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204P EVALUATION OF PTEN AND PIK3CA STATUS IN BREAST
CANCER FOR PATIENT SELECTION
V. Serra 1 , J. Rodon 2 , C.M. Aura 3 , A. Vivancos 4 , K. Stemke-Hale 5 ,
H. Hibshoosh 6 , Y. Wang 7 , S. Ramon Y Cajal 3 , J. Tabernero 8 , J. Baselga 9
1 Experimental Therapeutics Laboratory, Vall d’Hebron Institute of Oncology,
Barcelona, SPAIN, 2 Phase I Unit, Medical Oncology Service, VHIO, Barcelona,
SPAIN, 3 Molecular Pathology Laboratory, VHIO, Barcelona, SPAIN, 4 Cancer
Genomics Group, VHIO, Barcelona, SPAIN, 5 Systems Biology, MD Anderson
Cancer Center, Houston, TX, UNITED STATES OF AMERICA, 6 Department of
Pathology and Cell Biology, Columbia University, New York, NY, UNITED STATES
OF AMERICA, 7 Assay Application & Product Development, Mip, Affymetrix,
Santa Clara, CA, UNITED STATES OF AMERICA, 8 Medical Oncology /
Gastrointestinal Tumors Group, Vall d’Hebron University Hospital / VHIO,
Barcelona, SPAIN, 9 Hematology/Oncology, MGH Cancer Center, Massachusetts
General Hospital, Boston, MA, UNITED STATES OF AMERICA
PTEN and PIK3CA status are potential predictors of response to PI3K-pathway
inhibitors. Therefore, we searched the most reliable platform to assess both PTEN
expression and PIK3CA mutations in primary and metastatic breast cancer (BC).
We cross-validated the assays between different institutions. Studies were
performed using four different sample sets of formalin-fixed paraffin-embedded
(FFPE) breast cancers from VHUH/VHIO and from Columbia University. PTEN
alterations were genotyped in 14 TNBCs by Oncoscan TM platform (Affymetrix)
and were correlated to immunohistochemistry (IHC). PTEN loss of heterozygosity
(LOH, n = 4) with or without overlapping PTEN mutation (n = 5) was concordant
to PTEN protein loss (H-Score < 60) by IHC in 7 samples (7/8, 88%
concordance). PTEN protein loss by IHC was also cross-validated between two
institutions. In a cohort of 12 TNBCs containing eight PTEN low samples, only
two samples were discordant (2/12, 17% discordance). Paired primary versus
metastatic tumors were identified to transit either way, in the PTEN assessment
by IHC (2/8 paired samples, 25% transition). PIK3CA mutations by Oncoscan TM
were concordant to MassARRAY (Sequenom) in 4 out of 5 mutated samples
within the panel of 17 BCs. The discrepancy (1/17, 6%) was likely due to
differences in the sensitivity of the two assays. In another cohort of 21 BC
samples, PIK3CA mutational status was cross-validated by MassARRAY at two
institutions (MDACC and VHIO). Using identical, customized panels we found
that only two samples were discordant because of mutant allele frequency close to
the sensitivity of the assay (10%). Among 14 paired primary vs metastatic breast
cancers we detected two transitions from wild type (WT) to H1047R mutation
and one transition from E545K to WT (3/14, 21% transition), underscoring the
need to determine PIK3CA status in metastatic lesions. The divergence evaluating
PTEN and PIK3CA status between institutions was due to different scoring
methodologies and to sensitivity of the assays respectively. For patient
pre-screening purposes, MassARRAY and IHC can be performed at each
institution both in primary and metastatic breast cancer lesions. Oncoscan TM is a
valid, centralized platform for evaluation of PTEN and PIK3CA genomic
alterations in BC.
Disclosure: Y. Wang: Yuker Wang is employee of Affymetrix Inc. All other authors
have declared no conflicts of interest.
205P SIGNIFICANCE OF C-MET AS A THERAPEUTIC TARGET
IN TRIPLE-NEGATIVE BREAST CANCER
S. Kashiwagi, M. Yashiro, N. Aomatsu, H. Kawajiri, T. Takashima, N. Onoda,
T. Ishikawa, K. Hirakawa
Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka,
JAPAN
Background: The molecular and biological mechanisms of cancer proliferation and
metastasis are being elucidated. Therapies targeting the biological characteristics of
various cancers have been adopted. Hormone therapy, molecular target therapy, etc.,
are chosen depending on against estrogen receptor (ER), progesterone receptor (PR)
and human epidermal growth factor receptor 2 (HER2) expressions in breast cancer.
However, no effective therapy is available for ER-, PR-, and HER2-negative
triple-negative breast cancer (TNBC) because their targets are unknown. c-met
tyrosine kinase receptor for Hepatocyte growth factor (HGF) has attracted attention
as a novel molecular target of cancer therapy. The c-met receptor for HGF is
involved in the migration, invasion, and proliferation of cancer cells. The activation
mechanisms of c-met include the overexpression, amplification, and mutation of the
met gene, as well as ligand binding. Increased c-met protein expression, including
coexpression with its ligand HGF, is observed in various cancers. Reportedly, the
prognosis of breast cancer is correlated with HGF/c-met coexpression and c-met
overexpression. c-met signaling plays an important role in the proliferation of breast
cancer cells. However, few reports have been published regarding their correlation
with TNBC.
Material and methods: A total of 1,036 patients who had undergone resection of a
primary breast cancer at our institute were enrolled. ER / PR / HER2 status and
c-met expression were assessed by immunohistochemistry. In vitro study, TNBC cell
lines, MDA-MB 231 and OCUB-2, and non-TNBC cell lines, MCF-7 and OCUB-1,
were used. c-met mRNA expression was examined by RT-PCR. Then, the effects of
Annals of Oncology
HGF, c-met siRNA, and c-met inhibitors on the proliferation of breast cancer cell
lines were examined.
