) patients had low, intermediate, and high CTC-ETI scores, respectively. Interobserver concordance of CTC-ETI determination was from 94% to 95% (Kappa statistic, 0.90–0.91). Inter- and cell-to-cell intrapatient heterogeneity of expression of each of the CTC markers was observed. CTC biomarker expression was discordant from both primary and metastatic tissues. Conclusions: CTC expression of ER, BCL-2, HER2, and Ki67 can be reproducibly measured with high analytical validity using the CellSearch System. The clinical implications of CTC-ETI, and of the heterogeneity of CTC biomarker expression, are being evaluated in an ongoing prospective trial. Clin Cancer Res; 21(11); 2487–98. ©2014 AACR. See related commentary by Mathew et al., p. 2421

Importance of Photoperiodic Signal Quality to Entrainment of the Circannual Reproductive Rhythm of the Ewe

Abstract An endogenous circannual rhythm drives the seasonal reproductive cycle of a broad spectrum of species. This rhythm is synchronized to the seasons (i.e., entrained) by photoperiod, which acts by regulating the circadian pattern of melatonin secretion from the pineal gland. Prior work has revealed that melatonin patterns secreted in spring/summer entrain the circannual rhythm of reproductive neuroendocrine activity in sheep, whereas secretions in winter do not. The goal of this study was to determine if inability of the winter-melatonin pattern to entrain the rhythm is due to the specific melatonin pattern secreted in winter or to the stage of the circannual rhythm at that time of year. Either a summer- or a winter-melatonin pattern was infused for 70 days into pinealectomized ewes, centered around the summer solstice, when an effective stimulus readily entrains the rhythm. The ewes were ovariectomized and treated with constant-release estradiol implants, and circannual cycles of reproductive neuroendocrine activity were monitored by serum LH concentrations. Only the summer-melatonin pattern entrained the circannual reproductive rhythm. The inability of the winter pattern to do so indicates that the mere presence of a circadian melatonin pattern, in itself, is insufficient for entrainment. Rather, the characteristics of the melatonin pattern, in particular a pattern that mimics the photoperiodic signals of summer, determines entrainment of the circannual rhythm of reproductive neuroendocrine activity in the ewe.

Monitoring serial changes in circulating human breast cancer cells in murine xenograft models

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy.

Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance

Fulvestrant is a dose dependent selective estrogen receptor (ER) down‐regulator (SERD) used in ER‐positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance.

A pilot study to establish a clinical model to perform phase II studies of breast cancer chemopreventive agents in women at high risk with biomarkers as surrogate endpoints for activity.

Purpose: Use of surrogate end point biomarkers in phase II trials may help select agents that appear to have activity and might be evaluated in future phase III definitive trials of breast cancer prevention. We performed a pilot clinical trial to establish the logistics for a clinical model to perform phase II studies of breast cancer chemopreventive agents in women at high risk with novel imaging techniques and candidate surrogate end point biomarkers for activity. We chose tamoxifen to establish proof of principal with a known effective agent. Experimental Design: Women at a high risk of developing a new breast cancer and for whom tamoxifen was recommended were eligible. The women underwent baseline and 3 and 6 months mammogram and magnetic resonance imaging (MRI) of one breast to identify areas of water-like intensity (epithelial) and to determine the changes over time and MRI-directed core breast biopsies of these areas for surrogate end point biomarkers analysis. Results: From August 1999 to March 2001, 26 women underwent baseline imaging and core biopsies. Sixteen women took tamoxifen and 10 chose not to. Overall, 79% of the samples contained glandular tissue evaluable for histology, but only 66% of the samples were evaluable for marker analysis. Only 12 patients had specimens with glandular tissue sufficient for marker analysis both at baseline and in at least one follow-up. Because of the small number of women with matched samples, marker analysis was not informative. Conclusions: This study shows the feasibility of obtaining serial core breast biopsies from women at a high risk of developing a new breast cancer. Patient participation in this model is satisfactory, and such a model may provide indication of drug activity. MRI-directed biopsy did not provide a high yield of evaluable samples, and additional work on adequate collection of epithelial tissue for surrogate end point biomarker analysis is thus necessary.

