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cytomegalovirus (CMV). Analysis of the frequency of common virus infection in
different age groups showed the presence of two peaks in patients aged 18 to 35years
(HPVG-37,7%, HSV I + II-19, 1%, CMV-13, 9) and 65-78 years(HPVG-30,7%, HSV
I + II-29, 1%, CMV-17, 5). In the other age periods were detected less frequently:
36-45 years (HPVG-23.4%, HSV I + II-12, 1%, CMV-11, 9), 46-55 (HPVG-30.1%,
HSV I + II-171%, CMV-10, 9), with the lowest frequency in the age of 56-64 years
(HPVG-17,7%, HSV I + II-11, 1%, CMV-11, 9). From these, the total extent of
infection by age of patients was as follows: a high degree of infection ranged in age
from 18 to 35 years (97.2%) and over 65-78 years (86.7%), moderate - 46-55 years
(71.7%), low infection rates, 36-45 years (53.5%) and 56-64 years(54.5%). Thus, in
our study, the presence of a virus associated NHL revealed in all cases, with a
statistically significant increase in the frequency of occurrence of 18 to 44 years and
over 65 years. Our evidence suggests that virus infection may play an important role
in the pathogenesis of NHL, and the choice of treatment effectiveness and prognosis.
Disclosure: All authors have declared no conflicts of interest.
163P TESTING AND ANALYSIS OF FLUORESCENT MARKERS
PANEL FOR T-CELLS’ MULTIFUNCTIONALITY IN
GLIOBLASTOMA MULTIFORME PATIENTS
I. Ben-Horin 1 , I. Volovitz 2 , Z. Ram 3
1 Oncology Devision, Tel Aviv Sourasky Medical Center-(Ichilov), Tel Aviv, ISRAEL,
2 Cancer Immunotherapy Lab, Tel-Aviv Sourasky Medical Center, Tel-Aviv,
ISRAEL, 3 Neurosurgery, Tel-Aviv Sourasky Medical Center, Tel-Aviv, ISRAEL
Introduction: Immunotherapy had only limited success in brain tumors as the brain
is regarded as an immunologically privileged site. Recently published work on a rat
model showed that when live, non-attenuated brain tumor cells are injected
subcutaneously they are first accepted but are later spontaneously rejected. This
immunologically mediated rejection of the brain tumors cells in peripheral sites leads
to a similar intra-cranial tumor rejection. Objective: to characterize the different
immune cell populations found in human glioblastoma multiforme (GBM) and in
peripheral blood of GBM patients and to develop means to follow immune responses
in the CNS to anti-tumor therapy.
Methods: Tumors were broken down and tumor cells isolated. Cultures of tumor
cells with or without the respective patient’s activated or non-activated lymphocytes
were prepared, stained with a fluorescent markers panel for T-cells multifuncionality
and analyzed using flow cytometry. The panel was constructed in our lab with
markers for CD3 (total T-cells), CD4 (Th cells), CD8 (CTL), IFNγ, TNFα, IL-12 and
ViViD – a viability marker.
Results: Our panel succeeded in characterizing the different immune cell
populations. It also demonstrated that activation of CD4 T-cells increased IFNγ and
IL12 expression and not TNFα’s expression. CD8 T-cells demonstrated a smaller rise
in IFNγ and IL12 expression and no expression of TNFα, before or after stimulation.
Non-activated lymphocytes demonstrated no multifunctionality while activated cells
exhibited low levels of IFNγ and IL12 joint expression. The extent of
multifuncionality differed between CD4 and CD8 cells.
Conclusion: Our panel characterized the different T-cell populations in tumors. It
demonstrated that only a small percentage of cells exhibited multifuncionality. To the
best of our knowledge this is the first time T-cells multifunctionality in brain tumors
has been described. This phenomenon is of importance as it correlates with vaccines’
potency. Future work will characterize the difference between peripheral and
intratumoral T-cells and their interaction with other immune cells populations as we
aim to understand tumor induced immune system inhibition.
Disclosure: All authors have declared no conflicts of interest.
164 ISOLATION, PROLIFERATION, AND PHENOTYPING OF CANCER
STEM CELLS FROM PRIMARY BREAST CANCERS: A MODEL
FOR EVALUATING THE RESPONSE TO ANTI-TUMOR DRUGS
M.I. Salamoon 1 , M. Al Jamali 2 , L. Youcef 2 , I. Kasem 3 , M. Bachour 1
1 Medical Oncology, Al Bairouni University Hospital, Damascus, SYRIA, 2 Clinical
Pharmaceutics, Faculty of Pharmacy, Damascus, SYRIA, 3 Biotech, Technical
Institution, Damascus, SYRIA
Background: Recent years have witnessed an increasing interest in the role of cancer
stem cells (CSCs) because of selfrenewal, promoting metastasis, and resisting radioand
chemotherapies. In addition, the high levels of CSCs in tumor mass were linked
to a poor prognosis, relapse and metastasis. For the former reasons SCS is currently a
main focus of cancer research.
Objective: This study aims at establishing a CSC cellular model, which might enable
the testing of new chemotherapeutics and/or combinatorial therapies for currently
used agents.
