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CNIO_SYMP_MolecularMarkers_INT:CNIO_SYMP_MolecularMarkers_INT
21/11/06
CNIO Symposium on
Molecular markers
in cancer therapy:
Present use and future perspectives
November 27-29, 2006
Organisers:
Montserrat Sánchez-Céspedes
SPANISH NATIONAL CANCER RESEARCH CENTRE (CNIO), MADRID, SPAIN
Miguel Ángel Piris
SPANISH NATIONAL CANCER RESEARCH CENTRE (CNIO), MADRID, SPAIN
Centro Nacional de Investigaciones Oncológicas
Melchor Fernández Almagro, 3
E-28029 Madrid
www.cnio.es
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Contents
Foreword
5
Programme
7
Abstracts Sessions
13
Abstracts Posters
77
List of Participants
93
Forthcoming activities at the CNIO
99
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Foreword
Dear Colleague,
We would like to welcome you to the MOLECULAR MARKERS IN CANCER
THERAPY: Present Use and Future Perspectives Symposium, a multidisciplinary
meeting which is aimed to serve as a forum for the exchange of ideas and for the
discussion of candidate mechanisms and markers of drug resistance and therapeutic
efficacy.
Cancer therapy is quickly moving towards the design of novel and targeted drugs. As
a result of tremendous developments in the understanding of the molecular basis of
cancer during the last few years, numerous innovative agents have been discovered and
some of them are already in routine cancer clinical care or clinical trials. The search
for molecular markers identifying patients that may benefit from these treatments is
therefore becoming an urgent priority, and an exciting area of research.
Further advances in tailored therapies for cancer patients will be accomplished mainly
through further basic research in cancer biology, the conduct of translational research
projects, efficient new drug development, and the execution of large, prospective, randomised, multicentre cancer clinical trials. The different sessions of the meeting are
put together to address all these different aspects. We hope that the talks will be
thought provoking and lively with groundbreaking discussions. We also wish that the
Symposium contributes to encourage alliances that develop outstanding academic and
scientific collaborations to transcend boundaries in the goal of improving cancer treatments.
With our personal thanks for your interest,
Montserrat Sanchez-Cespedes
Miguel Angel Piris
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Programme
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CNIO Symposium on Molecular markers in cancer therapy 2006
MONDAY – NOVEMBER 27
9:15 - 9:30. Welcome address. Simposium Inauguration
Sra. Dña. Elena Salgado, Ministra de Sanidad y Consumo
9:30 - 10:00. Keynote lecture
Jose Baselga, Vall Hebron University Hospital, Barcelona, Spain
Incorporating molecular markers in early clinical trials with targeted agents
10:00 - 11:30. SESSION 1
TARGETING RECEPTORS AND SIGNAL TRANSDUCTION
Chair: Tito Fojo
10:00
Joan Albanell Hospital del Mar, Barcelona, Spain
Molecular markers of response to EGFR inhibitors
10:30
Alberto Bardelli IRCC, University of Torino Medical School, Candiolo, Italy
Mutational and functional profiling the kinome in cancer as a tool for directing targeted therapies
11:00
Manuel Hidalgo Johns Hopkins University, Baltimore, USA
Inhibitors of mTOR in Cancer Treatment. Clinical Implications
11:30-12:00 COFFEE BREAK AND POSTER VIEWING
12:00-13:10
SESSION II:
TARGETING RECEPTORS AND SIGNAL TRANSDUCTION
Chair: Alberto Bardelli
12:00
Luis Paz-Ares Hospital 12 de Octubre, Madrid, Spain
EGFR testing in the treatment of lung cancer
Short talk
12:30
Villanueva Augusto Mount Sinai School of Medicine, New York, USA
Activation of PI3K/AKT pathway in hepatocellular carcinoma and its abrogation with a
novel tyrosine kinase inhibitor (AEE788) and an mTOR inhibitor (RAD001, Everolimus.
Short talk:
12:50
Ignacio Casal Spanish National Cancer Centre (CNIO). Madrid, Spain
Antitumoral activity of FGFR3-specific immunotoxins in bladder carcinoma
13:10- 15:00 LUNCH AT CNIO CAFETERIA
13:15- 14:15 OPTIONAL LUNCH AT 2ND FLOOR SEMINAR ROOM
LUNCH TECHNICAL WORKSHOP:
“LATEST DEVELOPMENTS INTRODUCED BY AGILENT IN THE FIELD
OF MICROARRAY APPLICATIONS”
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Programme
15:00- 16:30
SESSION III:
DNA DAMAGING AGENTS AND MICROTUBULES
Chair: Manuel Hidalgo
15:00
Rafael Rosell Catalan Institute of Oncology, Barcelona, Spain
Molecular Understanding of Lung Cancer: A Need for Correct Treatment
15:30
Jean-Charles Soria Institut Gustave Roussy, Paris, France
DNA repair and prediction of sensitivity to chemotherapy in resected lung cancer
16:00
Tito Fojo National Cancer Institute Bethesda, USA
Molecular markers of response to taxans
16:30-17:00 COFFEE BREAK AND POSTER VIEWING
17:00 -17:50
SESSION IV:
DNA DAMAGING AGENTS AND MICROTUBULES
Chair: Jean C Soria
17:00
José Palacios Virgen del Rocío Hospital, Sevilla, Spain
Breast cancer response to nucleoside analogues and taxanes: a pharmacogenomic approach
Short talk
17:30
Miguel Quintela-Fandino Ontario Cancer Institute at the Princess
Margaret Hospital, Toronto, Canada
DNA-repair gene polymorphisms predict favorable clinical outcome among patients with
advanced squamous cell carcinoma of the head and neck treated with cisplatin-based
induction chemotherapy
Short talk
17:50
Manuel Collado Spanish National Cancer Centre (CNIO). Madrid, Spain
Genomic profiling of circulating plasma RNA for the analysis of cancer
Short talk
18:10
Ana María Rojas Spanish National Cancer Centre (CNIO), Madrid, Spain
Systems for the analyses of potential cancer target gene-lists
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CNIO Symposium on Molecular markers in cancer therapy 2006
TUESDAY – NOVEMBER 28
9:30-11:00
SESSION V:
PROTEOSOME INHIBITORS, APOPTOSIS AND INFLAMMATION
Chair: Rosario Perona Instituto de Investigaciones Biomédicas, Madrid, Spain
9:30
Elisabeth G.E. de Vries University of Groningen, Groningen,
The Netherlands
Clinical used agents to induce apoptosis especially targeting the TRAIL-receptors
10:00
Bharat Aggarwal University of Texas M. D. Anderson Cancer Center,
Houston, USA
Targeting inflammatory transcription factor pathway for prevention and treatment of cancer
10:30
Miguel Ángel Piris CNIO, Madrid, Spain
Molecular diagnosis in lymphoma: a windows towards the therapy
Short talk
11:00
María S. Soengas University of Michigan Comprehensive Cancer Center
and Department of Dermatology Medical Center, USA
c-MYC defines the tumor cell-selective killing of proteasome inhibitors by controlling the
expression of NOXA
11:20-11:50 COFFEE BREAK AND POSTER VIEWING
11:50-13:00
SESSION VI:
SIGNAL TRANSDUCTION KINASES AND STRESS-RELATED PROTEINS
Chair: Bharat Aggarwal
Short talk
11:50
Guillermo Velasco Complutense University, Madrid, Spain
Activation by cannabinoids of an ER stress-related pathway sensitizes tumor cells to apoptosis
Short talk
12:10
Angel R. Nebreda Spanish National Cancer Centre (CNIO). Madrid, Spain
p38α MAP kinase suppresses lung tumorigenesis by controlling self-renewal of stem/progenitor
cells
12:30
Juan L Steegmann Hospital Universitario de la Princesa, Madrid, Spain
Resistance to tyrosine kinase inhibitors in CML: a clinical perspective
13:00- 15:00 GROUP PICTURE AND LUNCH
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Abstracts-Sessions
15:00 - 15:30. Keynote lecture
Rene Bernards Netherlands Cancer Institute, Amsterdam, The Netherlands
Identification of biomarkers that predict therapy response in cancer
15:30- 17:00
SESSION VII:
EPIGENETIC AND TRANSCRIPTIONAL THERAPIES
Chair: Alfredo Carrato Presidente SEOM, Alicante, Spain
15:30
Manel Esteller CNIO, Madrid, Spain
Cancer epigenetics: from knowledge to therapy
16:00
Robert Brown Glasgow University, Glasgow, UK
DNA methylation as biomarker for old and new cancer therapies
16:30
Hugues de The Universite de Paris, Paris, France
Decrypting apl pathogenesis through therapy response
17:00-17:30 COFFEE BREAK AND POSTER VIEWING
17:30-18:30
SESSION VIII:
EPIGENETIC AND TRANSCRIPTIONAL THERAPIES
Chair: Hugues de The
17:30
Guillermo Garcia-Manero M.D. Anderson Cancer Center, Houston, USA
Epigenetic therapy of leukaemia
18:00
Stanley R. Frankel, Clinical Oncology, Merck & Co., Inc. North Wales, USA
Vorinostat for the treatment of cutaneous T cell lymphoma
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CNIO Symposium on Molecular markers in cancer therapy 2006
WEDNESDAY – NOVEMBER 29
9:30-11:30
SESSION IX:
PHARMACOGENOMICS AND PHARMACOGENETICS
Chair: Javier Benitez, Spanish National Cancer Centre
9:30
David Sidransky Johns Hopkins University School of Medicine,
Baltimore, USA
The Emerging Cancer “Methylome” in Primary Tumors and Cell lines
10:00
Montserrat Sanchez-Cespedes Spanish National Cancer Centre,
Madrid, Spain
Molecular pathways of lung carcinogenesis as potential markers for targeted therapy
10:30
Ramon Colomer Hospital de Girona Dr. Josep Trueta, Girona, Spain
Aromatase inhibitors, polymorphisms and breast cancer treatment
11:00
Lajos Pusztai, MD Anderson Cancer Center Houston USA
Gene expression profile-based predictors of response to chemotherapy
11:30-12:00 COFFEE BREAK AND POSTER VIEWING
12:00-13:10
SESSION X:
PHARMACOGENOMICS AND PHARMACOGENETICS
Chair: Lajos Pusztai
Short talk
12:00
Ana Gozalez-Neira Spanish National Cancer Centre, Madrid, Spain
Pharmacogenetics in cancer therapy: an overview
Short talk
12:20
Cristina Rodriguez-Antona Spanish National Cancer Centre, Madrid,
Spain
Pharmacogenetics of cytochromes p450 in oncology
12:50
Marco A. Pierotti Istituto Nazionale per lo Studio e la Cura dei Tumori,
Milan, Italy
Expression profiles to identify molecular markers of response to treatment in breast cancer
CLOSING REMARKS
13:20-13:35
Note:
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D. Francisco Gracia, Director del Instituto de Salud Carlos III
Talks: 20-minute / Short talk: 15-minute
Discussion: 10-minute after each talk / 5-minute after each short talk
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CNIO Symposium on Molecular markers in cancer therapy 2006
Molecular markers of response to EGFR inhibitors
Joan Albanell
Oncology Service, Hospital del Mar, Barcelona, Spain
The Epidermal Growth Factor Receptor (EGFR) is the target of novel anticancer agents
that have been recently approved in various countries for the treatment of several cancer types: erlotinib, an EGFR tyrosine kinase inhibitor (TKI), is approved for non-small
cell lung cancer (NSCLC) and pancreatic cancer; gefitinib, another EGFR TKI, was also
approved for NSCLC; and cetuximab, a monoclonal antibody directed at EGFR, is approved for colorectal cancer and head and neck cancer. In contrast, studies to date
have not shown sufficient activity of EGFR-targeted agents against many other epithelial cancers such as breast or prostate, to name two clear examples, despite promising
preclinical data. In addition, in diseases where EGFR-targeted agents are approved, only
a fraction of the patients obtain a clear benefit. Irreversible EGFR inhibitors, dual
EGFR/HER2 and pan-ErbB receptor inhibitors may also have greater antitumor activity
although the tolerance of these compounds compared to specific EGFR TKIs needs
further characterization. Lapatinib, a dual EGFR/HER2 TKI, is particularly promising in
breast cancer. In addition to the use of these agents as single agents, many clinical studies are addressing the role of combining them with hormonal agents, biological agents
or chemotherapy.
During the last few years, we have learned that we need to better understand biological and clinical criteria for patient selection and how to best use the different available agents. Optimizing patient selection based on clinicopathological as well as molecular criteria would most likely allow a more cost-efficient use of these agents and
also to find novel and more specific indications. There are two main translational approaches in this direction. A first one, is to find predictors of response such as EGFR
mutations, gene amplification, protein expression, polymorphisms, or phosphorylation
state of the target. Oncogene (EGFR) addiction appears to be an essential aspect of
the tumor cell to be sensitive to EGFR inhibition. In particular, EGFR mutations and,
to a lesser extent, EGFR gene amplification have been linked to dramatic responses to
erlotinib and gefitinib in NSCLC patients. These studies are commonly complemented
by combining EGFR assays with downstream markers, such as PI3K/Akt, Ras, p27 or
others, as well as by the coexpression of other receptors such as IGFR-1 or HER2.
There is also a great hope for proteomic and pharmacogenomic studies to find predictors of response. A second approach is to assay the biological effects of the agents
in vivo, in the so-called pharmacodynamic studies. These studies shed light on the effects of these agents on tumor EGFR and, perhaps more importantly, on downstream
elements, including Akt and MAPK. The analysis of these markers is useful to determine whether EGFR inhibition prevents downstream signalling events and points to clinically relevant mechanisms of response and resistance to these agents. An area of increasing interest is to find cooperative pathways of response/resistance and the
IKK/NF-kappaB pathway appears to be one of such novel avenues in the field.
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Abstracts-Sessions
SESSION I: Targeting Receptors and Signal Transduction
Notes
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CNIO Symposium on Molecular markers in cancer therapy 2006
Mutational and functional profiling the kinome in cancer as a tool
for directing targeted therapies
Alberto Bardelli
IRCC, University of Torino Medical School, Candiolo, Italy
The completion of the human genome project has marked a new beginning in biomedical sciences. Knowledge of the sequence and organization of the human genome
allows the systematic analysis of the genetic alterations underlying the origin and evolution of tumors. We previously completed the mutational analyses of gene families involved in signal transduction, including kinases and phosphatases, in a number of cancer genomes. We will discuss two applications of the results stemming out from these
mutational analyses.
