Annual Meeting Proceedings Part 1 - American Society of Clinical ...

10626 General Poster Session (Board #53C), Mon, 1:15 PM-5:15 PM
DNA damage responses in relation to late effects following radiotherapy for
early breast cancer. Presenting Author: Melvin Chua, University College
London Cancer Institute, London, United Kingdom
Background: In this study, we assessed if induction of apoptosis in G0 blood
lymphocytes following ex vivo irradiation correlated with clinical radiosensitivity
and DNA double-strand (DSB) repair in breast radiotherapy (RT)
patients. Using small molecule inhibitors, we examined the relationship
between DSB repair and radiation-induced apoptosis. Methods: Breast
cancer patients with minimal (controls) or marked late RT changes (cases)
were selected. DSB were quantified by �H2AX/53BP1 immunostaining 0.5
and 24 h after exposure of lymphocytes to 0.5 and 4 Gy, respectively.
Induction of apoptosis was assessed 48 h after 8 Gy using a fluorochrome
inhibitor of caspases (FLICA) assay. Apoptotic cells were defined by small
cell size and positive FLICA signal, measured using flow cytometry. Small
molecule DNA-PK (Nu7441) and ATM (Ku55933) inhibitors were used at
concentrations of 1 �M and 10 �M, respectively. Results: Despite similar
foci induction at 0.5 h, residual foci levels 24 h after 4 Gy differed
significantly between cases and controls (Foci per cell � 12.7 in cases, n �
8 vs 10.3 in controls, n � 8, p � 0.002). In contrast to residual foci levels,
comparable levels of apoptosis were observed between cases and controls
48 h after 8 Gy. Mean proportion of apoptotic cells was 37.2% in cases and
34.7% in controls (p � 0.413). Residual foci levels were not correlated
with levels of apoptosis in lymphocytes of the same patients (R � -0.059, p
� 0.785). To determine if apoptosis is modulated by DSB repair, apoptosis
measurements were repeated in lymphocytes treated with Nu7441 to
inhibit non-homologous end-joining. We observed that Nu7441-treated
cells had higher levels of residual foci and apoptosis compared to
mock-treated cells, 48 h after 1 Gy. This effect was wholly dependent on an
active ATM function as cells treated simultaneously with Nu7441 and
Ku55933 had extremely low levels of apoptosis, comparable to levels
observed in cells treated with Ku55933 alone. Conclusions: Higher levels of
residual DSB observed among radiosensitive patients suggest a role for
DSB repair in the pathogenesis of late radiation-induced effects. Induction
of apoptosis appears to be dependent on DSB end-joining following
exposure to ionising radiation.
10628 General Poster Session (Board #53E), Mon, 1:15 PM-5:15 PM
In silico identification of an epithelial core signature in human tumors.
Presenting Author: Chirayu Pankaj Goswami, Center for Computational
Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis,
IN
Background: Gene expression analysis is performed on grossly selected
specimens often without any microscopic analysis of tumor content. In
studies where histological analyses have been performed, cases having
80% or more tumor content are used for microarray analysis. The variability
in amount of epithelial and stromal cells may generate to misleading
differential expression analysis and selection for wrong targets for therapeutics.
It is also often unclear, whether the genes identified are stromal or
epithelial in origin. The goal of this study was to identify genes that define
core epithelial phenotype; these genes could provide means of normalization
of expression data. Methods: The CABIG GSK microarray (HG-
U133_plus_2) data consisting of 950 cell lines from carcinoma (n�562),
non-carcinoma (n�385) and normal tissue (n�3) was analyzed to identify
epithelial specific genes. 10 carcinomas each from 11 sites (n�110) and
an equal number of non-carcinomas were randomly selected. In silico
analyses were performed by 1) identifying genes differentially expressed
between carcinoma and non-carcinoma samples using a one way ANOVA;
2) identifying gene signature associated with carcinoma using Predictive
Analysis of Microarrays (PAM) and 3) a weighted gene coexpression
network analysis (WGCNA) was performed to identify co-expression modules.
A similar analysis was also performed on tissue samples (E-GEOD-
12360) from carcinomas and non-carcinomas. Venn-diagram was generated
to identify intersecting set. Results: Comparison of the carcinoma and
non-carcinoma samples using ANOVA identified 1455 differential expressed
gene probes in cell lines and 540 gene probes in tissues
(FDR�1E-10). The cell lines analysis identified 5 modules and a 65-gene
signature (43 core and 22 accessory set) that was specific for epithelial
cells. In the tissue analysis a 188-gene signature was similarly identified.
Cross-comparison identified a smaller 31 gene intersecting set; this was
not associated with loss of discriminatory power. Conclusions: A 31 geneset
which can be used to determine the epithelial content of heterogeneous
tumors, was identified. This study has the potential to significantly impact
the use of microarray based gene expression data.
