Abstract P4-01-15: Assay Development for detection of estrogen responsive gene histone acetylation in breast cancer circulating tumor cells
Authors: Litherland S A , Reynolds Robert , Barr Louis , Almodovar Alvin JO , Decker David A 
Published: May 2015

Research Objectives: To develop a tumor-specific assay for assessment of histone acetylation and histone deacetylase (HDAC) effects in circulating tumor cells (CTC).

Rationale: Therapy-resistant tumor cells arise in breast cancer patients during hormone therapy. Anti-deacetylation drugs such as Entinostat, Vorinstat, and Romidepsin block histone deacetylases (HDAC), preventing removal of epigenetic acetyl chromatin modifications which allow for expression of estrogen receptor (ER). Clinical trials such as ECOG E2112 are investigating a histone acetylase inhibitor (HDACi) therapy in combination with standard endocrine therapy to overcome hormone resistance. A drawback for HDACi clinical trials in the past has been the lack of a sensitive assay to identify tumor specific HDAC effects contributing to hormone resistance.

Methods: We testing a histone acetylation assay using Fluorescence Activated Cell Sorting (FACS) peripheral blood CTC isolation and histone acetylation/HDAC specific chromatin immunoprecipitation (ChIP) with realtime PCR analysis to identify ERalpha and GREB-1 gene activation. We report the initial findings from 11 consented subjects (IRB approved protocol# 372522).

Results: Our findings indicate that GREB-1, a gene essential to breast cell ER expression in response to estrogen, can be regulated via a histone acetylation epigenetic control mechanism. Using our CTC-ChIP analysis, we found both ER+ and ER- CTC arising from ER+ tumors and that the proportion of ER- CTC mirrored clinical hormone resistance. GREB-1 activation and expression is detectable in both ER+/ER- CTC when enough CTC could be collected for ChIP analysis; however, ERalpha acetylation and mRNA expression was below the level of detection.

Table of Preliminary ResultsPatient Group/NTumor statusHT StatusCTC ER+/million (mean±SD)CTC ER-/million(mean ± SD)CTC ER+GREB-1 Acetylation+(mean ± SD)CTC ER- GREB-1 Acetylation(mean ± SD)PREDICTED ESTROGEN RESPONSEER+ HST sensitive/4MetastaticSensitive130379±260109336±5940.95*±0.080.015*±0.02Sensitive ER+&gt;ER-ER+ HST resistant/2MetastaticResistant824±29.77334±64210.87*±0.131*Resistant ER+&lt;ER-ER- metastatic/1MetastaticResistant18758214981*NDResistant ER+&lt;ER-ER- primary/2PrimaryUnknown277251±379512112754±1594513400000089500000000±126600000000Sensitive ER+&gt;ER- active GREB-1Healthy controls/2NoneNone0.5±0.707100*0*-*Insufficient cells for accurate ChIP analysis

Conclusions: Our preliminary data suggest that by using a combination of CTC based epigenetic biomarker analyses; it is possible to detect the presence of hormone resistant ER+ and ER- CTC subpopulations arising from ER+ breast cancer tumors. Monitoring of longitudinal changes in ER+/ER- CTC and the epigenetic activation of GREB-1 and ERalpha responsiveness could provide support in deciding which patients may benefit from the use of anti-deacetylation drug therapy in combination with hormonal therapy to promote ER alpha expression, reinstating estrogen dependency in both ER- and ER+ subpopulations; and thereby, re-sensitizing them to hormonal therapy.

Citation Format: S A Litherland, Robert Reynolds, Louis Barr, Alvin JO Almodovar, David A Decker. Assay Development for detection of estrogen responsive gene histone acetylation in breast cancer circulating tumor cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-15.