On one side we have attempted to couple specific molecular lesion affecting kinase
genes to the therapeutic response to specific cancer drugs. This strategy was used to
identify the patients that benefit from drugs targeting the EGF receptor, a well known
tyrosine kinase. Specifically we found that in patients with metastatic colorectal cancer,
the response to monoclonal antibodies targeting the EGFR is associated with increased copy number of the corresponding gene. This observation represents the first paradigm for personalized targeted therapy of colorectal cancer based on a specific molecular alteration.
The second approach has been the construction of cellular models recapitulating genetic alterations is a prerequisite to translate these findings into the clinic. So far this
has been achieved mainly by (over)expressing the corresponding mutated cDNA at
non-physiological levels in mouse or human cells. To build cellular models more closely recapitulating the genetic alterations identified in human tumours we introduced
a spectrum of cancer mutations in human, non transformed cells via homologous recombination. We suggest that this strategy is an effective approach to build tumor progression models that can be used to dissect oncogenic signaling pathways and to evaluate anti-cancer drugs. The combined mutational and functional profiling of gene families
is expected to have an important impact on future diagnostic and therapeutic strategies of cancer.
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Abstracts-Sessions
SESSION I: Targeting Receptors and Signal Transduction
Notes
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Inhibitors of mTOR in cancer treatment. Clinical implications
Manuel Hidalgo
The Johns Hopkins University School of Medicine, Baltimore, USA
The mammalian target of rapamycin (mTOR) is a serine treonine kinase which is involved in the control of cellular growth and proliferation. Abnormal activation of signaling pathways both proximal and distal to this protein occurs frequently in human
cancer, making mTOR an attractive target for anticancer drug development. mTOR is
downstream from Akt in the PI3K/Akt signaling pathway and controls de translation
of key mRNAs involved in cell cycle progression through 4EBP1 and p70S6k. More recently, TSC and Rheb have been found to be implicated in the regulation of mTOR.
Importantly, genes involved in the PI3K/Akt pathway such as PTEN and PI3K are frequently mutated in common cancers. Recent studies have elucidated the intracellular
interaction of mTOR with Raptor, forming a rapamycin sensitive pathway and with Rictor, in the so called rapamycin insensitive pathway.
Rapamycin and its analogs inhibit mTOR, and have showed potent antitumor activity
in vitro and in xenografts models. Pharmacologically, these agents inhibit mTOR resulting in inhibition of the cell cycle though decrements in cyclin D1 and increment in
p27 levels. In selected models, the agents also induce apoptosis. More recently, several
studies indicate that mTOR inhibition leads to antiangiogenesis effects by down regulation in HIF-1a production. This effect is particularly relevant in tumors with defective VHL such as renal cell cancer.
In Phase I clinical trials, these agents were well tolerated and induced antitumor effects in patients with different solid tumors and lymphomas. An important problem at
the end of Phase I trials is that there was no a clear dose-related toxicity profile with
these agents. Indeed, the clinical development of mTOR inhibitors exemplifies the challenges in developing targeted agents. mTOR inhibitors have been well tolerated at a
wide range of doses in clinical trials difficulting the selection of phase II doses based
solely on toxicity criteria. Assessment of pharmacodynamic effects in surrogate normal
tissues and tumor tissues has been used to determine pharmacodynamically active doses. Lack of parallel assessment of tumor tissue effects as well as the intrinsically high
interpatient variability has limited the value of these studies, however.
In Phase II studies, the agent resulted in tumor responses in a low percentage of patients with renal and breast cancer. In preclinical studies, these agents appear to be
more effective in tumors with mutations in the PTEN gene that result in activation of
the PI3/Akt signaling pathway. This information has not been exploited in the clinic in
detail yet. A better understanding of determinants of response to mTOR inhibitors
could be used for patient selection in clinical trials. In conclusion, mTOR inhibitors are
promising anticancer agents. Future studies are needed to properly development of
these drugs as current cancer treatment.
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Abstracts-Sessions
SESSION I: Targeting Receptors and Signal Transduction
Notes
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EGFR testing for the treatment of lung cancer
Luis Paz-Ares
Hospital Universitario Doce de Octubre, Madrid, Spain
According to 2004 estimates, 1.4 million people were diagnosed with and 1.2 million
died of lung cancer worldwide. Recently, a new class of medications targeting signal
transduction pathways has come into focus in the treatment of various malignancies.
In lung cancer, the molecules which target the intracellular kinase domain of the epidermal growth factor receptor (EGFR), such as erlotinib and gefitinib, cause significant
tumour responses and, in the case of erlotinib, a survival benefit in patients with previously treated cancers. Responses were most pronounced in females, asians and nonsmokers with adenocarcinoma histology. These patients were found more likely to harbour mutations of the receptor kinase domain, including in-frame deletions in exon 19
(such as deletions of codons 746-750) and point deletions in exon 21 (such as L858R).
Other EGFR kinase domain mutations have been found to confer resistance (T790M)
or differential susceptibility to erlotinib and gefitinib (E884K). Now, we now that the
biology and natural history of this subset of lung cancers is different. A somatic/germline egfr intron 1 CA repeat sequence polymorphism has shown to have an important
role in the control of EGFR protein expression, and has been linked to an increased
risk of familial breast cancer, a worse outcome in patients with colorectal cancer, and
anti-EGFR treatment efficacy in preclinical models. Gene amplification and protein expression of EGFR also may predict sensitivity, and has been suggested that these test
may have lower positive predictive value, but may be more valuable tools to exclude
which patients may not benefit from EGFR TKIs. Other potentially useful negative predictor of erlotinib/gefitinib activity is the presence of a Ras mutation. In this talk, we
will review efforts that have been undertaken to identify determinants of drug susceptibility to EGFR tyrosine kinase inhibitors and their potential clinical applications,
with particular focus on the role of gene mutations.
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SESSION II: Targeting Receptors and Signal Transduction
Notes
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Activation of PI3K/Akt pathway in hepatocellular carcinoma
and its abrogation with a novel tyrosine kinase inhibitor (AEE788)
and an mTOR inhibitor (RAD001, everolimus).
Villanueva Augusto1, Peix J1, Di Feo A2, Lemmer E1 , Van Laarhoven S1, Fiel MI1,
Thung S1, Yea S1, Roayaie S1, Chen Y1, Schwartz M1, Martignetti J2, Friedman SL1, Llovet
JM1,3.
1. Division of Liver Diseases and Liver Cancer Program
2. Human Genetics
3. Mount Sinai School of Medicine, New York, USA. BCLC Group. Liver Unit. IDIBAPS
Hospital Clinic, Barcelona, Spain
Background: There is no first-line treatment for advanced hepatocellular carcinoma
(HCC).The knowledge of the signaling pathways involved in the initiation/progression
of the disease may provide molecular targets for new therapies.
Aim: Study PI3K/Akt/mTOR pathway activation in human HCV-hepatocarcinogenesis
and the effect of its abrogation with a novel tyrosine kinase inhibitor and an mTOR
inhibitor.
Methods. We investigated by qRT-PCR the expression of 8 genes of the Akt/mTOR
pathway (EGF, EGFR, IGFII, IGFBP3, Akt, PTEN, mTOR and RICTOR) in 78 human samples and the immunostaining status of pAkt and pEGFR. Huh7, Hep 3B and Hep G2
(liver cancer cell lines) were incubated with increasing concentrations of an
EGFR/Her2/VEGFR inhibitor (AEE788, Novartis) and an mTOR inhibitor (RAD001, everolimus: Novartis-Pharma AG, Basel, Switzerland). We assessed cell viability (MTT assay) and proliferation (3[H] Thymidine Incorporation Assay). We performed FACS for
cell cycle studies and immunoblot to evaluate protein dysregulation.
Results: EGF and IGF-II were 5-fold and 3-fold respectively upregulated in human
HCCs (p<0.01), whereas tumor suppressors PTEN and IGFBP3 were significantly downregulated (p<0.01). HCCs showed positive staining for p-Akt compared with non-tumoral tissues (10/20 vs 2/33, p=0.002). In vitro studies showed that AEE788 [0.5µM10µM] decreased cell viability by 65-83%, RAD001 [20nM] decreased it by 28-44% and
the combination by 66-88% (all p<0.02). Regarding proliferation, AEE788 decreased
thymidine incorporation by 97% (p<0.002). AEE788 induced significant increase in apoptosis and arrest in G2/M phase compared with controls (59% vs 4%, and 39% vs 15%,
respectively) and time-dependent PARP cleavage. AEE788 abolished phosphorilation of
EGFR, Akt, ERK and p70S6.
Conclusions: PI3k/Akt signaling pathway is activated in a subset of HCV-related HCCs.
Blocking this pathway with a novel combination therapy (AEE788 and RAD001) decreases cell viability, proliferation and increases apoptosis. This therapeutic approach
should be tested in experimental models, and might be of therapeutic use in HCC patients.
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SESSION II: Targeting Receptors and Signal Transduction
Notes
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Antitumoral activity of FGFR3-specific immunotoxins in bladder
carcinoma.
JL Martinez, LH Cheung, P Lopez, S Ferreiro, M Cañamero, MG Rosemblum
and Ignacio Casal
CNIO, Madrid, Spain and MDACC, Houston, USA
The receptor for fibroblast growth factor 3 (FGFR3) appears to be up-regulated in a
number of neoplastic states including multiple myeloma, hepatocellular carcinoma (HCC)
and bladder carcinoma. Two “naive” semi-synthetic human scFv libraries were used to
select antibodies against the extracellular domain of FGFR3alpha(IIIc). The reactivity of
the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon resonance analysis, showing the antibodies an affinity
in the nanomolar range. They reacted with native FGFR3 on the membrane of RT112
bladder carcinoma cells by a fluorescence-activated cell sorter and confocal microscopy. Two antibodies (designated 3C and 7D) were able to inhibit proliferation of RT112
cells in vitro. In order to study the ability of gelonin to enhance this inhibitory effect,
both scFvs were fused to the N-terminus of rGel toxin. The resulting fusion immunotoxins were expressed as soluble proteins in E. coli, purified by IMAC chromatography
and functionally characterized. Immunofluoresence studies demonstrated that both the
3C/rGel and 7D/rGel fusion constructs internalize well into RT112 bladder cancer cells
within 1 h of exposure. The cytotoxicity of 3C/rGel and 7D/rGel against non-target
A375 melanoma cells was identical to that of native rGel. In contrast, the IC50 of both
7D/rGel and 3C/rGel were approximately 200 nM on RT112 cells compared to ~1200
nM for rGel. Preliminary tumor xenograft studies in SCID mice with 3C/rGel at a dose
of 15 mg/kg showed a significant decrease in tumor size after scFv/rGel administration.
Further studies are currently ongoing.
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Notes
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Molecular understanding of lung cancer: a need for correct treatment
Rafael Rosell1, David Gandara2, Manuel Cobo3, Dolores Isla4, Bartomeu Massuti5,
Ana Montes6, Jose Miguel Sanchez7, Mariano Provencio8, Nuria Viñolas9, Teresa Moran1
1.
2.
3.
4.
5.
6.
7.
8.
9.
Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Ctra Canyet, Badalona, Spain
UC Davis Cancer Center, Sacramento, CA, USA
Hospital Carlos Haya, Malaga, Spain
Hospital Lozano Blesa, Zaragoza, Spain
Hospital General de Alicante, Alicante, Spain
Catalan Institute of Oncology, Hospital Duran i Reynals, Barcelona, Spain
Hospital Alcorcon, Madrid, Spain
Hospital Puerta de Hierro, Madrid, Spain
Hospital Clinic, Barcelona, Spain
To date, no diagnostic tests to identify subgroups of patients who can respond more
favorably to chemotherapy have neither been developed nor made available. Non-smallcell lung cancer (NSCLC) is one of the most common cancers and, at the time of diagnosis, 50% of the cases have metastases (stage IV) with a gloomy outcome. It is said
that all cisplatin-containing chemotherapy regimens yield the same clinical outcomes.
However, there are several hints that indicate that cisplatin partners could be chosen
based on tumor genetic abnormalities. We have carried out the first individualized phase III randomized trial in stage IV NSCLC patients.
Purpose: Although current treatment options for metastatic NSCLC rely on cis-based
chemotherapy, low response rates and short progression-free survival times suggest that
individualized approaches to therapy are required to improve results. ERCC1, involved
in nucleotide excision repair, has been associated with cis resistance. We hypothesized
that customizing cis based on pretreatment ERCC1 mRNA levels would improve response. Methods: Randomized trial of 444 stage IV NSCLC patients (p), enrolled at 24
study sites from August 2001 to October 2005. RNA was isolated from pretreatment
biopsies, and quantitative real-time reverse transcriptase PCR assays were performed to
determine ERCC1 mRNA expression. p were randomly assigned in a 1:2 ratio to either the control arm or the genotypic arm before ERCC1 assessment was performed.
p in the control arm received docetaxel (doc)/cis. In the genotypic arm, p with low
ERCC1 levels received doc/cis, and p with high levels received doc/gemcitabine. The
primary endpoint was the overall objective response (OR) rate (complete plus partial).
Secondary endpoints were progression-free survival (PFS) and overall survival (OS). Results: Of the 444 p enrolled in the study, 78 (17.6%) went off-study prior to receiving
one cycle of chemotherapy. The main reason for withdrawal was insufficient tumor tissue for ERCC1 assessment (42 p [9.4%]). Of the 366 p who completed the study, 346
were evaluable for OR and 366 for PFS and OS. OR was observed in 53 p (39.3%; 95%
CI, 31.4-47.8%) in the control arm and 107 p (50.7%; 95%CI, 44-57.5%; P = 0.019) in
the genotypic arm. Conclusion: Assessment of ERCC1 mRNA expression in patient tumor tissue is feasible in the clinical setting and predicts response to doc/cis. Further
studies are warranted to optimize methodologies for ERCC1 analysis in small tumor
samples and to refine a multi-biomarker profile predictive of patient outcome.
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Abstract: DNA repair and prediction of sensitivity to
chemotherapy in resected lung cancer
Jean-Charles Soria
Institut Gustave Roussy, in behalf of IALT-Bio investigators, Paris, France
Background Adjuvant cisplatin-based chemotherapy improves survival among patients
with completely resected non-small-cell lung cancer, but no clinical or biological predictor of the benefit of chemotherapy has yet been validated in large-scale studies.
Methods We immunohistochemically analyzed ERCC1 expression in operative specimens from patients who had been enrolled in the International Adjuvant Lung Trial
(IALT) to examine this marker’s ability to predict a survival benefit from adjuvant cisplatin-based chemotherapy. Overall survival was analyzed with a Cox model adjusted
on clinical and pathological factors.