Abstract 4154: Multi-parameter molecular characterization of circulating tumor cells (CTC): Development of a CTC-endocrine therapy index (CTC-ETI)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction. CTC can be reproducibly and reliably enumerated using a commercially-available, automated immunomagnetic system (CellSearch®; Veridex LLC). High levels of CTC predict rapid progression in patients with metastatic breast cancer (MBC). Only ∼ 50% of patients with estrogen receptor (ER) positive MBC benefit from endocrine therapy (ET). Patients with endocrine-refractory MBC are better palliated with chemotherapy. We have developed a multi-parameter assay using CellSearch® that may identify patients with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. Methods. CellSearch® has four fluorescence channels. Three of these are used to distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4th “empty” channel was used to measure ER, Bcl-2, HER2, and Ki67 expression with antigen-specific fluorescent-labeled antibodies. These four markers were chosen because of their associations with sensitivity (ER, Bcl-2) or resistance (HER2, Ki-67) to ET. Cultured human breast cancer cells (MCF-7: ER +, BCL-2 +, HER2 -, Ki67 +; MDA-MB-231: ER -, Bcl-2 -, HER2 -, Ki67 +; BT-474: ER +, Bcl-2 +, HER2 +, Ki67 +) were spiked into 7.5 ml human whole blood from normal donors and separated and characterized using the CXC CellSearch® kit. Results. Each cell line stained appropriately for the respective markers, with appropriate negative controls, although staining was heterogeneous, even within a single cell line. Using these preliminary data, we have developed a CTC-ETI in which scores are assigned to the individual categories consisting of cell counts coupled with the relative percent and degree of cell positivity of each marker. We predict that lower scores (low or no CTC, or CTC with high % and intensity of ER and Bcl-2; low HER2 and Ki-67) will be associated with favorable response to ET. Conclusions. CellSearch® can be used to measure the expression of 4 clinically useful and proven breast cancer biomarkers. A clinical trial is underway at U. Michigan to permit calculation of CTC-ETI in patients with MBC, which will lead to a prospective trial to determine if high CTC-ETI predicts resistance and rapid progression on ET. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2011-4154

P4-07-16: Development of Circulating Tumor Cell-Endocrine Therapy Index in Metastatic Breast Cancer Patients.

Introduction: Only ∼ 50% of patients (pts) with estrogen receptor (ER) positive metastatic breast cancer (MBC) benefit from endocrine therapy (ET). Currently only clinical judgment can be used to identify pts with endocrine-refractory MBC, who are better palliated with chemotherapy. Circulating Tumor Cells (CTC) are reliably enumerated using an automated immunomagnetic system (CellSearch®; Veridex LLC). High CTC levels predict rapid progression in pts with MBC. We have developed a multi-parameter assay, the CTC-Endocrine Therapy Index (CTC-ETI) using CellSearch® that may identify pts with ER positive MBC who are unlikely to benefit from ET and may be better served with chemotherapy. CTC-ETI scores are assigned based on CTC levels coupled with the relative percent and degree of marker positivity on the CTC. We report preliminary results from a pilot single institutional study. Methods: CellSearch® has 4 fluorescence channels. Three distinguish CTC from WBC (DAPI, anti-cytokeratin, anti-CD45). The 4 th “empty” channel was used to measure ER, BCL-2, HER-2, and Ki-67 expression with fluorescent-labeled antibodies. These 4 markers reflect sensitivity (ER, BCL-2) or resistance (HER-2, Ki-67) to ET. Forty ml of blood was drawn into 4 CellSave® tubes from pts with progressive MBC. Whole blood from 4 tubes was pooled and divided into 4 different 7.5 ml aliquots of blood, which were processed and characterized for CTC counts and each of the four molecular markers using the CXC CellSearch® kit. Results: 21 pts have been accrued to the feasibility study. One patient was ineligible. Five of 20 pts had low CTC counts ( Conclusions: ER, BCL-2, HER-2, and Ki-67 can be accurately determined on CTC using the 4 th channel in the CellSearch® system to calculate CTC-ETI. We predict that lower CTC-ETI scores (low or no CTC, or CTC with high CTC ER and BCL-2 and low CTC HER-2 and Ki-67) could be associated with favorable response to ET. Successful completion of the feasibility study will lead to a prospective trial to determine if high CTC-ETI at baseline predicts resistance and rapid progression on ET in women starting a new endocrine therapy for MBC. Supported by Veridex, LLC, Fashion Footwear Charitable Foundation of New York/QVC Presents Shoes on Sale ™ (DFH), Associazione Sandro Pitigliani and by a studentship from FIRC (CP). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-16.

Abstract P2-02-19: Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients

Introduction: Somatic mutations, including those in TP53 , PIK3CA , and estrogen receptor alpha ( ESR1 ), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch