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

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664s Tumor Biology<br />

10532 General Poster Session (Board #41E), Mon, 1:15 PM-5:15 PM<br />

VEGF polymorphism may be associated with overall survival in lapatinibtreated<br />

breast cancer patients with brain metastases. Presenting Author:<br />

Colin F. Spraggs, GlaxoSmithKline, Stevenage, United Kingdom<br />

Background: Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor (TKI)<br />

approved in combination with capecitabine or letrozole for patients with<br />

HER2� metastatic breast cancer (MBC). Consistent with TKI therapies,<br />

patient response is variable and suggestive <strong>of</strong> additional determinants <strong>of</strong><br />

sensitivity and resistance. This exploratory pharmacogenetic study sought<br />

to identify germline genetic variants that associate with lapatinib treatment<br />

outcomes in HER2� MBC patients. Methods: Fifty five functional single<br />

nucleotide polymorphisms (SNPs) in 24 candidate genes were evaluated in<br />

a subset <strong>of</strong> MBC patients participating in two clinical trials: EGF105084:<br />

lapatinib monotherapy in HER2� MBC patients with recurrent brain<br />

metastases following trastuzumab and cranial radiotherapy (n�120) and<br />

EGF104900: lapatinib plus trastuzumab (n�92) and lapatinib monotherapy<br />

(n�103) in HER2� MBC patients with disease progression<br />

following trastuzumab. Testing for associations <strong>of</strong> SNPs with progression<br />

free survival (PFS) and overall survival (OS) during lapatinib treatment was<br />

performed using Cox proportional hazards methods, with covariate adjustment.<br />

Markers were considered to be significantly associated if they<br />

achieved a predefined multiple testing threshold <strong>of</strong> p�0.0003. Results: No<br />

SNPs were significantly associated with PFS in either study. A SNP in<br />

VEGFA (rs3025039, 936C�T) was significantly associated with improved<br />

OS (p�0.0002) for the T allele carriers, with an allelic hazard ratio <strong>of</strong> 0.21<br />

(0.08-0.52) in EGF105084. The association was not seen in EGF104900.<br />

This SNP is located in the 3’ UTR gene region, the T allele is associated<br />

with lower serum VEGFA levels and reduced breast cancer risk [Krippl et al,<br />

2003, Int J Cancer 106: 468; Kataoka et al, 2006, Cancer Epidemiol<br />

Biomarkers Prev, 15:1148]. Conclusions: A germline variant in VEGFA may<br />

be associated with survival outcome in MBC patients with brain metastases<br />

who are treated with lapatinib. This may represent activation <strong>of</strong> VEGF<br />

angiogenic pathways to overcome HER2 inhibition in patients carrying the<br />

higher expression genotype. These associations are considered exploratory<br />

and require confirmation in an independent dataset.<br />

10534 General Poster Session (Board #41G), Mon, 1:15 PM-5:15 PM<br />

Molecular characterization <strong>of</strong> circulating tumor cells in human metastatic<br />

colorectal cancer. Presenting Author: Jorge Barbazan, Translational Oncology<br />

Laboratory, Medical Oncology Department, Santiago de Compostela,<br />

Spain<br />

Background: Metastatic colorectal cancer (mCRC) relies on the detachment<br />

<strong>of</strong> aggressive malignant cells from the primary tumor into the bloodstream,<br />

being this Circulating Tumor Cells (CTC) the principal source <strong>of</strong> the further<br />

metastasis. <strong>Clinical</strong>ly, the presence <strong>of</strong> CTC is associated with poor<br />

prognosis and there exists a clear necessity for more specific and efficient<br />

chemotherapies in the treatment <strong>of</strong> mCRC. Our aim was to overcome this<br />

adverse scenario through the massive molecular pr<strong>of</strong>iling <strong>of</strong> the CTC<br />

population isolated from mCRC patients. Methods: CTC’s were magnetically<br />

isolated by using anti-EpCAM coupled magnetic beads from 6 mCRC<br />

patients and 3 healthy controls. The presence <strong>of</strong> CTCs was evaluated by<br />

citokeratins 8, 18 and 19 staining. RNA from CTCs was amplified by a<br />

whole transcriptome amplification system (WTA) and resulting material was<br />

hybridized onto gene expression arrays. Bioinformatics were used to array<br />

analysis. Selected genes were validated by RT-qPCR. Results: 410 transcripts<br />

were found to specifically characterise the CTC population after<br />

array signals comparison between mCRC patients and controls. Gene-gene<br />

interaction analysis generated networks related with cell migration, adhesion<br />

or apoptosis resistance. Gene ontology revealed similar functions.<br />

Signalling pathways such as RhoA, PKA, ILK, integrins or actin cytoskeleton<br />

signalling were found as relevant in CTCs. Eleven genes (TGFB1, APP,<br />

TIMP1, CD9, CLU, ITGB5, LIMS1, RSU1, VCL, BMP6 and TLN1) were<br />

validated by real-time PCR in 20 mCRC patient and 10 control samples. All<br />

genes showed a significantly higher expression in patients (p�0.0001).<br />

Except from TLN1 and CD9, all genes had a prognostic value in terms <strong>of</strong><br />

progression free survival (p�0.05). Conclusions: We described the molecular<br />

pr<strong>of</strong>iling <strong>of</strong> CTCs in stage IV CRC patients, characterized by a migratory<br />

and invasive phenotype. Specific and sensitive diagnostic/prognostic markers<br />