Results Among 761 patients, ERCC1 expression was positive in 335 patients (44 percent). The benefit of cisplatin-based adjuvant chemotherapy was correlated with the
ERCC1 status (test for interaction, P=0.009). Patients with ERCC1-negative tumors
who were randomized to adjuvant chemotherapy had significantly prolonged survival
compared with patients with ERCC1-negative tumors who were randomized to observation (adjusted hazard ratio for death, 0.65; 95 percent confidence interval [0.50
to 0.86], P=0.002). Conversely, there was no evidence for a survival benefit from adjuvant chemotherapy among patients with ERCC1-positive tumors (adjusted hazard ratio for death, 1.14; 95 percent confidence interval [0.84 to 1.55], P=0.40). In patients
randomized to observation, the subgroup with ERCC1-positive tumors had better survival compared to those with ERCC1-negative tumors (adjusted hazard ratio for death, 0.66; 95 percent confidence interval [0.49 to 0.90], P=0.009).
Conclusions Patients with completely resected non-small-cell lung cancer and ERCC1negative tumors gain a substantial benefit from adjuvant cisplatin-based chemotherapy.
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Breast cancer response to nucleoside analogues and taxanes: a
pharmacogenomic approach
José Palacios
Virgen del Rocío Hospital, Sevilla, Spain
A better knowledge of the molecular mechanisms of action of common drugs used in
chemotherapy would improve the design of treatment protocols and the prediction of
resistance and clinical response in individual patients. A comprehensive molecular characterization of modifications of gene expression was performed by cDNA microarrays in breast cancer cell lines after treatment with the nucleoside analogues 5-Fluorouracil (5-FU), Gemcitabine (GMB) and 5DFUR, and the taxanes paclitaxel (PTX) and
docetaxel (DTX). 5-FU induced the expression of P53 target genes involved in cell
cycle and apoptosis. Most of the genes regulated with 5-FU were also upregulated with
equivalent concentrations of 5DFUR, including many p53 targets. However, this effect
was almost completely reverted when MCF7 cells were simultaneously exposed to a
specific inhibitor of the nucleoside transporters ENT1 (NBT1), and this was associated with a higher resistance to 5DFUR. These data suggest that both, p53 status and
the expression of ENT1, may determine capecitabine response in breast cancer.
GMB is able to induce DNA damage, producing cell cycle arrest and of apoptosis. The
induction p53, TNF or NFkB pathway genes depend on the cellular context. Our results indicate NFkB activation as a mechanism of resistance to GMB.
As expected, taxanes induced G2/M arrest and microarray analysis showed overexpression of some genes related with mitosis (survivin, STK6, and BUB1). However, PTX
also induced a group of genes involved in the activity of the proteasome. Moreover,
there was an additive effect when the proteasome inhibitor MG132 was used in combination with PTX, in a sequential manner. These data suggest that a combination of
PTX and proteasome inhibitors should be tested in the clinical setting.
A neoadjuvant phase II trial for stage II-III breast cancer patients was specifically designed to test the ability for gene signature on the prediction of clinical and pathological responses to treatment with GMB and PTX. Resistant tumors are correctly classified with an accuracy of 90%, although for sensitive tumors, misclassifications are
about 35%. Some of predictor genes are involved in drug metabolism, DNA biosynthesis,
and extracellular matrix remodelling. Gene signatures could allow the development of
clinical tests for reducing unnecessary treatment for women suffering from breast cancer.
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DNA-repair gene (DRG) single nucleotide polymorphisms (SNPs)
influence on response and survival in stage IV SCCHN patients
treated with CDDP-based therapy.
Miguel Quintela-Fandino, R. Hitt, P. P. Medina, S. Gamarra, M. Amador, L. Manso, H.
Cortes Funes, M. Sanchez-Cespedes
Ontario Cancer Institute at the Princess Margaret Hospital, Toronto, Canada
Background: CDDP kills tumor cells by DNA cross-linking. SNPS at DRG may influence
the ability of patients to respond to CDDP. We studied the role of the SNPs XPDAsp312Asn, XPD-Lys751Gln, ERCC1-C8092A and XRCC1-Arg399Gln to influence the
response to CDDP-based therapy and to predict survival in SCCHN patients Methods:
SNPs were genotyped in DNA from peripheral lymphocytes using PCR-restriction fragment length polymorphism in 103 stage IV (0% M1) SCCHN patients followed prospectively. Treatment schedules were concurrent CDDP/RT (n = 28), CDDP + fluoropyrimidine (FP) CDDP/RT (33) or CDDP + FP + taxane CDDP/RT (42). Endpoints: to assess
the impact of the SNPs in OS (Kaplan Meier and Cox’s model) and in response to chemotherapy (multivariate multinomial regression). The covariables gender, age, treatment
type, number of polymorphic (poly.) variants (0-8), smoking and alcohol consumption and
amount (mg) of CDDP were included in the multivariate analysis Results: Median age 60
yrs (39-94); 97 (94%) male. After 78 months of follow-up, 24% had died, 22% had relapsed and 51% were free of disease. The allele frequencies for the homozygous common
allele, heterozygous and homozygous poly. variant were: ERCC1: 53, 40 and 7%; XPD312:
50, 42 and 8%; XPD751: 35, 57 and 8%; XRCC1: 35, 51 and 13%. Overall, 9%, 16%, 22%,
20%, 24%, 6%, 1% and 1% of the patients harbored 0, 1, 2, 3, 4, 5, 6 and 7 poly. variants
simultaneously. Table shows OS according to genotype. Patients with only common alleles had a median OS of 5.1 months (4.3-6.0) vs not reached for patients with >1 variant
(p = 0.0000)(mean 6.2 (4-8.4) vs 61.3 (53-59.8). Each variant allele decreases the probability of dying 2.1 fold (p = 0.0000) on the Cox’s analysis (7 variants = 175 fold protection) and increases 2.94 fold (multinom. reg.)the probability of achieving a CR vs PD (p
= 0.041) Conclusions: In our series the poly. variants at DRG are the strongest prognosis factors and accurately predict clinical response among SCCHN patients.
Overall Survival in Months
(Kaplan Meier) According
to Each Genotype
XPD 751
XPD 312
XRCC1
ERCC1
Patients with 2
wild type alleles
Median 20.7 (5.4-36)
Median 30.1 (9.3-50.9) Median not reached
Median not
(Mean 23.0) (17.0-29.1) (Mean 29.5) (22.3-33.7) (Mean 46.4) (34.0-58.9) reached
Patients with 1
or 2 variant alleles
Median not reached
Median not reached
Median not reached
Median not
(Mean 65.6) (57.4-73.9) (Mean 68.2) (59.1-77.3) (Mean 50.3) (42.5-58.1) reached
Log Rank p value
0.0012
0.0012
0.0044
Patients with 1 and 2 variant alleles were grouped due to the low (<10%) freq. of homozygous patients
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Genomic profiling of circulating plasma RNA for the analysis
of cancer
Manuel Collado1, Vanesa Garcia2, Jose M Garcia2, Isabel Alonso2, Luis Lombardia1,
Ramon Diaz-Uriarte1, Luis A Lopez-Fernandez3, Angel Zaballos3, Felix Bonilla2,
Manuel Serrano1
1. Spanish National Cancer Centre (CNIO), Madrid, Spain
2. Department of Oncology. Hospital Universitario Puerta de Hierro, Madrid, Spain
3. National Centre of Biotechnology (CNB-CSIC), Universidad Autónoma, Madrid, Spain
Background. The blood of cancer patients is known to contain fragmented RNA released from the tumor. This material has the potential to yield non-invasive cancer markers. However, so far, the analysis of plasma RNA has been restricted to a few genes
based on educated guesses. Unbiased selection of markers by genomic profiling of plasma RNA is necessary for the clinical use of this novel type of markers.
Methods. We profiled plasma RNA from colorectal cancer (CRC) patients (12) and
from healthy donors (8) by cDNA microarray hybridization. Genes significantly enriched in CRC plasma were identified and further analyzed by quantitative RT-PCR in a
new set of plasma samples from CRC patients (29) and healthy donors (36), including
paired plasma samples from the same CRC patients before and after surgical resection of the tumor (11 pairs).
Results. Gene expression analysis selected 40 candidate markers. Three of them, EPAS1,
UBE2D3 and KIAA0101, were significantly increased in the plasma of CRC patients.
Importantly, two of the markers, EPAS1 and UBE2D3, showed a significant decrease after surgery, returning to normal levels. Finally, class prediction using the levels of EPAS1
from the pre- and post-surgery plasma samples was able to correctly assign the majority of samples (86%) to their corresponding class, that is, pre-surgery samples to
the CRC group and post-surgery samples to the healthy group.
Conclusions. Gene expression profiling of circulating plasma RNA is a valuable tool
to find cancer markers of potential clinical value.
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Systems for the analyses of potential cancer target gene-lists
Ana María Rojas
Spanish National Cancer Centre (CNIO), Madrid, Spain
The current trends of cancer candidate genes detection heavily relies on large-scale
genotyping efforts. The recent publication (Sjoblom et al, Science, 2006) of a screening
of more than 13,000 genes in 11 colorectal and 11 breast cancer tissues provides a
good example of what cancer genomic data deluge will be (see the ongoing projects
at the Sanger Centre, http://www.sanger.ac.uk/ and NCI, http://www.cancer.gov/). Needless to say is that the impact of these studies depends largely on the capabilities of
experimental biologists to mine and use this information.
In realistic terms in would be impossible to use the information derived from the HT
efforts for the formulation of significant research without a consistent strategic data
integration and analysis. Unfortunately this task is complicated by the diversity and heterogeneity of the biological databases, repositories and computational methods.
Therefore it is important to create specialized bioinformatics environments that would
facilitate the interaction of the users with this type of HT and enable the effective use
of the information. This type of analysis environment should be able to systematize and
optimize the tedious and time-consuming process of analyzing the functions and relations of candidate genes and proteins.
Here we present a prototype of such a system, where it is easy to visualize and analyze the above-mentioned lists of breast and colon cancer related genes. On the surface the system uses a “Google”-like querying system that is build on top of a cuttingedge web server technology based on distributed specialized services following the
“biomoby” standard (www.inab.org). The available widgets in the system provide information of associated diseases, potential protein interactions automatically extracted
from papers or from curated databases, protein function annotations, and SNP’s in coding regions, which are mapped in the available structures.
A description of the current capabilities of the prototype and planned future developments will be presented.
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Clinically used agents to induce apoptosis especially targeting the
TRAIL-receptors.
Elisabeth G.E. de Vries, S. de Jong, C.H. Mom, J.A. Gietema.
Dept Med Oncol, University Medical Center Groningen, the Netherlands.
Apoptosis induction can result in tumor shrinkage. It can be executed through a mitochondria-dependent (instrinsic) and a mitochondria independent (extrinsic) pathway.
The ”extrinsic” pathway is initiated by activation of cell membrane death receptors.
The TRAIL pathway is currently explored in the clinic. The natural occurring death receptor ligand TRAIL, can induce apoptosis in tumor cells and not in normal cells by
binding to its cell membrane death receptors (DR) 4 and DR5. Soluble, zinc-stabilized
recombinant human (rh)TRAIL without His-tag showed no preclinical toxicity. In a phase 1 study with rh Apo2L/TRAIL, which activates both DR4 and DR5, patients received once daily for 5 days every 3 weeks doses of up to 15 mg/kg. No drug-related,
dose limiting toxicities were seen. Pharmacokinetic analysis showed a half-life of 36
min. One partial response occurred in a patient with chondrosarcoma at 8 mg/kg. Currently also agonistic DR4 and DR5 antibodies against DR4 or DR5 are studied in the
clinic as another option to induce apoptosis. The phase 1 studies with agonistic DR4
antibody mapatumumab showed that it is well tolerated at doses up to 20 mg/kg per
cycle. Mean terminal half-life was 17 days. Phase 2 studies with single agent mapatumumab administered in pretreated NSCLC and colorectal cancer patients showed stable disease in resp. 29% and 32%. In a non-Hodgkin’s lymphoma study in 3 out 40 patients tumor responses have been observed, all in refractory or relapsed follicular
lymphoma patients. In the preclinical setting a synergistic effect was observed between death receptor ligands and chemotherapy. There are 2 phase 1b studies in which
mapatumumab is combined with gemcitabine/cisplatin (ongoing) and paclitaxel/carboplatin. The maximum tolerated dose is not yet reached. No pharmacokinetic interaction between the drugs was seen. Data on DR5 antibodies are only available for one
antibody, lexatumumab. Two phase 1 studies have been performed. A dose of 10 mg/kg
per cycle was considered the maximum tolerated dose. Based on preclinical data, combinations with numerous targeted agents are also of interest. Hopefully choices for
specific (modified) death receptor ligand treatment can in the future be rationally made
based on tumor characteristics.
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Targeting Inflammatory Transcription Factor Pathway for
Prevention and Treatment of Cancer
Bharat B. Aggarwal
Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of
Texas M. D. Anderson Cancer Center, Houston, USA.
NF-κB, a transcription factor, is present normally in the cytoplasm as an inactive heterotrimer consisting of p50, p65 and IkBa subunits. When activated, NF-kB translocates to the as a p50-p65 heterodimer. This factor regulates the expression of various
genes that control apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation. NF-kB has been linked to the development of carcinogenesis for several reasons. First, various carcinogens and tumor promoters have been shown
to activate NF-kB. Second, activation of NF-kB has been shown to block apoptosis and
promote proliferation. Third, the tumor microenvironment can induce NF-kB activation. Fourth, constitutive expression of NF-kB is frequently found in tumor cells. Fifth,
NF-kB activation induces resistance to chemotherapeutic agents. Fifth, several genes involved in tumor initiation, promotion, and metastasis are regulated by NF-kB. Sixth, various chemopreventive agents have been found to downregulate the NF-kB activation.
All these observation suggest that NF-kB could mediate tumorigenesis and thus can
be used as a target for chemoprevention and for the treatment of cancer. Besides NFkB, we have also targeted AP-1 and STAT3, other transcription factors that mediate
tumorigenesis. We will present the data which shows that phytochemicals derived from
fruits, vegetables and spices are important inhibitors of NF-kB activation, and can suppress the expression of genes involved in carcinogenesis and tumorigenesis in vivo.
To be presented CNIO Symposium on “Molecular markers in cancer Therapy: present
use and future perspectives” Madrid, on November 27-29, 2006.
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Molecular diagnosis in lymphoma: a windows towards the therapy
Miguel Ángel Piris
Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas (CNIO),
Madrid, Spain
High-throughput molecular studies have demonstrated that the common forms of cancer carry on a large variety of changes in the expression of relevant genes codifying
for the most essential cell functions.