Results: The 1,036 patients included 190 TNBC patients, whose prognoses were
poorer than those of non-TNBC patients. In the TNBC patients, the c-met
expression-positive group showed a poorer prognosis than the control group. c-met
was expressed in the TNBC cell lines, whose proliferation was enhanced by HGF.
c-met kinase inhibitors and c-met siRNA inhibited the proliferation of TNBC cell
lines.
Conclusion: c-met expression is a potential molecular target and useful in classifying
TNBC.
Disclosure: All authors have declared no conflicts of interest.
206P PREDICTION OF DISEASE OUTCOME WITH QUANTITATIVE
MEASUREMENT OF HER-2 RECEPTOR EXPRESSION AND
DIMERIZATION IN PATIENTS WITH BREAST CANCER
H. Bazin 1 , F. Andre 2 , M. Mathieu 3 , A. Ho-Pun-Cheung 4 , E. Lopez-Crapez 4 ,
G. Mathis 1 , P. Garnero 1
1 Research, Cisbio Bioassays, Codolet, FRANCE, 2 Department of Medical
Oncology, Institut Gustave Roussy, Villejuif, FRANCE, 3 Department of Pathology,
Institut Gustave Roussy, Villejuif, FRANCE, 4 Translational Research Unit, CRLC
Val d’Aurelle Paul Lamarque, Montpellier, FRANCE
Introduction: Expression of HER2 is commonly assessed by immunohistochemistry
(IHC) and IHC-HER2 positive patients with breast cancer are candidate for
anti-HER2 therapy. However IHC is not quantitative, does not allow to detect subtle
changes in HER2 expression and cannot assess HER2 dimerization which is critical
for its activation. The aim of this study was to quantify the expression and
dimerization of HER2 in patients with breast cancer and to relate these
measurements to disease outcome.
Methods: Using a novel microtiter plate based Time Resolved Fluorescence
(TR-FRET) assay we quantify HER2 receptor expression and dimerization on frozen
tumor samples from 100 patients with breast cancer. Normalized fluorescence signals
allowed a quantitative measure of the overall receptors/dimers expression. Disease
free (DFS) and overall survival (OS) was assessed in each subject.
Results: Among the 100 patients, 82 were IHC-HER2 negative, including 60 subjects
who were ER+ and treated with hormonal therapy. Using Cox proportional hazard
analyses we showed that in IHC-HER2 negative, ER+ subjects, the presence of HER2
dimer was significantly associated with both reduced DFS (p = 0.0001) and OS
(p = 0.00237). Quantitative measure of HER2 expression was also associated with
DFS (p = 0.0005) and OS (p = 0.03).
Conclusion: Quantitative measurement of expression and dimerization of HER2 by
the novel TR-FRET assay predicts disease outcome in IHC-HER2 negative, ER+
breast cancer patients. These new biomarkers may be useful to identify failure
patients to hormonal treatment who may benefit from adjuvant therapy with
anti-HER therapy. Validation series is ongoing in 200 FFPE-samples and will be
presented.
Disclosure: F. Andre: research funding. M. Mathieu: research funding. All other
authors have declared no conflicts of interest.
207P KRAS MUTATIONAL STATUS AND OXALIPLATIN SENSITIVITY:
THE OTHER SIDE OF THE MOON?
A. Orlandi, M. Di Salvatore, M. Basso, C. Bagalà, A. Strippoli, A. Calegari,
A. Astone, C. Pozzo, A. Cassano, C. Barone
Unit of Medical Oncology, Catholic University of Sacred Heart, Rome, ITALY
Background: Oxaliplatin is a milestone of colorectal cancer therapy, but it is still
lacking of a validated predictive biomarker of response. In a recent retrospective
study we found a greater efficacy of oxaliplatin in KRAS mutated patients with
metastatic colorectal cancer. Aim of the present study is to investigate “in vitro” the
molecular basis of this finding and the possible role of ERCC1, the main mechanism
of oxaliplatin resistance.
Methods: We selected four colorectal cancer cell lines, two KRAS wild type (wt)
(HCT-8, HT-29) and two KRAS mutated (mt)(SW620, SW480). The sensitivity of
these cell lines to oxaliplatin was evaluated by MTT-test. ERCC1 levels before and
after exposure to oxaliplatin were determined by RT-PCR. KRAS was silenced in a
KRAS mt cell line (SW620) in order to evaluate the effect on oxaliplatin sensitivity
and on ERCC1 levels. ERCC1 was also silenced in all cell lines to confirm his role in
the KRAS-mediated oxaliplatin resistance pathway.
Results: KRAS mt cell lines were more sensitive to oxaliplatin (OR 2,68; IC 95%
1.511-4.757 p < 0.001). KRAS mt and wt cell lines did not show significant
differences in ERCC1 basal levels, however, after 24h exposure to oxaliplatin, only
resistant KRAS wt cell lines showed a statistically significant upregulation of ERCC1
compared to KRAS mt cell lines (OR 42.9; IC 95% 17.260-106.972 p < 0.0005).
Silencing of KRAS demonstrated to reduce Oxaliplatin sensitivity and to restore the
ability to induce ERCC1 in KRAS mt cell lines. Finally, silencing of ERCC1 increased
Oxaliplatin citotoxicity only in those cells able to upregulate ERCC1 after exposure to
ix84 | Abstracts Volume 23 | Supplement 9 | September 2012