were obtained. Our strategy represents an innovative and promising<br />

approach in the clinical management <strong>of</strong> mCRC patients.<br />

10533 General Poster Session (Board #41F), Mon, 1:15 PM-5:15 PM<br />

Isolation <strong>of</strong> circulating tumor cells using a novel EMT-based capture<br />

method. Presenting Author: Rhonda Lynn Bitting, Duke Cancer Institute,<br />

Durham, NC<br />

Background: Circulating tumor cells (CTCs) have potential prognostic,<br />

predictive and surrogate implications in oncology. In patients with metastatic<br />

castration-resistant prostate cancer (CRPC) and breast cancer (BC),<br />

we have shown that CTCs isolated using epithelial cell adhesion molecule<br />

(EpCAM) ferromagnetic capture express mesenchymal markers, including<br />

N- and O-cadherin, suggesting phenotypic plasticity and the presence <strong>of</strong><br />

epithelial-mesenchymal transitions (EMT). Therefore, we postulate that<br />

during metastasis, tumor cells exist as a spectrum <strong>of</strong> epithelial to<br />

mesenchymal phenotypes and may not be captured with existing EpCAMbased<br />

CTC technology. The goal <strong>of</strong> this study is to identify CTCs in CRPC<br />

and BC patients using a novel mesenchymal-based capture method.<br />

Methods: In patients with advanced CRPC and BC, two EDTA and one<br />

CellSave tube <strong>of</strong> blood are collected. Using the CellSearch system (Veridex,<br />

USA), CTCs are captured with either anti-N-cadherin (N-cad) or anti-Ocadherin<br />

(O-cad) ferr<strong>of</strong>luid and detected cells (events) are defined as<br />

beta-catenin and DAPI positive, CD45 negative intact cells. We evaluated<br />

the performance <strong>of</strong> these EMT-based ferr<strong>of</strong>luids in healthy volunteers,<br />

control cells, and in a pilot study <strong>of</strong> patients with CRPC, and compared<br />

enumeration <strong>of</strong> cells using novel vs. standard EpCAM-based capture<br />

methods. Results: In healthy volunteers, rare events were detected using<br />

the novel capture methods. In CRPC patients, O-cad capture detected more<br />

events in 3 <strong>of</strong> 5 subjects than EpCAM-based capture, and the majority <strong>of</strong><br />

captured cells were cytokeratin negative. See table below. Conclusions:<br />

These preliminary results suggest that a novel CTC phenotype exists in<br />

CRPC based on EMT properties, particularly overexpression <strong>of</strong> O-cadherin.<br />

Further characterization <strong>of</strong> these cells from patients with advanced PC, BC,<br />

and other solid tumors will provide insight into EMT and metastasis biology.<br />

O-cad capture, beta-catenin� N-cad capture, beta-catenin� EpCAM capture, cytokeratin�<br />

Healthy volunteers 0-51 events (mean 5.95) n�21 0-4 events (mean 0.28) n�25 NA<br />

CRPC patients 0-465 events (mean 138.4) n�5 0-2 events (mean 1.2) n�5 1-123 CTCs (mean 50.8) n�5<br />

10535 General Poster Session (Board #41H), Mon, 1:15 PM-5:15 PM<br />

Pr<strong>of</strong>iling <strong>of</strong> circulating tumor cells isolated from 105 metastatic gastric<br />

cancer patients revealed HER2 overexpression/activation for potential use<br />

in clinical setting. Presenting Author: Saswati Hazra, Prometheus Laboratories<br />

Inc., San Diego, CA<br />

Background: Gastric cancer (GCA) is the second leading cause <strong>of</strong> cancer<br />

mortality in the world. Survival <strong>of</strong> patients with advanced GCA treated with<br />

chemotherapy remains low. New targeted therapies are urgently needed.<br />

There is mounting evidence <strong>of</strong> the role <strong>of</strong> HER2 overexpression in patients<br />

with GCA, and it has been highly correlated to poor outcomes with more<br />

aggressive disease. The ability to accurately determine HER2 status by<br />

testing circulating tumor cells (CTCs) may improve patient treatment by<br />

allowing ongoing assessment <strong>of</strong> HER2 status during treatment and/or<br />

identifying additional patients who could potentially benefit from HER2targeted<br />

therapy. Methods: The Collaborative Enzyme Enhanced Reactiveimmunoassay<br />

(CEER) was utilized to determine the expression and activation<br />

(phosphorylation) levels <strong>of</strong> HER2 in CTCs isolated from blood specimens<br />

obtained from 105 metastatic GCA patients. Results: Utilizing the CEER<br />

platform, the levels <strong>of</strong> HER2 expression and phosphorylation were determined<br />

for CTCs isolated from metastatic GCA patients. Evaluable CTCs<br />

were found in 33% (35/105) <strong>of</strong> enrolled patients. Out <strong>of</strong> 35 patients, 7<br />

patients (20%) have high HER2 over expression, 6 patients (17%) have<br />

moderate HER2 expression and 11 patients (31%) have HER2 activation<br />

(phospho positive) with no HER2 over-expression. Conclusions: When CTCs<br />

were present, the CEER assay identified varying levels <strong>of</strong> HER2 involvements<br />

in 68% <strong>of</strong> metastatic GCA patients. HER2 positive CTCs could serve<br />

as a prognostic and/or predictive marker in patients with advanced GCA and<br />

CTC-HER2 pr<strong>of</strong>ile shifts can be utilized to monitor the treatment efficacy.<br />

Visit abstract.asco.org and search by abstract for the full list <strong>of</strong> abstract authors and their disclosure information.

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