Our effort, working in lymphomas, is currently focused into the recognition of molecular signatures of key pathways and functions, which allow redrawing the sequence of
events that induce the neoplastic transformation and progression. Pathways which are
being recognised as highly relevant in Non-Hodgkin’s Lymphoma are BCR signalling, somatic hypermutation, glycolysis, NF-kB activation and others. Each one of them is identified through a characteristic signature, which allows recognizing its functional and biological relevance.
At the same time, the accumulation of information on multiple markers is allowing to
propose predictive systems, mainly based in biological data, which may complement the
current standard clinical predictive systems. These biological-based predictor models
identify the specific weight of each marker, and associate the most relevant into a single formula. Our group is now proposing predictive systems for Hodgkin’s Lymphoma,
B-CLL, CTCL, MCL and Large B-cell lymphoma. The validation of these results is being
performed through the analysis of disease-specific genes printed on low-density arrays,
in a design focused to specific clinical decisions.
These studies are also making possible the development of Pharmacogenomics projects, aimed to identify expression profiles associated with sensitivity of resistance to
specific drugs. Finally they are pinpointing to the presence of potential therapeutic targets.
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c-MYC defines the tumor cell-selective killing of proteasome
inhibitors by controlling the expression of NOXA.
Nikiforov M., Tang W-H., Gractchouk V., Riblett M., Manava S. and María S. Soengas
University of Michigan Comprehensive Cancer Center and Department of Dermatology,
Ann Arbor, USA
The 26S proteasome is ubiquitously expressed in all mammalian systems analyzed, and
plays a central role in the control of cell division and cell survival. Thus, it is puzzling
that proteasome inhibitors such as bortezomib can preferentially kill a variety of malignant cells, sparing normal cell compartments. Here we show that a main component
of the preferential selectivity of bortezomib towards cancer cells relies on the oncogene c-MYC. This function of c-MYC was unexpectedly identified in a search for mechanisms of activation of NOXA (a proapoptotic factor we and others have previously
shown to be induced by bortezomib in a tumor-cell restricted manner). The requirement of c-MYC was found in a variety of tumor cell types in marked contrast with a
dispensable role of p53, HIF-1 or E2F-1, main known regulators of NOXA mRNA expression. Conserved MYC-binding sites were identified in the NOXA promoter and validated by chromatin immunoprecipitation. Importantly, interfering with the endogenous
levels of c-MYC abrogated the ability of bortezomib to induce NOXA. Conversely, induction of MYC allowed normal cells to activate NOXA in response to proteasome
inhibition. c-MYC is itself a proteasomal target whose levels or function are invariably
upregulated during tumor progression. Therefore, our studies provide a new role of cMYC in the control of the apoptotic machinery of tumor cells and identify a longsought tumor-associated factor that is necessary and sufficient to confer sensitivity to
proteasome inhibition.
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Activation by cannabinoids of an ER stress-related pathway
sensitizes tumor cells to apoptosis
Arkaitz Carracedo1, Mar Lorente1, Ainara Egia1, Cristina Blázquez1, Stephane García2,
Valentin Giroux2, Cedric Malicet2, Raquel Villuendas3, Meritxell Gironella2,
Luis González-Feria4, Miguel Ángel Piris3, Juan L. Iovanna2, Manuel Guzmán1
& Guillermo Velasco1
1. Department of Biochemistry and Molecular Biology I, School of Biology, Complutense
University, Madrid, Spain
2. U624 INSERM, Marseille, France
3. Centro Nacional de Investigaciones Oncológicas, Instituto, Madrid, Spain
4. Department of Neurosurgery, University Hospital, Tenerife, Spain
Investigations performed during the last few years have shown that cannabinoids inhibit the survival of tumor cells in culture and the growth of tumor xenografts in rodents, an effect that is selective of tumor cells. These investigations have led to the
promotion of a clinical trial aimed at studying the effect of 9-tetrahydrocannabinol,
the major active ingredient of marijuana, on the growth of recurrent brain tumors,
which first stage has been very recently completed.
The antitumoral action of cannabinoids relies, at least in part, on the ability of these
compounds to induce apoptosis of tumor cells. However, the molecular mechanism
responsible for this effect remains unraveled. By analyzing the gene expression profile
of several tumor cell lines (sensitive and resistant to cannabinoid-induced apoptosis)
here we identify the stress-regulated protein p8 (also designated as candidate of metastasis 1) as an essential mediator of cannabinoid pro-apoptotic and anti-tumoral action. p8 mediates its apoptotic effect via up-regulation of the endoplasmic reticulum
(ER) stress-related genes ATF-4, CHOP and TRB3. Results obtained with primary cultures obtained from human brain tumor samples as well as with different astrocytoma cell lines show that activation of this pathway is reduced in cannabinoid-resistant
tumor cells. Interestingly, administration of ER stress inducers together with 9-tehtrahydrocannabinol acts in a synergic fashion to induce apoptosis of tumor cells. Our
findings suggest that activation of the p8-regulated pathway may constitute a new therapeutic strategy for inhibiting tumor growth.
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p38α MAP kinase suppresses lung tumorigenesis by controlling
self-renewal of stem/progenitor cells
Juan José Ventura1, Stephan Tenbaum1, Eusebio Perdiguero1, Carmen Guerra1,
Mariano Barbacid1, Manolis Pasparakis2 and Angel R. Nebreda1
1. CNIO (Spanish National Cancer Center), Madrid, Spain
2. EMBL-Mouse Biology Unit, Monterotondo, Italy
Cell renewal is essential to maintain tissue homeostasis and repair throughout the life
of an organism. These functions rely on the presence of stem cells that have the potentiality to differentiate into specialized cell types. Deregulation of stem cell self-renewal and differentiation results in pathological states. We have used a conditional knockout approach to investigate the function of the stress-activated p38α MAP kinase in
adult mice. The most striking phenotype observed upon genetic deletion of p38α is a
serious defect in lung homeostasis. In particular, p38α inactivation results in increased
proliferation and defective differentiation of lung stem/progenitor cells both in vivo and
in vitro. We found that p38α positively regulates factors, such as C/EBP, required for
lung cell differentiation. In addition, p38α controls self-renewal of the lung stem/progenitor cell population by inhibiting proliferation-inducing signals, most notably EGFR.
As a consequence, the inactivation of p38α leads to an immature and hyperproliferative lung epithelium, which is highly sensitized to K-RasG12V-induced tumorigenesis.
Our results indicate that, by coordinating proliferation and differentiation signals in lung
stem/progenitor cells, p38α plays a key role in the regulation of lung cell renewal and
tumorigenesis.
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Identification of biomarkers that predict therapy response in cancer.
René Bernards
Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam,
The Netherlands
Unresponsiveness to therapy is remains a significant problem in the treatment of cancer, also with the new classes of targeted therapeutics. In my laboratory, we use functional genetic approached identify biomarkers that can be used to predict responsiveness to clinically-relevant cancer therapeutics. We focus on the well-established breast
cancer drug trastuzumab (Herceptin) and the lung and colorectal cancer drug Gefitinib (Iressa). These drugs have in common that they target a specific molecule that is
either over-expressed or mutated in cancer. Nevertheless, it remains poorly explained
why a significant number of tumors, which express the drug target, do not respond to
the therapy. We aim to elucidate the molecular pathways that contribute to unresponsiveness to targeted cancer therapeutics using a functional genetic approach. This
will yield biomarkers that can be used to predict how individual patients will respond
to specific drugs. Furthermore, this work may allow the development of drugs that act
in synergy with the established drug in the treatment of cancer.
To identify biomarkers that control tumor cell responsiveness to cancer therapeutics,
we use both genome-wide gain-of-function genetic screens (with cDNA expression libraries) and genome wide loss-of-function genetic screens (with RNA interference libraries) in cancer cells that are sensitive to the drug-of-interest. We search for genes
whose over-expression or whose down-regulation in cultured cancer cells confers resistance to the drug-of-interest. Once we have identified such genes, we ask if their
expression is correlated with clinical resistance to the drug-of-interest by using tumor
samples of cancer patients treated with the drug in question, whose response to therapy is documented.
Examples of gain-of-function genetic screens to identify mechanisms of resistance to
Histone Deacetylase Inhibitors and loss-of-function genetic screens to identify mechanisms of resistance to Herceptin will be discussed.
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Cancer Epigenetics: from knowledge to therapy
Manel Esteller
Director, Cancer Epigenetics Laboratory, CNIO, Madrid, Spain
We are in an era where the potential exists for deriving comprehensive profiles of
DNA alterations characterizing each form of human cancer. DNA methylation is the
main epigenetic modification in humans. Tumor cells show aberrant methylation of several CpG islands, but global demethylation versus the counterpart normal cells. We
have combined a candidate gene and biochemical approach to determine the overall
aberrant DNA methylation in transformed cells. Our results show that CpG island
promoter hypermethylation has a tumor-type specific pattern, where each gene tends
to be methylated in the cancer cells driven from a particular tissue but not from others.
Epigenetic silencing affects all cellular pathways: DNA repair (hMLH1, MGMT, BRCA1),
cell cycle (p16INK4a, p14ARF, p15INK4b, p73), apoptosis (DAPK, TMS1), hormone receptors
(ER, PR, AR, RARB2, CRBP1), cell adherence (CDH1, TIMP3), detoxifiers (GSTP1) and
many more (APC, LKB1, SOCS-1…). Promoter hypermethylation of particular genes
have important consequences for the biology of that particular tumor. This is for example the case of the DNA repair gene MGMT which methylation-mediated silencing leads to transition mutations, but, at the same time, “marks” those neoplasms that are
going to be more sensitive to the chemotherapy with alkylating drugs. Hypermethylation can be observed in hereditary tumors, where it may account for the “second hit”
of the tumor suppressor gene. We have also developed massive genomic screenings to
find new hypermethylated genes in cancer cell. From these assays we have identified
new candidate tumor suppressor genes with important potential roles in the pathogenesis of human cancer.
Second, we have studied the global methylcytosine content of a large collection of normal tissues and sporadic and hereditary primary tumors. The picture that emerges
shows that 85% of human cancer cells are hypomethylated when compared to the original normal cells. We have also found that the 5-methylcytosine DNA content and the
number of CpG islands hypermethylated in a given tumor is not random, but it involves environmental factors and genetic predisposition.
Overall, our data demonstrates that human tumors suffer a profound, but specific, disturbance in their DNA methylation and chromatin patterns.
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DNA methylation as biomarker for old and new cancer therapies
Robert Brown
Centre for Oncology and Applied Pharmacology, University of Glasgow, Cancer Research UK
Beatson Laboratories, Glasgow, UK
Standard therapy of ovarian cancer patients consists of a platinum agent in combination
with a taxane. Individual variability in outcome and toxicity to these chemotherapy agents
makes the identification of pharmacogenetic markers that can be used to pre-screen
patients prior to therapy selection an attractive prospect. We have been assessing genetic polymorphisms and aberrant CpG island methylation to attempt to identify biomarkers which are associated with toxicity, biological efficacy or clinical outcome.
We assessed 29 polymorphisms in 18 key genes from pathways that may influence cellular sensitivity to taxanes and platinum in PBMC DNA from 914 ovarian cancer patients,
but no significant associations between genotype and outcome or toxicity were found for
any of the genes analysed. Also, we have determined the methylation frequencies of 24
CpG-islands of genes associated with DNA damage responses in 106 stage III/IV epithelial ovarian tumours. Methylation of at least one of the group of genes involved in DNA
repair / drug detoxification (BRCA1, GSTP1, MGMT) was associated with improved response to chemotherapy. Currently we are using an unbiased discovery approach of differential methylation hybridisation to identify aberrant methylation of further novel loci
which may be associated with survival of patients following chemotherapy.
Unlike DNA mutations, which are inherited passively through DNA replication, epimutations require an active mechanism for their maintenance during cell proliferation.
They are therefore, amenable to pharmacological manipulation by small molecule inhibitors. Hypomethylating agents such as 2’-deoxy-5-azacytidine (decitabine) have been
examined in clinical trials of solid tumours, but with disappointing results. However,
many of these studies have used the hypomethylating agents at their maximum tolerated dose, rather than their maximum biologically effective dose based on pharmacodynamic responses of biomarkers. Within a Phase I trial of the combination of decitabine and carboplatin we have shown a good correlation between plasma levels of
decitabine and demethylation in PBMCs and are now using these biological responses
to guide the dose and schedule of these combinations in further clinical studies.
Chemotherapy can exert a selective pressure on epigenetically silenced drug sensitivity
genes present in subpopulations of cells, leading to acquired chemoresistance. Patterns
of acquired aberrant DNA methylation, particularly from tumour DNA released into accessible body fluids, may help to identify those patients who will particularly benefit from
epigenetic therapies such as hypomethylating agents at the time of relapse and treatment
failure. We have previously shown that methylation of hMLH1 detected in plasma DNA
is increased at relapse after carboplatin/taxoid chemotherapy, with 25% (34/138) of relapse samples having hMLH1 methylation which is not detected in matched pre-chemotherapy plasma samples. Acquisition of hMLH1 methylation in plasma DNA at relapse
predicts poor overall survival of patients, independent from time to progression and age
(HR1.99, 95%ci 1.20-3.30 p=0.007). Currently we are examining further methylation biomakers in plasma DNA to aid early detection and prognosis of ovarian cancer.
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Decrypting apl pathogenesis through therapy response
Hugues de Thé, Jun Zhu, Valérie Lallemand-Breitenbach, Marie-Claude Guillemin,
Jun Zhou, Dominique Vitoux, Dima Kamashev, Rihab Nasr.
Hôpital St. Louis, Paris, France
Expression of the PML/RARA fusion underlies pathogenesis of acute promyelocytic leukemia (APL), as well as its clinical response to retinoic acid (RA) and arsenic trioxide.
Both agents target PML/RARA for proteasome-mediated degradation, RA targeting the
RARA moiety of the fusion, while arsenic targets its PML part. Arsenic-induced degradation involves enhancement of sumolation on a specific lysine K160 in PML or PML/RARA.
While previous models of PML/RARA oncogenesis were exclusively focused on enforced RARA dimerisation and enhanced corepressor recruitment, the K160 sumolation site
was paradoxically shown to be absolutely required for the APL-specific differentiation
block and leukemogenesis in vivo (1). Ex vivo studies suggest that the function provided
by this sumolation site is transcriptional repression, providing a clue as to why PML, rather than any self-dimerizing protein, is the recurrent translocation partner of RARA.