Tumor Biology
687s
10627 General Poster Session (Board #53D), Mon, 1:15 PM-5:15 PM
Correlation between in vitro chemotherapy sensitivity and PARP-1 expression
in patients with breast cancer. Presenting Author: Cornelia Liedtke,
University of Muenster, Muenster, Germany
Background: Based on its potential use as a therapeutic target through
PARP inhibitors expression of Poly-A-Ribose-Polymerase-1 (PARP-1) has
come into scientific focus. Furthermore, it could be demonstrated that
cytoplasmic PARP-1 expression varies depending on molecular breast
cancer subtypes and that it correlates with an increased response to
neoadjuvant taxane-anthrazykline containing chemotherapy (von Minckwitz
et al., J Clin Oncol 2011). In-vitro-chemotherapy sensitivity and
resistance assays (CSRAs) are a means to directly measure chemotherapy
sensitivity in a given tumor uninfluenced by host factors. Methods: We
conducted a systematic immunohistochemical tissue-microarray (TMA)based
analysis of 550 samples of invasive breast cancers to evaluate the
expression of a set of molecular markers including estrogen receptor (ER),
progesterone receptor (PR) and HER2 as well as PARP-1. Triple negative
breast cancers (TNBC) were identified through lack of (over-)expression of
ER, PR and HER2. All carcinomas were analyzed in an in vitro CSRA
protocol for epirubicin/docetaxel (ED) and epirubicin/cyclophosphamide
(EC). In-vitro-chemotherapy sensitivity was analyzed using an adenosine
triphosphate (ATP) bioluminescence assay. Results: Moderate/high expression
of PARP-1 was found in 48 and 33% of cases with TNBC and
non-TNBC, respectively (p�0.015). No correlation between TNBC phenotype
and cytoplasmic expression was observed. However, increased expression
of both cytoplasmic and nuclear PARP-1 was correlated with an
increased in-vitro sensitivity against ED (p�0.012 and 0.025, respectively)
but not EC (p�0.27 and 0.62, respectively). Conclusions: Our
results support previous observations demonstrating a significant correlation
between expression of PARP-1 and an increased sensitivity against
taxane-anthracycline chemotherapy independent of tumor phenotype.
10629 General Poster Session (Board #53F), Mon, 1:15 PM-5:15 PM
Intestinal HIF-2 to protect against radiation-induced gastrointestinal syndrome.
Presenting Author: Cullen M. Taniguchi, Stanford University
Medical Center, Stanford, CA
Background: Gastrointestinal (GI) toxicity accounts for significant morbidity
during systemic chemotherapy or abdominopelvic radiation therapy. Unfortunately,
there are no effective preventative treatments. Hypoxic signaling
through hypoxia-inducible factor-1 (HIF-1) and -2 (HIF-2) is critical for
intestinal homeostasis during inflammatory challenges and was posited to
play a role in radioprotection. Methods: We stabilized both HIF-1 and HIF-2
pharmacologically with the prolyl hydroxylase inhibitor DMOG and with
transgenic mice. Male C57/Bl6 mice eight weeks of age were pretreated
with saline or 8mg DMOG 16hrs and 4hrs prior to being irradiated with a
single fraction of 20Gy to the abdomen using a modified TLI jig that shields
the upper body of the mouse to reduce hematopoietic toxicity. After XRT,
the mice got daily doses of saline or DMOG for 5 days. Results: Mice treated
with DMOG had a 1.5-fold enrichment of crypt regeneration in the colon as
determined by microcolony assay (13.3�3.4 vs 21.2�1.7 crypts/section,
saline vs DMOG, n�10/group, p�0.005). Death from GI radiotoxicity can
result from compromised epithelial integrity. We tested epithelial and
barrier function by gavaging irradiated mice with FITC-dextran, which
undetectable in the blood unless the GI epithelia is compromised.
Treatment with DMOG decreased FITC-dextran uptake by 4-fold over
controls (52.6�14.7 vs 12.4�3.4 mcg/ml, n�6/group, p�0.02). These
physiologic improvements translated to improved overall survival with
DMOG treatment after being exposed to 20Gy of abdominal radiation
(p�0.03). To determine if the radioprotective effect of DMOG was specific
to HIF-1 or HIF-2, we overexpressed stabilized HIF1 or HIF2 in the
intestinal epithelia using a lox-stop-lox (LSL) system and Villin-Cre. The
overexpression of HIF1 had no effect on survival (p�0.22), while mice with
intestinal-specific HIF2-overexpression had significantly improved survival
after receiving 20Gy of abdominal radiation (p�0.001). Conclusions:
Activation of HIF-2 is sufficient to protect mice from lethal doses of
abdominal radiation may provide a novel class of treatments to protect the
GI epithelium from the deleterious side effects of chemotherapy and
radiation.
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