Material and methods: We culture tumor cells obtained from primary breast tumors
of Syrian patients. This was followed by isolating stem cell population existing in the
primary tumor by means of serial dilution of cells, and the characterization of these
cells by flow cytometry based on their morphology, surface antigen profile (CD44
+ /high/CD24-/low), and their growth and enrichment in both adherent and
Annals of Oncology
non-adherent conditions. Finally, the viability of CSCs was tested after freezing cells
in liquid nitrogen.
Results and discussion: In this study, we were able to isolate CSCs from primary
breast tumor with high purity exceeding 90%, and we showed their CSC
characteristics, which included: i) CD44high/CD24-/low profile, ii) their ability to
form tumorspheres in non-adherent/suspension conditions, iii) and their ability of
resisting chemotherapeutic agents in comparison with non-purified/non-stem breast
tumor cells.
Conclusions: Our results confirm the establishment of a cellular model representing
the characteristics of cancer stem cell population. This breast CSC model could be
useful in conducting research enabling the identification of responsible mechanisms
behind breast cancer resistance to chemotherapeutics. In addition, this model might
enable the examining of new combinatorial therapeutics by evaluating the effects of
drug combinations targeting the different and heterogeneous cell populations inside
breast tumors, both tumor non-stem as well as tumor stem cells, thus, achieving the
desirable therapy outcomes.
Disclosure: All authors have declared no conflicts of interest.
165 EFFECTS OF IL6 ON STATIN INDUCED APOPTOSIS IN HUMAN
MELANOMA CELLS
C. Minichsdorfer 1 , C. Wasinger 2 , E. Sieczkowski 3 , B. Atil 2 , M. Hohenegger 2
1 Department of Medicine I, Medical University of Vienna, Vienna, AUSTRIA,
2 Pharmacology, Medical University of Vienna, Vienna, AUSTRIA, 3 Neurology,
Medical University of Vienna, Vienna, AUSTRIA
Statins trigger apoptosis in primary cells and tumour cells. In particular, melanoma
cells have been found to be susceptible to statin-induced apoptosis, although only
after longer incubation times. Cells derived from metastatic melanoma cell lines
(A375, 518A2) secrete high amounts of IL-6 in contrast to WM 35 cells which derive
from an early lesion. However, IL-6 did not ameliorate the statin induced apoptosis
in A375 and 518A2 cells. Conversely, in WM35 cells IL-6 led to a marked decrease
in Cyclin D1 levels and enhanced the simvastatin induced apoptosis. Melanoma cells
from early growth stage are more resistant to simvastatin induced apoptosis than
metastatic melanoma cells. IL-6 plays a major role in the progression of melanoma.
Interestingly, IL-6 has no cytostatic effect on metastatic melanoma cells but primes
WM 35 cells for statin induced apoptosis. These pro-apoptotic stimuli confirm
possible therapeutic potentials and may guide feasibility for more potent statins in
anti-cancer strategies and help to understand the dualistic effect of IL-6 during
melanoma tumorigenesis. This work was supported by Herzfeldeŕsche
Familienstiftung and FWF (grant P22385).
Disclosure: All authors have declared no conflicts of interest.
166 EFFECT OF NEURAL CELL ADHESION MOLECULE
EXPRESSION ON GLYCOLYTIC PATHWAY IN MELANOMA
H.J. Cho1 , M. Choi2 , J.D. Lee2 , C.H. Kim1 1
Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, KOREA,
2
Department of Nuclear Medicine, Yonsei University College of Medicine, Seoul,
KOREA
Background: Neural cell adhesion molecule (NCAM), a member of the
immunoglobulin superfamily, has been well characterized in cell-cell adhesion,
neurite outgrowth, and synaptic plasticity. Recent studies have also shown that the
expression of NCAM affects tumor progression. Indeed, the expression of NCAM has
been implicated in the cellular invasion and metastasis of melanoma. Like many
other malignant tumors, melanomas are dependent on aerobic glycolysis for ATP
production. Many studies have consistently correlated poor prognosis and increased
tumor aggressiveness with increased glucose uptake. In this study, we tested whether
the expression of NCAM influences glycolytic pathways in mouse melanoma cell
line.
Methods: We constructed a recombinant viral vector derived from adeno-associated
virus serotype 2 (rAAV2) that expresses NCAM. Mouse melanoma cell line B16F10
was transduced with rAAV2. We performed in vitro fluorine-18 fluorodeoxyglucose
( 18 F-FDG) uptake assay in a gamma counter. In addition, western blot analysis was
carried out for glucose transporters (GLUT) and hexokinases (HK).
Results: Transduction of B16F10 cells with rAAV2 NCAM resulted in increased
18 F-FDG uptake. Western blot analyses demonstrated that the 180-kDa isoform of
NCAM (NCAM 180) was up-regulated. Expression levels of GLUT1, GLUT2 and
HK II were elevated.
Conclusions: Overexpression of NCAM was associated with elevated levels of
GLUT1, GLUT2, and HK II, which contributes to increased glycolysis. We
demonstrated that 18 F-FDG uptake might be used as a surrogate marker for assessing
NCAM expression in melanoma. In terms of monitoring NCAM expression, the
utility of 18 F-FDG uptake as a prognostic indicator requires further elucidation.
Disclosure: All authors have declared no conflicts of interest.
ix72 | Abstracts Volume 23 | Supplement 9 | September 2012