Activation of cAMP signaling can trigger differentiation of APL cells both ex vivo and in
vivo and greatly enhances the differentiation mediated by RA or arsenic (2). Cyclic AMP
can also reverse RA-resistance conferred by a point mutation in PML/RARA (3). Only
the combination of RA and cAMP activates PML/RARA-dependent transcription in a
RA-resistant leukemia, directly demonstrating a functional cooperation of these two
signaling molecules on PML/RARA (3). Activation of cAMP signaling, which can be achieved in patients, therefore constitute a third PML/RARA-targeted therapy.
Enforced RARA dimerization induces not only the tighter binding of corepressors, but
also a dramatic extension of the repertoire of DNA-binding sites and hence of target
genes (3). Assessing the role of RARA dimer formation in immortalization of mouse
hematopoietic progenitors, we have shown that dimerization-induced extension of the
target gene repertoire is absolutely essential for transformation, demonstrating how
translocation-induced dimerization transforms an unessential transcription factor into
a dominant oncogene (4).
1. Zhu J, Zhou J, Peres L, et al. A sumoylation site in PML/RARA is essential for leukemic transformation. Cancer Cell 2005;7(2):143-53.
2. Guillemin MC, Raffoux E, Vitoux D, et al. In Vivo Activation of cAMP Signaling Induces Growth Arrest and Differentiation in Acute Promyelocytic Leukemia. J Exp Med
2002;196(10):1373-80.
3. Kamashev DE, Vitoux D, De Thé H. PML/RARA-RXR oligomers mediate retinoidand rexinoid- /cAMP in APL cell differentiation. J Exp Med 2004;199:1-13.
4. Zhou J, Peres L, Honore N, Nasr R, Zhu J, de The H. Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization. Proc Natl Acad Sci U S A 2006;103(24):9238-43.
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Epigenetic therapy of leukemia
Guillermo Garcia-Manero
Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, USA
Epigenetic repression of gene expression is a common feature of human leukemia. Two
epigenetic alterations are currently of clinical interest: aberrant DNA methylation of
promoter associated CpG islands; and biochemical modifications of histone code, in
particular histone H3/H4 acetylation. From a prognostic perspective, work by several
groups has demonstrated that profiling of the methylation status of multiple CpG islands allows the segregation of groups of patients with leukemia and different prognostic risks. Furthermore, aberrant DNA methylation patterns are stable at the time
of relapse, an residual methylation can be detected in patients in morphological remission but at high risk for relapse. This information is allowing the development of
new methylation-based assays for minimal residual disease detection. From a therapeutic perspective, there is a significant interest in developing new agents with the capacity to reverse aberrant epigenetic alterations. These includes drugs with hypomethylating activity and histone deacetylase (HDAC) inhibitors. Two hypomethylating
agents, 5-azacitidine and 5-aza-2’-deoxycitidine (decitabine), are currently approved in
the US for patients with myelodysplastic syndrome (MDS). Several HDAC inhibitors
are currently being evaluated in multiple clinical trials in leukemia, lymphoma and solid tumors. Based on preclinical models, it is now accepted that optimal clinical activity in leukemia and MDS is observed when the hypomethylating agent is used at lower doses. We have tested this concept using decitabine in a series of sequential phase
I and randomized phase II studies. The results of these studies will be presented. In
parallel with this work, we have also performed a number of phase I clinical trials with
different HDAC inhibitors in leukemia. Our experience with vorinostat, a panHDAC
inhibitors, and MGCD0103, a class 1 selective inhibitor, indicates that this class of drugs
is safe and active in leukemia. This data will also be presented. In vitro the combination of a hypomethylating agent and a HDAC inhibitor has synergistic antileukemia activity. We have tested this concept using valproic acid (VPA) as the HDAC inhibitor. In
two consecutive studies, we have demonstrated that the combination of decitabine and
VPA or 5-azacitidine with VPA and all-trans retinoic acid are associated with complete remission rates close to 50% in both studies in older patients with advance acute
myelogenous leukemia or high-risk MDS. Importantly, the administration of these combinations is well tolerated and associated with a very low early induction death rate,
making them a very attractive alternative to intensive chemotherapy in this age group.
Recent data from cellular systems is now emerging that suggests that specific methylation patterns may predict response to epigenetic therapy, in the future this type of
information may allow the targeted use of this type of therapy in leukemia. In summary, the understanding of epigenetic alterations in leukemia is having a profound impact by allowing us to develop new molecular classifications of the disease and therapeutic strategies.
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Vorinostat for the treatment of cutaneous T cell lymphoma
Stanley R. Frankel
Merck Research Labs, North Wales, USA
Introduction: Vorinostat (formerly known as suberoylanilide hydoxamic acid, SAHA; ZOLINZA™) is a hydroxamic acid and is an orally active, potent inhibitor of both Class
I (HDAC 1 and 3) and Class II (HDAC 6)HDACs. Antitumor activity in vitro and in
vivo, increase in acetylation of both histones and non-histone proteins, and subsequent
cell cycle arrest, differentiation and apoptosis have been observed.
Methods: Vorinostat was tested in 13 phase I or phase II clinical trials in over 350 patients at doses ranging from 200-900 mg per day—administered either once, twice or
three times per day, either continuously or on intermittent schedules. In two studies
of 107 CTCL patients, cutaneous responses, pruritus, and safety were assessed. In the
pivotal study, response was measured by a rigorous severity weighted assessment tool
score and supported by annotated body diagrams and digital photography. Responding
patients also were evaluated by CT scan. Pruritus was evaluated at each visit on a 010 scale. Response required a 50% decrease in SWAT score maintained for a minimum
of 4 weeks. Pruritus response required a decrease of 3 or more points maintained for
a minimum of 4 weeks.
Results: In the pivotal CTCL study, 74 patients were enrolled, including 61 with ≥ stage IIB disease. Patients received vorinostat 400 mg p.o. daily until disease progression
or intolerable toxicity. The ORR was 29.7% overall; 29.5% in ≥ stage IIB patients. Median TTR in patients with ≥ stage IIB was 56 days. DOR was not reached but ≥ 185
days (34+ to 441+). Median TTP was 4.9 months overall, and 9.8+ months for ≥ stage IIB responders. Overall, 32% of patients had pruritus relief. Twelve patients have continued on treatment for more than one year. The most common adverse events reactions (incidence ≥20%) are diarrhea, fatigue, nausea, thrombocytopenia, anorexia and
dysgeusia. No adverse event occurred at grade 3 or higher severity in more than 6%
of patients. Less than 25% of patients had either dose interruption, dose reduction, or
discontinuation due to adverse events. The most common serious adverse event was
pulmonary embolism in 4% of patients.
Discussion: Vorinostat is safe and effective for the treatment of the cutaneous manifestations in patients with CTCL who have progressive, persistent or recurrent
disease on or following two systemic therapies. This study supported approval for this
indication by the US FDA in October 2006. Vorinostat continues to be evaluated both
alone and with other classes of anti-cancer therapies in other cancers. Vorinostat may
have clinical activity in other lymphomas, mesothelioma, lung cancer, acute myeloid leukemia, and multiple myeloma.
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The emerging cancer “methylome” in primary tumors and cell
lines
Mohammad Obaidul Hoque, Myoung Soo Kim, Kimberly Ostrow, Junwei Liu1,
G. Bea A. Wisman, Wim Van Criekinge, David Sidransky
Johns Hopkins University School of Medicine, Baltimore, USA
Identification of all gene promoters methylated in cancer cells “the cancer methylome”
would greatly advance our understanding of gene regulatory networks in tumorigenesis. We describe a robust approach that couples probabilistic search algorithms in the
entire human genome with an established pharmacologic unmasking strategy in cancer
cell lines for unbiased and precise global localization of tumor-specific methylated genes. Through this approach, we identified a set of 200 candidate genes that cluster
throughout the genome of which 25 were previously reported as harboring cancer
specific promoter methylation. The remaining genes 175 were tested for promoter
methylation by bisulfite sequencing (BS) or methylation-specific PCR (MSP). 82 of
175(47%) genes were found to be methylated in cell lines and 53 of these 82 genes
(65%) were methylated in primary tumor tissues. From these 53 genes, cancer specific methylation (methylation in neoplastic but absent in normal tissue) was identified
in 28 genes (28/53) (53%). Furthermore, we tested 8 of the 28 newly identified cancer specific methylated genes with quantitative methylation specific PCR (QMSP) in a
panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large cluster of genes with cancer-specific methylation supports the hypothesis that methylation is occurring non-stochastically and that “methylation proneness”
is at least partially encoded in the primary genomic sequence.
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Molecular pathways of lung carcinogenesis as potential markers
for targeted therapy.
Montserrat Sanchez-Cespedes
Lung Cancer Group, Spanish National Cancer Centre (CNIO), Madrid, Spain
Lung cancer is currently the leading cause of cancer-related death in many countries.
Most lung tumors are attributed to cigarette smoking, for which reason special efforts
are being made to prevent initiation and encourage cessation of this habit. During the
past two decades, considerable research has been devoted to the identification of the
genes that drive lung cancer development. The complete catalogue of such genes and
the understanding of their biological functions will undoubtedly guide the design of future lung cancer treatments.
The genes that are widely acknowledged to contribute to lung cancer development include those encoding growth factor receptors, and molecules implicated in signal transduction (i.e. BRAF, EGFR, ErbB2, KRAS, LKB1, PIK3CA, PTEN), transcription factors (i.e.
MYC, P53), and cell cycle regulators (i.e. P16, RB). In lung cancer, EGFR inhibitors have
proven their effectiveness in specific therapies within selected groups of patients. Moreover, therapeutic agents against some of these proteins and downstream targets thereof are being developed or already in clinical trials.
According to the frequency of inactivating mutations, a key tumor suppressor gene in
non-small cell lung cancer is LKB1, mutated in 30-50% of lung tumors. LKB1 encodes
a serine/threonine kinase that activates the AMP-activated protein kinase (AMPK). This
promotes catabolism and inhibits mTOR signalling through TSC2. We report here that
LKB1-mutant lung cancer cells are deficient for AMPK activity and refractory to mTOR
inhibition upon glucose depletion but not growth-factor deprivation. The requirement
for wild-type LKB1 to properly activate AMPK is further demonstrated in genetically
modified lung cancer cells and in LKB1-deficient lung primary tumours with diminished
AMPK activity. LKB1 wild-type cells are more resistant to cell death upon glucose withdrawal than their mutant counterparts. Finally, our data also indicates that modulation
of AMPK activity did not affect PI3K/AKT signalling, an advantage for the potential use
of AMPK as a target for cancer therapy in LKB1 wild-type tumours. Thus, sustained
abrogation of cell energetic checkpoint control, through alterations at key genes, appear to be an obligatory step in the development of some lung tumors and thus, may
be a suitable target for therapeutic intervention.
The search for oncogenes is becoming increasingly important in cancer genetics as
they constitute targets for therapeutic intervention, in addition to the common proto-oncogene activation through gene amplification. To identify novel oncogenes altered
by gene amplification in lung cancer we performed high-resolution CGH (Comparative
Genome Hybridization) analysis on cDNA microarrays and compared DNA copy number and mRNA expression levels in lung tumors. Several candidate oncogenes have
been identified and are currently being tested for their ability to modify cell growth
and their relevance for lung carcinogenesis.
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Aromatase inhibitors, polymorphisms and breast cancer treatment
Ramon Colomer
Catalan Institute of Oncology, Girona, Spain
Purpose: To evaluate the efficacy of treatment with the aromatase inhibitor letrozole in breast cancer patients segregated with respect to DNA polymorphisms of the
aromatase gene CYP19
Patients and methods: Postmenopausal patients (n=67) with hormone receptor-positive metastatic breast cancer were treated with the aromatase inhibitor letrozole.
PCR allelic discrimination was used to examine three single nucleotide polymorphisms
(SNPs) in DNA obtained from breast carcinoma tissue. Two SNPs analyzed (rs 10046
and rs 4646) were located in the 3’ untranslated region (UTR) and one (rs 727479)
in the intron of the aromatase CYP19 gene. The primary endpoint of treatment efficacy was time to progression (TTP).
Results: Median age was 62 years and median number of metastatic sites was 2. Of
the 67 patients, 65 were evaluable for efficacy. Median TTP was 12.1 months. Observed allelic SNP frequencies were: rs10046 = 71%, rs4646 = 46%, rs727479 = 63%. We
observed no relationship between TTP and the rs10046 or rs727479 variants. In contrast, we found that TTP was significantly improved in patients with the rs4646 variant,
compared to the wild type gene (17.2 vs. 6.4 months; p=0.02).
Conclusion: In patients with hormone receptor-positive metastatic breast cancer treated with the aromatase inhibitor letrozole, the presence of a single nucleotide polymorphism in the 3’UTR of the CYP19 aromatase gene is associated with improved
treatment efficacy. Testing for the CYP19 rs4646 SNP may be useful as a prognostic
marker and as a tool for selection of breast cancer patients for anti-aromatase therapy.
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Gene expression profile-based predictors of response to
chemotherapy.
Lajos Pusztai
D.Phil UT M.D. Anderson Cancer Center, Houston, USA
A molecular test that could help selecting the most effective chemotherapy for a particular individual could save patients from unnecessary toxicity and the right choice of
drugs may save lives particularly in the adjuvant treatment of breast cancer. Administration of chemotherapy before surgery provides an attractive opportunity to discover predictors of response (1). Pathologic complete eradication of cancer from the
breast and lymph nodes (pCR) represents an extreme form of chemotherapy sensitivity and invariable heralds excellent long-term survival. We adopted pCR as an early
surrogate of clinically meaningful benefit from therapy and as an outcome that is worth
predicting. There are simple clinical and histological parameters including, grade, ER-status, tumor size that can be combined into powerful prediction scores (2). However,
these clinical variables do not yield treatment regimen-specific predictions and cannot
be used to select one therapy over another. Assessment of traditional single gene markers of chemotherapy sensitivity has not yet resulted in clinically useful tests. Gene expression profiling that enables simultaneous measurement of thousands of genes represents a promising new tool that may be applied to this clinical problem. It is currently
unknown what the best strategy is to discover response predictors from high dimensional gene expression data. The simplest approach may be to search for differentially
expressed genes between responders and non-responders and combine these into a
multi-gene prediction score (3,4). These genes may also lead to new mechanistic insights into the biology of chemotherapy response (5). Another approach is to recognize the different molecular subtypes of breast cancer and attempt to develop distinct
predictors for each subtype (6). This approach assumes that by focusing on the molecularly more homogenous subgroups, more accurate predictors could be developed
than by analyzing all breast cancers together. We will present results from our own research program illustrating the success and limitations of each of these approaches.
1. Pusztai L, Rouzier R, Wagner P, Symmans WF. Individualizing chemotherapy treatment
for breast cancer: is it necessary, can it be done? Drug Resistance Updates 7:325-331,
2005.
2. Rouzier R, Pusztai L, Delaloge S, Gonzalez-Angulo AM, Andre F, Hess KR, Buzdar
AU, Garbay JR, Spielmann M, Mathieu MC, Symmans WF, Wagner P, Atallah D, Valero V,
Berry DA, Hortobagyi GN. Nomograms to predict pathologic complete response and
metastasis-free survival after preoperative chemotherapy for breast cancer. J Clin Onc
23:8331-8339, 2005.
3. Hess KR, Anderson K, Symmans W, Valero V, Ibrahim N, Mejia JA, Booser D, Theriault
RL, Buzdar AU, Dempsey PJ, Rouzier R, Sneige N, Ross JS, Vidaurre T, Gomez HL, Hortobagyi GN, Pusztai L. Pharmacogenomic predictor of sensitivity to preoperative che-
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motherapy with paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide in breast cancer. J Clin Oncol 24:4236-4244, 2006.
4. Anderson K, Hess KR, Kapoor M, Tirrell S, Courtemanche J, Wang B, Wu Y, Gong Y,
Hortobagyi GN, Symmans WF, Pusztai L. Reproducibility of gene expression signature based predictions in replicate experiments. Clin Cancer Res 12(6):1721-1727, 2006.
5. Rouzier R, Rajan R, Hess KR, Gold D, Stec J, Ayers M, Ross JS, Zhang P, Wagner P,
Buchholz TA, Kuerer H, Buzdar AU, Hortobagyi GN, Symmans WF, Pusztai L. Microtubule associated protein tau is a predictive marker and modulator of response to paclitaxel-containing preoperative chemotherapy in breast cancer. Natl Acad Sci USA
102(23):8315-8320, 2005.
6. Rouzier R, Anderson K, Hess KR, James S, Ayers M, Gold D, Ross JS, Wagner P, Meric-Bernstam F, Valero V, Symmans F, Perou CM, Hortobagyi GN, Pusztai L. Basal and
luminal types of breast cancer defined by gene expression patterns respond differently
to neoadjuvant chemotherapy. Clin Cancer Res 11(16):5678-5685, 2005.
Notes
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Applicability of pharmacogenetics in cancer therapy: an overview
Anna Gonzalez-Neira
Genotyping Unit, Spanish National Cancer Centre, Madrid, Spain
Pharmacogenetics is the study of variation in patient responses to drugs due to hereditary traits, which is suggested as the area of genetics with the most potential to provide public health benefits. The main objective is the improvement of drug safety and
efficacy in order to implement “tailor made” or “personalized” medicine, specially, in
cancer drugs where there is usually a fine line between a dosage that has a therapeutic
effect and on that produces toxicity.
Single nucleotide polymorphism (SNPs) account for 90 per cent of the variability and
are observed one every 500-1000 base pairs. The focus of pharmacogenetics has largely been on this type of biallelic markers, not only because they are the most common, but also because they are the most technically accessible class of genetic variation. The new generation of high throughput genotyping technologies is allowing to
undertake more ambitious projects in order to identify this genetic variability between patients. The characterization of a large number of genetic polymorphism (biomarkers) in drug transporters, drug metabolizing enzymes, drug receptors and drug targets has provided knowledge about the mechanisms of interindividual differences in
anticancer drug response in terms of both toxicity and efficacy. These molecular markers that are likely to affect the outcomes of anticancer drug therapy could be integrated into the routine care to allow the choice of the right drug for the right patient
in the right disease at the right dose.
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Pharmacogenetics of cytochromes p450 in oncology
Cristina Rodríguez-Antona
Hereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National
Cancer Center (CNIO), Madrid, Spain
The inter-individual variability in the efficacy and toxicity of drugs has crucial implications in Oncology. The survival of cancer patients being much influenced by the drugs
efficacy and by the frequent severe/fatal toxicities caused by the anticancer drugs, usually
with narrow therapeutic indexes. In this context, Pharmacogenetics aims to individualize pharmacotherapy by the identification of DNA inter-individual variations and/or
gene expression profiles able to predict the drug response of the patient, which could
be assayed before therapy rendering it safer and more efficient.
Cytochromes P450 (CYPs) are key enzymes for the hepatic biotransformation and elimination of drugs. The highly polymorphic nature of these enzymes impacts the pharmacokinetics of drugs and is able to influence the clinical outcome of pharmacotherapy, both in terms of adverse drug reactions and efficacy. This is illustrated by some
therapeutic drugs already labelled for CYP genetic testing. In addition to the inter-individual CYP variation, cancer cells can somatically alter CYP expression, and an increased tumoral expression of a drug inactivating enzyme can result in resistance to
the drug. Thus, we show that the tumoral expression levels of CYP3A4, which inactivates drugs used in peripheral T-cell lymphomas (PTCLs) standard chemotherapy, are
inversely associated with the survival of PTCL patients. FISH and MPA data indicate
that changes in CYP3A4 copy number could be the tumoral mechanism underlying the
overexpression of CYP3A4 in the cancer cells. On the other hand, CYPs with a tumor-specific expression could represent novel cancer targets by activation of cytotoxic pro-drugs specifically within the tumor cells. In conclusion, activity alterations in
drug metabolizing enzymes, and specifically in CYPs, through changes in drug hepatic
disposition and in drug metabolism in cancer cells can modify drug response.
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Expression profiles for novel therapies in breast cancer: the model
of the heredo-familial tumors
Marco A. Pierotti
Scientific Director, IRCCS Foundation, Istituto Nazionale dei Tumori – Milan, Italy
The molecular characterizations of the five to ten per cent of breast carcinomas with
a hereditary origin have revealed that some of them display germ-line mutations in the
BRCA1 and BRCA2 susceptibility genes. However, the majority of the cases fulfilling the
criteria of a genetic, or familial, predisposition are due to genes still unknown. They have
been therefore classified as BRCAX. Since the mutation of a cancer-associated gene is
assumed to change the expression pattern of several other genes, an analysis of the
gene expression profiles in this peculiar type of tumors is expected to provide invaluable information on their biology and sub-classification. Moreover, a new class of small
non-coding RNAs that modulate the expression of target mRNAs has been discovered.
These molecules, named MiRNAs, may have potential as a therapeutic target to control
cancer growth. In this context we integrated mRNA profile with a MiRNA analysis on
the same cases to identify MiRNA dependent mechanisms involved in these tumors.
We studied 48 familial breast cancer cases consisting in 17 samples from patients characterized by carrying germ-line mutations of BRCA1 gene, 11 samples with BRCA2
mutations and 20 samples from BRCAX patients, respectively. As a control, 12 tumors
from patients with no family history of breast cancer were similarly analyzed.
We identified genes differentially expressed between the BRCA groups by means of a
linear model that included, and therefore adjusted for, the ER status, to avoid the influence of the latter in the observed clusters. We found 80 genes modulated between
groups (F test P value<0.005) and reduced the differences in expression between
BRCA1 and the other classes.
BRCAX cases cluster into two distinct sub-groups, one mixed with BRCA2 and sporadic
cases, the other very tight to BRCA1 cases. The distinct biological identity of the two subgroups is further supported by the fact that when re-clustering the samples with the 33
genes common between our and Hedenfalk et al’s classifier which defines BRCAX subgroups (PNAS, 2003; 100: 2532) we perfectly reproduced the same structure. Clinical characteristics of the tumors show that those belonging to the group with only BRCAX cases are similar to BRCA1 tumors. Samples mixed to BRCA2 and sporadic cases are more
heterogeneous, suggesting a possible involvement of low penetrance genes.
Analysis of the same cases by miRNA expression profile highlighted that miRNA deregulation is a frequent event in sporadic breast cancer and distinguishes it from the
heredo-familial cases. We were also able to identify MiRNAs that were differentially
expressed between the two BRCAX sub-groups.
We are now integrating gene expression and miRNA data to identify the targets of
the miRNAs found as differentially expressed in the groups in analysis, which could
have a potential therapeutic application.
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Notes
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1
Transcriptome analysis of the major types of B-cell Non-Hodgkin
Lymphoma
Mohit Aggarwal1, Raquel Villuendas1, Fatima Al-Sharour3, Abel Sanchez-Aguilera1,
Nerea Martínez1, Elena Ruiz-Ballesteros2, Francisca I. Camacho1, Alberto Pérez1,
Paloma de la Cueva1, María Jesús Artiga1, Jose A.Garcia-Marco4, Joaquín Dopazo3,
Miguel A. Piris1
1. Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid,
Spain
2. Genetics and Pathology Departments, Hospital Virgen de Salud, Toledo, Spain
3. Prince Felipe Research Centre, Valencia, Spain
4. Hematology Department, Hospital Universitario Puerta de Hierro, Madrid, Spain
B-cell lymphomas are presently diagnosed according to the WHO criteria based on
morphologic, immunophenotype and cytogenetic findings. However, the precise distinction among common lymphoma types is frequently a difficult task, and there are
areas of overlapping and heterogeneity between them.
Here we have analyzed whether gene expression profiling (GEP) data, solely considered, could be used to validate the currently used B-cell lymphoma classification, or
proposing new lymphoma types, and for identifying functional signatures or genes defining these GEP-based lymphoma classification.
To this aim, we collected Gene Expression Profiling (GEP) for 173 cases of B-cell NHL,
including BL (9), DLBCL (36), MALT (3), MCL (20), CLL (38), FL (33), MZL (6) and
SMZL (29). The gene expression data for lymphoma cases was normalized against an
average gene expression of reactive lymph nodes, except the SMZL which was normalized against normal spleen (3 cases).
The functional signatures that were identified as distinguishing between these lymphoma types were defining cell cycle, apoptosis, cytokine-cytokine receptor interaction, Tcell receptor, B-cell receptor, cell adhesion, NF-kB activation, and other significant interactions. Moreover these signatures reveal sub-classes for diagnosed lymphoma types,
suggesting the existence of a distinct functional heterogeneity among CLL, MCL and
DLBCL. Comparison between these lymphoma clusters following this definition yielded large number of genes distinguishing them, this list including already known genes
and a number of new potential markers and therapeutic targets.
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2
Quantitative hypermethylation of a small panel of genes augments the
diagnostic accuracy in fine-needle aspirate washings of breast lesions
Carmen Jerónimo1,4,5, Paula Monteiro2, Rui Henrique2,5, Mário Dinis-Ribeiro3,
Vera L. Costa1, Luísa Filipe1, André L. Carvalho6, Mohammad O. Hoque6, Irene Pais2,
Isabel Costa2, Conceição Leal2, Manuel R. Teixeira1,5, and David Sidransky6
1. Departments of Genetics, 2. Pathology and 3. Gastroenterology, Portuguese Oncology
Institute, Porto, Portugal
4. Bioengineering and PharmacoClinical Research Group; Fernando Pessoa University School
of Health Sciences, Porto, Portugal
5. Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences
Abel Salazar, University of Porto, Portugal
6. Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer
Research Division, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Purpose: Promoter methylation of cancer-related genes is a common event in breast
cancer. We hypothesized that a comprehensive breast cancer methylation profile might
provide clinically useful cancer biomarkers. Because fine needle aspiration (FNA) is widely used in diagnostic assessment of breast lesions, we tested whether quantitative
hypermethylation analysis of a panel of genes would distinguish benign from malignant
breast FNA washings.
Experimental design: Twenty-three gene promoters were surveyed by quantitative methylation-specific PCR (QMSP) in bisulfite-modified DNA from 66 primary breast carcinomas
(BCa), 31 fibroadenomas (FB) and 12 normal breast tissues (NT) to define a set of genes
differentially expressed in malignant and non-malignant breast tissues. Then, this set of genes was tested in a series of paired FNA washings obtained pre-operatively from some of
those patients (66 malignant and 12 benign). Receiver operator characteristic (ROC) curves was used to identify a gene panel which might distinguish between cancer and noncancerous lesions. Finally, this panel was validated in an independent series of FNA washings (45 cases) in which cytomorphology was unable to reach definitive diagnosis.
Results: In tissue samples, 14-3-3- , DAPK, CCND2, RASSF1A, CALCA, APC, HIN-1, RAR 2,
TIG1, and GSTP1 methylation levels differed significantly among BCa, FB, and NT. ROC
curve analysis identified a panel of 4 gene loci (CCND2, RASSF1A, APC, and HIN-1) which
discriminated BCa from benign lesions in a set of 78 FNA washings from histologically
confirmed breast lesions. When this panel was tested in 45 FNA washings in which a
definitive cytomorphological diagnosis could not be rendered, breast cancer was identified with 67% sensitivity and 78% specificity, when 2 out of 4 gene loci were hypermethylated, providing estimated added information of 58% over cytomorphology alone.
Conclusions: Our data provide evidence that multigene methylation analysis (i.e., CCND2,
RASSF1A, APC, and HIN-1) augments diagnostic accuracy of cytological assessment of suspicious breast lesions, and might be a valuable ancillary tool for breast cancer diagnosis.
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3
Hypermethylation of Tumor Supressor Genes in Astrocytomas
Aiala Lorente1, Paula Lázcoz1, Wolf Mueller2, Edurne Urdangarín1, Andreas von
Deimling2, Javier S. Castresana1
1. Brain Tumor Biology Unit, University of Navarra, Pamplona, Spain
2. Institute for Neuropathology, Charité, Berlin, Germany
Introduction: Astrocytomas constitute the most common type of brain tumors. They
mainly develop in adults and are characterized by diffuse invasion of adjacent brain tissues. WHO classifies them from grade I to grade IV according to their malignancy.
Hypermethylation of CpG islands in gene promoter regions can lead to gene silencing.
Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes (TSG). The following TSGs have been selected for this study: RASSF1A
(3p21.3), NORE1A (1q32.1) and BLU (3p21.3), which have been shown to be downregulated by hypermethylation, and PTEN (10q23.3) and MGMT (10q26.1) that are located in an area commonly deleted in astrocytomas
Material and methods: Fifty three astroytoma samples and 10 glioma cell lines have
been analysed (LN-405, CCF-STTG1, U87MG, SW1088, SW1783, GOS3, A172, MOGG-CCM, T98G, U118). Gene expression levels were assessed by RT-PCR. Bisulfite sequencing and MSP were performed to detect hypermethylation. Treatments with 1 M
5-Aza-2-deoxicitidine (5´-Aza-dC) for 3 days were applied to restore gene expression
in cell lines.
Results and conclusions: RASSF1A, NORE1A and MGMT genes were re-expressed after the 5´-Aza-dC treatments, associated with promoter hypermethylation. 92% of tumor samples were methylated for RASSF1A gene and 47% for MGMT, respectively, suggesting their promoter hypermethylation to be a common event in the tumorigenesis
of astrocytomas. Only 4% of the tumors revealed a hypermethylated promoter for
NORE1A. No association between hypermethylation and loss of expression could be
established for BLU and PTEN.
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4
Amphiregulin mediates resistance of glioma cells to cannabinoidinduced apoptosis
Mar Lorente, Arkaitz Carracedo, Francesco Natali, Ainara Egia, Cristina Blázquez,
Manuel Guzmán and Guillermo Velasco
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense
University, Madrid, Spain
Gliomas are one of the most malignant forms of cancer, mostly due to their high resistance to conventional therapies such as chemotherapy and radiation. The identification of the molecular mechanisms responsible for this resistance could be extremely
interesting in order to improve the specificity and efficiency of current therapies. 9Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids have been shown to reduce the growth of several types of tumor xenografts
including gliomas. This anti-tumoral effect of cannabinoids relies, at least in part, on the
ability of these compounds to induce apoptosis of transformed cells.
In order to identify the molecular markers associated with the resistance of tumor
cells to cannabinoid treatment, we analyzed the gene expression profile of two subclones of the rat C6 glioma cell line: C6.9, which is THC-sensitive, and C6.4, which is
THC-resistant. We found differences in the expression of several genes involved in cell
proliferation, differentiation and adhesion. Subcutaneous tumor xenografts generated
from C6.9 and C6.4 glioma cells showed a similar pattern of THC sensitivity and gene
expression profile than cultured cells.
Interestingly, the EGFR ligand amphiregulin (AREG) was found to be highly over-expressed in the cannabinoid-resistant cell line C6.4. Moreover, basal ERK phosphorylation was also increased in these cells both in vitro and in tumor xenografts. In addition, silencing of AREG expression decreased the basal level of ERK phosphorylation
and increased the sensitivity of C6.4 cells to cannabinoid treatment, suggesting that
EGFR activation is associated with the resistance of C6.4 cells to cannabinoid-induced
apoptosis. In line with this notion, reduction of AREG levels increased the expression
of p8 and TRIB3 –two genes that have been previously implicated in the mechanism
of cannabinoid-induced apoptosis.
In summary, our data support that increased AREG levels mediates resistance of glioma cells to cannabinoid treatment, at least in this model, and suggest that inhibition of
the EGFR pathway may help to improve the efficiency of a potential antitumoral therapy with cannabinoids.
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5
Molecular markers in colorectal cancer: differences between
mutator and suppressor phenotypes and clinical applications
Alberto Morán1, Paloma Ortega1, Carmen de Juan1, Cristina Frías1,
Antonio Díaz-López1, Jose-Antonio García-Asenjo2, Andrés Sánchez-Pernaute3,
Antonio Torres3, Pilar Iniesta1, Manuel Benito1
1. Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, UCM, Madrid,
Spain, and Servicios de 2. Anatomía Patológica y 3. Cirugía, Hospital Clínico San Carlos,
Madrid, Spain
Colorectal cancers from the mutator phenotype pathway (MSI-H) display distinctive
pathological features and confer a lesser aggressiveness than colorectal adenocarcinomas originated by the suppressor pathway (MSS/MSI-L). Molecularly, both types of tumour are quite different: mutation profile differs in mutator tumours from suppressor
ones, but also overexpression and downregulation of several molecules are not the
same in both types of tumours.
Our laboratory is really concerned about these molecular differences existing between mutator and suppressor tumours and its possible clinical usefulness. During the last
years we have studied the upregulation, downregulation and mutation of several molecules in relation to the mutator phenotype and their possible value as prognostic or
diagnostic factor.
Overall, our studies demonstrate important differences in adhesion molecules, such as
E-cadherin, proteases (MMP-9 and MMP-3) and proteins related to Wnt pathway such
as beta-catenin, Dvl2, Kremen2, subunit A of the phosphatase 2A, TLE2, casein kinase,
ICAT, FZD2, TLE3 and WNT1.
Among these molecules, the most remarkable findings were: 1) E-cadherin integrity is
maintained in MSI-H tumours versus MSS/MSI-L tumours, where integrity is lost; 2)
MMP-9 expression is higher and its activity is lower in MSI-H tumours than in MSS/MSIL tumours; 3) MMP-3 promoter is mutated in a certain region in mutator tumours but
not in suppressor ones; 4) According to array analysis of the Wnt pathway, the previously named molecules are overexpressed in MSI-H versus MSS/MSI-L. These data are
being now confirmed by real time PCR analysis.
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Acetylcholinesterase Biogenesis is Impaired in Lung Cancer Tissues
P. Martínez-Moreno1,2, Susana Nieto-Cerón1, F. Ruiz-Espejo1, J. Torres-Lanzas3,
I. Tovar-Zapata1, P. Martínez-Hernández1, C.J. Vidal-Moreno2 and J. Cabezas-Herrera1.
1. Unidad de Investigación. Servicio Análisis Clínicos. Hospital Universitario Virgen de la
Arrixaca. El Palmar, Murcia. Spain.
2. Departamento de Bioquímica y Biología Molecular A. Universidad de Murcia, Espinardo,
Murcia, Spain.
3. Servicio de Cirugía Torácica. Hospital Universitario Virgen de la Arrixaca. El Palmar,
Murcia. Spain.
Lung cancer is a leading cause for cancer death worldwide and is projected to cause
2,000,000 deaths per annum by 2020-2030. The lung cancer 5-year survival rate is still
very low (13-15%) and it is essential a better knowledge of the biological processes
accounting for carcinogenesis and tumour progression. Non-neuronal cholinergic systems
are widespread expressed in human tissues. Cholinergic systems are composed of several components, (i) the machinery for acetylcholine synthesis, transport and secretion, (ii) cholinergic receptors, and (iii) the choline-esters hydrolyzing enzymes (ChEs),
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Several findings strongly
support the existence of an autocrine and/or paracrine loop in which ACh may stimulate cell growth.
AChE could modulate the effective level of ACh at the extracellular space. In order to
gain insights into the effect of cancer on human lung AChE, the content and properties of AChE in healthy and malignant specimens have been analysed and the results
show that normal lung contains a significant amount of AChE activity (0.60±0.33 U/mg
protein) which decreases to 0.31±0.21 U/mg protein by cancer. The pattern of AChE
components is not affected by lung cancer, but the oligossacharides moieties of AChE
species differed in normal and cancerous lung, as shown by lectin interaction assays.
Finally, the presence of size differences between AChE subunits in normal and tumour
samples, as well as the occurrence of catalitically inactive AChE in them, have been tested by Western-blot.
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7
The effect of non-selective cyclooxygenase-2 (COX-2) inhibitor:
indomethacin on the response of colorectal cancer (CRC) cell
lines and xenografts on 5-fluorouracil (5-FU) treatment
Andrea Réti, B. Budai, V. Komlósi, V. Adleff, A. Zalatnai1, A. Jeney1, J. Kralovánszky
National Institute of Oncology, (1) 1st Institute of Pathology and Experimental Cancer
Research, Semmelweis University, Budapest, Hungary
Background: COX-2 has an important role in tumor development and its high expression in tumours is an unfavourable prognostic factor, however, the influence of
COX-2 expression levels on tumour response to chemotherapy has been relatively little studied.
Aim of the study: Investigation of the effect of 5-FU combined with the non-selective COX-2 inhibitor, indomethacin (INDO), on HT-29 and HCA-7 human CRC cell lines and on HCA-7 and HT-29 xenografts bearing SCID mice.
Materials and methods: Sulphorhodamine B proliferation assay was used to measure the effect of (48 hours) treatment with 5-FU ± INDO on HT-29 (low COX-2
expression) and HCA-7 (high COX-2 expression) cells. In both cell lines COX-2 expression was characterized with Western blot analysis. COX-2 positivity of HCA-7 xenograft was confirmed with immunohistochemistry. Both, HCA-7 and HT-29 xenograft
bearing SCID mice were treated s.c. with 6 or 30 mg/kg 5-FU for 5 days ± 2.5 mg/kg
INDO for 20 days. Control mice received 0.9% NaCl or 2.5 mg INDO for 20 days.
The tumour volume and weight were measured.
Results: 5-FU+INDO combination compared to 5-FU treatment on HCA-7 cells significantly (p=0.0082) decreased the proliferation rate, while on HT-29 cells there was
no significant difference.
On HCA-7 xenografts the 5-FU+INDO combination compared to the 5-FU treatment
caused a significant decrease of relative tumour volume and weight (p=0.0236 and
p=0.0081, respectively), while this tendency on HT-29 xenografts has not been observed.
Conclusion: INDO enhanced the cell growth inhibition of 5-FU in high COX-2 expressing HCA-7 cell lines and xenografts.
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Production of matrix metalloproteinases by Th-1 cytokineactivated lymphocytes in breast cancer patients
Sandra Stankovic, G. Konjevic, T. Srdic, S. Jezdic, Z. Tomasevic, N. Borojevic
Institute of Oncology and Radiology of Serbia, Belgrade,Serbia
Activation of peripheral blood lymphocytes (PBL) to a locomotive state requires production of proteases regulated by cytokines to invade target tissue. Matrix metalloproteinases (MMPs), aside from being involved in process of angiogenesis and invasion,
participate in transmigration of lymphocytes to the tumor site.
In this study we investigated the activity levels of MMP-2 and MMP-9 released by PBL
and effects of different cytokines on their levels in breast cancer patients and healthy
controls. PBL (5x106/ml) of patients in clinical stage I, II,IV and controls were treated
in vitro with culture medium (CM) alone and with IFN
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Quantitative promoter methylation analysis of multiple
cancer-related genes in kidney neoplasias
Vera L. Costa1, Rui Henrique2,4, Franclim R. Ribeiro1, Mafalda Pinto1, Jorge Oliveira3,
Francisco Lobo3, Manuel R. Teixeira1,4, Carmen Jerónimo1,4,5
Departments of 1. Genetics, 2. Pathology, and 3. Urology, Portuguese Oncology Institute,
Porto, Portugal
4. Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences
Abel Salazar, University of Porto, Portugal
5. Fernando Pessoa University School of Health Sciences, Portugal.
Purpose: Aberrant promoter hypermethylation of several cancer-associated genes occurs frequently during carcinogenesis and is a promising marker for cancer detection.
Renal tumors are genetically, histopathologically, and clinically heterogeneous. Renal cell
carcinomas (RCCs) are curable if organ-confined and surgically resectable, but patients
with advanced disease have a poor prognosis. Because renal tumors are frequently clinically silent, novel strategies for early detection are warranted. The development of
methylation-based markers might provide valuable tools for cancer detection and accurate preoperative diagnosis of suspicious renal lesions. Thus, we aimed at determine
the clinical and pathological value of epigenetic markers through quantitative methylation profiling of the major types of renal cell epithelial neoplasms.
Patients and methods: A panel of 18 gene promoters were assessed by quantitative methylation-specific PCR (QMSP) in 85 primary renal tumors representing the four
major histologic types (52 clear cell (ccRCC), 13 papillary (pRCC), and 10 chromophobe (chRCC) carcinomas and 10 oncocytomas) and 62 paired normal renal tissue
samples, from patients who consecutively underwent radical nephrectomy. Genomic
DNA was isolated and sodium bisulfite modified. Methylation levels were calculated
and correlated with standard clinicopathological parameters.
Results: Significant differences in methylation levels among the four types of renal tumor were found for CDH1 (P=0.0007), PTGS2 (P=0.002), and RASSF1A (P=0.0001).
PTGS2 and RASSF1A methylation levels were significantly higher in ccRCC and pRCC,
respectively, when compared with paired normal tissue (P=0.04 and P=0.035). CDH1
methylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (P=0.00016 and P=0.0034, respectively). In pRCC, CDH1 methylation levels
correlated negatively with tumor stage (P=0.031) and RASSF1A methylation levels were
significantly higher in low nuclear grade tumors (P=0.022).
Conclusions: Major subtypes of renal epithelial neoplasms display differential aberrant
promoter methylation levels at some cancer-related genes. A gene panel that includes
CDH1, PTGS2, and RASSF1A might provide a more accurate discrimination among common renal tumors and serve as a valuable tool for preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.
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Blockade of Hedgehog Pathway signaling inhibits growth of human
hepatocellular carcinoma cell lines.
Lemmer E1, Augusto Villanueva1, Yea S1, Van Laarhoven S1, Di Feo A2, Peix J1,
Schwartz M1, Narla G1, Martignetti J2, Waxman S1, Friedman SL1, Llovet JM1,3
1. Division of Liver Diseases and Liver Cancer Program
2. Human Genetics
3. Mount Sinai School of Medicine, New York, USA. BCLC Group. Liver Unit. IDIBAPS.
Hospital Clinic, Barcelona, Spain
There is currently no first line systemic treatment for advanced hepatocellular carcinoma (HCC), and new therapeutic targets are urgently needed. We recently examined
the expression of key components of the Hedgehog (Hh) pathway in progressive stages of HCV-associated liver cancer, and identified pathway activation in a subgroup of
HCV-cirrhotic HCC patients (J Hepatol 2006;44:S105). Tumor growth in these patients
appears to be driven by Indian Hedgehog (IHH) ligand (≥ two-fold upregulation in 60%
of patients). This was preceded by a progressive loss of Human Hedgehog Interacting
Protein (HHIP) expression (0.03-fold change in advanced HCC), suggesting that silencing of HHIP activity may be a key event during hepatocarcinogenesis.
Aim: Define the role of Hh pathway signaling in human HCC cell lines.
Methods: Expression levels of Hh pathway genes in human HCC cell lines were analyzed by qRT-PCR, and the effects of cyclopamine (a Hh signaling blocker) on growth of
HCC cells were assessed by MTT viability assay, 3H-thymidine incorporation, Gli reporter luciferase assay and flow cytometry.
Results: Untreated Huh-7, Hep-3B and Hep-G2 cells showed upregulation of IHH
(p<0.0001), PTCH (p<0.0001), and GLI oncogene (p<0.0001), together with virtually
complete loss of expression of HHIP (p<0.0001). Cyclopamine [10 M] decreased Hh
pathway activity by 30 – 40% (assessed using a Gli reporter) and also resulted in ~40%
decrease in 3H-thymidine incorporation in Huh-7 cells. Tomatidine 10 M (inactive analogue) had no significant effect. Similarly, MTT assay elicited a 50 – 60% decrease in
viability of HCC cells treated with cyclopamine. Flow cytometry showed no increased
apoptosis .
Conclusion: (1) IHH-driven activation of the Hh pathway occurs in human HCC cell
lines; (2) Blockade of Hh pathway inhibits growth of these cells through an antiproliferative effect; (3) These data indicate that Hh blockade merits evaluation in experimental HCC animal models and possibly clinical trials.
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11
Cutaneous T-cell lymphoma sensitivity to IFN and/or PUVA treatment
is dependent of the expression of a signature of genes associated
with MAPK signaling, T cell receptor signaling and NF-kB activation
Magdalena Wozniak1, Pablo Ortiz2, Lorraine Tracey1, Jose L Rodriguez-Peralto3,
Monica Alvarez4, Miguel A Piris1 and Raquel Villuendas1.
1.
2.
3.
4.
Molecular Pathology Programme, CNIO, Madrid, Spain
Dermatology Department, Hospital 12 de Octubre, Madrid, Spain
Pathology Department, Hospital 12 de Octubre, Madrid, Spain
Pathology Department, Hospital La Paz, Madrid, Spain
Mycosis fungoides (MF) is a low-grade cutaneous T-cell lymphoma, in which malignant
T cell clones (mostly CD4+) arise in the skin from the early disease stages. IFN-a is widely used in the treatment of MF being able to induce a positive response in over 50%
of patients and when used in combination with PUVA both response and its duration
have been reported to be improved. However, in spite of encouraging results of the initial studies, currently there is no information available on specific prognostic factors enabling prediction of IFN-a and/or PUVA treatment response. The aim of the present
study was to find the molecular signature associated with lack of remission and resistance to IFN-a/PUVA treatment. In the present study, the gene expression profile of the
pre-treatment samples from 30 patients subjected to a randomised clinical trial of IFNa and/or PUVA treatment has been analyzed by use of cDNA microarrays. Following
the treatment outcome, the patients have been divided into two groups depending on
the response to IFN-a and/or PUVA and the time of remission. The two groups included: good responders (23 patients that have achieved complete remission in the time
of 24 weeks or less) and bad responders (7 patients that have not acquired completed
remission or have shown progression of the disease during treatment. Four genes associated with cell cycle regulation, tumor microenvironment (antigen presentation and
B cell signaling) have been identified to predict good response by the significance analysis of microarrays correlating expression data with survival time using SAM Stanford tools. Moreover, 38 genes involved in T cell receptor signaling pathway, NF-kB activation
and Jak-Stat signaling have been found to be associated with worse response to treatment by use of the same method. Using SAM, t-statistics and Cox-model a set of significant genes deregulated in the two groups of patients has been found. FDR significance levels were also used when appropriate, for adjusting the associated p-value.
Expression of 26 genes has been found to be significantly associated with remission. Finally, the model of 2 genes distinguishing two groups of MF patients with remission
at 24 weeks of 15%, and 60% (log-rank test, p:0.007) has been generated by use of
FCMS method (Filter, Cluster and Stepwise model selection) provided by SignS, a tool
for gene selection and molecular signature finding. The genes associated with MAPK signaling, T cell signaling pathway and activation of NF-kB survival pathway have been found
to be of importance in prognosis of response to IFN-a and PUVA treatment. A subsequent study on paraffin embedded tissue sections demonstrated that the genes selected were expressed not only by the tumoral cells, but also by specific subpopulations
of macrophages, dendritic cells and other non-neoplastic cell types.
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12
HSP 90 as a novel therapeutic agent in treating Peripheral T cell
Lymphoma
Magdalena Zajac1, Marta Cuadros1, Emiliano Honrado1, Javier Alves Ferreira2,
María Victoria Moneo Ocaña3, Beatriz García-Serelde3, Amancio Carnero3,
Javier Benítez1, Beatriz Martínez-Delgado1.
1. Departamento de Genética Humana. Centro Nacional de Investigaciones Oncológicas
(CNIO), Madrid, Spain
2. Departamento de Patología y Medicina Interna. Fundación Jiménez Díaz. Madrid, Spain
3. Grupo de Desarrollo de Ensayos. Centro Nacional de Investigaciones Oncológicas (CNIO),
Madrid, Spain
Peripheral T cell lymphomas comprise a heterogeneous group of rare diseases accounting for approximately 10-15% of all non-Hodgkin”s lymphomas. To date these tumors remain as a complex entity showing great heterogeneity regarding morphology,
immunophenotype and clinical behaviour. In general PTCL are considered as clinically
aggressive tumors, generally demonstrating a much poorer response to treatment and
a shorter survival than B-cell lymphomas, with less than 30% 5-years overall survival.
Standard therapy for PTCL has not been defined, yet. First line anthracycline-based
chemotherapy has been uniformly disappointing so novel treatment approaches are urgently needed.
Heat shock protein (HSP 90) is a chaperone for several client proteins involved in
transcriptional regulation, signal transduction, and cell cycle control. Inhibition of HSP90
function disrupts the multichaperone HSP complex and paves the way for client proteins to proteasome degradation. Recent findings with gene expression profiling in T
cell lymphomas revealed a correlation between HSP 90 expression and proliferation.
The objective of the study was to determine the role of HSP 90 in promoting growth
and survival of PTCL and to verify the sensitivity of the T cell lymphoma cell lines to
inhibitor of the HSP 90 17-allyloamino-17-demethoxy-geldanamycin (17-AAG).
HSP 90 expression was determined by immunohistochemistry, western blot analysis
and further validated by quantitative real–time PCR (qRT-PCR) on T cell lymphoma patient tumor samples, reactive lymph nodes. On top of that we have included in our
studies T cell lymphoma derived cell lines and stimulated and non-stimulated normal
T lymphocytes as a control.
Cell viability after treating the T cell lymphoma cell lines and normal T lymphocytes
with 17-AAG inhibitor was assessed by the MTT test.
In all T lymphoma derived cell lines (Peer, Karpas 45, Molt 13, Jurkat, Hut78, Karpas
299) the HSP 90 was clearly overexpressed compared to the normal both stimulated
and non-stimulated cultured T cells. Taking T cell lymphoma samples
HSP 90 was moderately to strongly expressed according to IHC. Further evaluation
by qRT-PCR revealed on average 30% of the tumors overexpressing HSP90.
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Abstracts-Posters
With MTT test the T cell lymphoma cell lines showed differential yet higher sensitivity to HSP90 inhibitor compared to normal lymphocytes, what correlates to common
knowledge that 17-AAG has higher affinity to tumoral cells, so preferably inhibits tumor growth.
Our results suggest that selective inhibition of HSP90 may be a favorable target for investigational therapy in PTCL patients and further studies are planned to investigate
molecular effects of the inhibition.
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List of invited speakers and participants
Abstracts-Sessions
with posters and oral presentations
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List of invited speakers and participants with posters and oral presentations
Bharat B. Aggarwal
aggarwal@mdanderson.org
MD Anderson Cancer Center
Texas, USA
Mohit Aggarwal
maggarwall@cnio.es
CNIO
Madrid, Spain
Joan Albanell
JAlbanell@imas.imim.es
Oncology Service
Hospital del Mar, Barcelona, Spain
Alberto Bardelli
a.bardelli@ircc.it
University of Torino Medical School
Torino, Italy
José Baselga
jbaselga@agendeshuvh.cs.vhebron.es
Vall d’ Hebron University Hospital
Barcelona, Spain
René Bernards
r.bernards@nki.nl
Netherlands Cancer Institute
Amsterdam, The Netherlands.
Robert Brown
R.Brown@beatson.gla.ac.uk
Beatson Institute
Glasgow, UK
Ignacio Casal
icasal@cnio.es
CNIO
Madrid, Spain
Dharminder Chauhan
Dharminder_Chauhan@dfci.harvard.edu
Dana-Farber Cancer Institute and
Harvard Medical School
Boston USA
Manuel Collado
mcollado@cnio.es
CNIO
Madrid, Spain
Ramon Colomer
'rcolomer@icogirona.scs.es
Hospital de Girona Dr. Josep Trueta
Girona, Spain
Vera Lúcia Costa
veralmcosta@sapo.pt
Portuguese Oncology Institute
Porto, Portugal
Manel Esteller
mesteller@cnio.es
CNIO
Madrid, Spain
Tito Fojo
tfojo@helix.nih.gov
National Cancer Institute
Bethesda, USA
Stanley Frankel
stanley_frankel@merck.com
M.D., Clinical Oncology, Merck & Co., Inc.
North Wales, USA
Guillermo García-Manero
ggarciam@mdanderson.org
MD Anderson Cancer Center
Texas, USA
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Anna Gonzalez-Neira
agonzalez@cnio.es
CNIO
Madrid, Spain
Manuel Hidalgo
mhidalg1@jhmi.edu
Johns Hopkins University School of Medicine
Baltimore, USA
Carmen Jerónimo
cjeroni@ufp.pt
Portuguese Oncology Institute
Porto, Portugal
Paula Lázcoz
paula.lazcoz@unavarra.es
University of Navarra
Pamplona, Spain
Mar Lorente
lorentem@bbm1.ucm.es
Complutense University
Madrid, Spain
Alberto Morán
amoran@farm.ucm.es
Universidad Complutense de Madrid
Madrid, Spain
Angel Nebreda
anebreda@cnio.es
CNIO
Madrid, Spain
Susana Nieto Cerón
susananc2003@yahoo.es
Hospital Universitario Virgen de la Arrixaca
Murcia, Spain
José Palacios
jose.palacios.sspa@juntadeandalucia.es
CNIO
Madrid, Spain
Luís Paz-Ares
Ontario Cancer Institute at the Princess
Margaret Hospital
Toronto, Canada
lpazares@hotmail.com
Marco A. Pierotti,
marco.pierotti@istitutotumori.mi.it
Istituto Nazionale per lo Studio e la Cura
dei Tumori
Milano, Italy
Miguel Ángel Piris
mapiris@cnio.es
CNIO
Madrid, Spain
Lajos Pusztai
lpusztai@mdanderson.org
UT MD Anderson Cancer Center
Houston, USA
Miguel Quintela-Fandino
quintelamiguel2000@yahoo.es
Ontario Cancer Institute at the Princess
Margaret Hospital
Toronto
Andrea Réti
retiandi@freemail.hu
National Institute of Oncology
Budapest, Hungary
Cristina Rodriguez-Antona
crodriguez@cnio.es
CNIO
Madrid, Spain
Ana María Rojas
arojas@cnio.es
CNIO
Madrid, Spain
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List of invited speakers and participants with posters and oral presentations
Rafael Rosell
rrosell@ico.scs.es
Catalan Institute of Oncology
Barcelona, Spain
Montserrat Sánchez-Céspedes
msanchez@cnio.es
CNIO
Madrid, Spain
David Sidransky
Sidransky
Johns Hopkins University
Baltimore, USA
Maria S. Soengas
soengas@med.umich.edu
University of Michigan Comprehensive
Cancer Center and Department of
Dermatology Medical Center
USA
Jean C Soria
soria@igr.fr
Institut Gustave-Roussy
Paris, France
Sandra Stankovic
stankovics@lycos.com
Institute of Oncology and Radiology of
Serbia
Belgrade, Serbia
Hugues de Thé
dethe@jupiter.chu-stlouis.fr
Université de Paris
France
Guillermo Velasco
gvd@solea.quim.ucm.es
Complutense University
Madrid, Spain.
Agusto Villanueva
augusto.villanueva@mssm.edu
Mount Sinai School of Medicine
New York, USA
Elisabeth G. de Vries,
egedevries@hotmail.com
University of Groningen
The Netherlands
Magdalena Wozniak
mwozniak@cnio.es
CNIO
Madrid, Spain
Magdalena Zajac
mzajac@cnio.es
CNIO
Madrid, Spain
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Forthcoming
Abstracts-Sessions
Activities at the CNIO
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Forthcoming Activities at the CNIO
FORTHCOMING ACTIVITIES AT THE CNIO
CNIO CANCER CONFERENCES (CCCS):
2007
MOLECULAR MECHANISMS IN LYMPHOID NEOPLASM
19/02/2007 - 21/02/2007
ORGANISERS: E. CAMPO (HOSPITAL CLINIC, BARCELONA), R. DALLA
FAVERA (COLUMBIA UNIVERSITY, NEW YORK), E. JAFFE (NCI, BETHESDA),
M. A. PIRIS (CNIO, MADRID)
MYC
TRANSCRIPTIONAL CONTROL OF PROLIFERATION AND ONCOGENESIS
11/06/2007 - 13/06/2007
ORGANISERS: R. N. EISENMAN (FRED HUTCHINSON CANCER RESEARCH
CENTER, SEATTLE), M. EILERS (UNIV. MARBURG), J. LEÓN (UNIV.
CANTABRIA, SANTANDER)
AND THE
LINKS
BETWEEN CANCER, REPLICATION STRESS AND GENOMIC INTEGRITY
07/11/2007 - 09/11/2007
ORGANISERS: O. FERNÁNDEZ-CAPETILLO (CNIO, MADRID), J. LUKAS
(DCS, COPENHAGEN), J. MÉNDEZ (CNIO, MADRID), A. NUSSENZWEIG
(NCI/NIH, BETHESDA)
SCIENTIFIC MEETINGS
II WORKSHOP ON SNPS ANALYSIS, TOOLS AND APPLICATIONS
01/12/2006 - 02/12/2006
ORGANISERS: MERCEDES ROBLEDO, JAVIER BENÍTEZ
DOPAZO
AND JOAQUIN
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CNIO Symposium on Molecular markers in cancer therapy 2006
SYMPOSIA
MOLECULAR MARKERS IN CANCER THERAPY: PRESENT USE AND FUTURE PERSPECTIVES
27/11/2006 - 29/11/2006
ORGANISERS: MONTSERRAT SÁNCHEZ-CÉSPEDES AND MIGUEL ÁNGEL
PIRIS, MOLECULAR PATHOLOGY PROGRAMME, CNIO
NATURE AND CNIO SYMPOSIUM ON ONCOGENES AND HUMAN CANCER: THE
25 YEARS
03/10/2007 - 06/10/2007
ORGANISERS: CNIO AND NATURE PUBLISHING GROUP
SCIENTIFIC ORGANISING COMMITTEE: M. BARBACID, E.HUTCHINSON,
D. LANE, C. MARSHALL, B. MARTE, F. MCCORMICK, B. PULVERER, C. SAWYERS,
B. VOGELSTEIN, K. VOUSDEN, R. WEINBERG.
DATES: OCTOBER 3-6 2007
NEXT
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Sponsors
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© Fundación Centro Nacional de Investigaciones Oncológicas Carlos III. Madrid 2004, Spain.
Co-ordinated and compiled by Sara Bertrand (sbertrand@cnio.es)
Dept. Scientific Events
Design: Equipo de maquetación de Grafilia, SL
Print: Grafilia, SL
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