relat act 2005

RELATÓRIO DE ACTIVIDADES
DO CIBO/IPATIMUP
2005
1
ÍNDICE
PÁG.
1.
INTRODUÇÃO
2
2.
INVESTIGAÇÃO CIENTÍFICA
4
2a. Cancer Biology
5
2b. Cancer Genetics
11
2c. Carcinogenesis
21
2d. Genetics, Evolution and Pathology
29
2e. Population Genetics
34
2f. Tumour Evolution and Development
39
3.
EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA
42
4.
SERVIÇO À COMUNIDADE
44
4a. UPS
45
4b. UPSi
50
4c. UPSs
53
5.
RECEM-DOUTORADOS
55
6.
ESTAGIÁRIOS ESTRANGEIROS
65
7.
TRABALHOS PUBLICADOS
66
8.
REVISTAS CIENTÍFICAS INTERNACIONAIS COM MEMBROS DO
9.
I P A T I M U P NOS SEUS EDITORIAL BOARDS
71
NÚCLEO DE AMIGOS DO I P A T I M U P
72
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1. INTRODUÇÃO
Dando sequência ao procedimento seguido nos últimos anos por sugestão dos avaliadores externos do IPATIMUP,
o relatório de actividades de 2005 inclui uma Introdução que resume os elementos mais expressivos da actividade
no ano passado, os relatórios científicos, em inglês, dos seis grupos de investigação do Instituto, o relatório da
Unidade de Divulgação Científica e Educação Continua e das três Unidades de Prestação de Serviços (UPS, UPSi
e UPSs), as listas dos recém-doutorados, das publicações científicas, dos corpos editoriais de revistas
internacionais indexadas em que participam investigadores do Instituto, e dos estagiários estrangeiros e a lista das
21 Empresas que integram nesta altura o Grupo de Amigos do IPATIMUP.
Em 2005 continuámos a instalação de equipamento e a ocupação da ala do IPATIMUP, recém-construída (A área
do Instituto passou de 2000 para cerca de 4000m2).
Os investigadores do IPATIMUP publicaram 99 artigos científicos 94 dos quais em revistas internacionais com
Factor de Impacto. Cerca de 40% desses artigos foram publicados em revistas com Factor de Impacto entre 3 e
10,4. Também em 2005, atingiu-se o limiar dos 20 artigos publicados por investigadores do IPATIMUP com mais de
100 citações.
O número de doutoramentos foi, em 2005, de 10. Os 10 recém-doutorados encontram-se a trabalhar em
instituições que se estendem de Moçambique à Califórnia.
Continuaram-se em 2005 os programas de Mestrado e Doutoramento e realizaram-se, na Fundação Gulbenkian e
em Serralves, as sessões do segundo Ciclo de Colóquios sobre Medicina Preventiva do Cancro para mais de mil
professores de Biologia e Físico-Química. Em Outubro o IPATIMUP organizou e realizou, em colaboração com a
Associação de Estudantes da Faculdade de Medicina da Universidade do Porto, um ciclo de Introdução à Medicina
Molecular do Cancro para 150 alunos dos 1ºs anos das licenciaturas em Medicina na U.Porto. De Novembro de
2003 a Setembro de 2005, alguns elementos do IPATIMUP fizeram divulgação prática e experimental de biologia
aplicada às ciências da saúde, usando um verdadeiro Auto-Laboratório, a cerca de 9000 alunos do 1ª e 2ª ciclo de
64 Escolas do Ensino Básico do Norte do País (360 sessões).
Mantiveram-se as actividades de apoio à comunidade e de consultadoria diagnóstico. Os cerca de 200 casos
nestas condições (2005) foram enviados ao IPATIMUP por hospitais e institutos de cancro de 19 países.
Em 2005, o Laboratório de Histopatologia e Citopatologia do IPATIMUP terminou o processo de acreditação pelo
American College of Pathologists, ficando a ser um dos três laboratórios europeus com esta credencial.
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Os investigadores do IPATIMUP integram os Corpos Editoriais de 23 revistas científicas internacionais. O
IPATIMUP realizou, em 2005, como nos anos anteriores, três reuniões internacionais – uma sobre Genética
Populacional e Genética Forense (Portugaliae Genética – 9º edição), outra sobre Cancro (Porto Cancer Meeting –
15ª edição) e a terceira sobre Ciência e Cultura (Conferências do Equinócio – 10ª edição).
Em 2005 continuámos o reforço da ligação ao IBMC e INEB com a consolidação do Program’s Office comum aos
três Institutos, concursos interintitucionais a programas nacionais e internacionais, e uma política de implementação
de partilha de equipamentos, para além da continuação da colaboração na divulgação científica, no Programa
GABBA e em outros tipos de formação pós-graduada.
Nota: O IPATIMUP voltou a contar, em 2005, com o apoio excepcional dos seus Associados Efectivos
(Universidade do Porto, Câmara Municipal do Porto, Fundação Luso-Americana Para o Desenvolvimento, Liga
Portuguesa Contra o Cancro, Comissão de Coordenação e Desenvolvimento da Região Norte, Instituto de
Genética Medica Dr. Jacinto de Magalhães e Cruz Vermelha Portuguesa) e Aderentes (Faculdade de Medicina,
Faculdade de Ciências, Instituto de Ciências Biomédicas Abel Salazar, Faculdade das Ciências da Alimentação e
Nutrição, Faculdade de Medicina Dentária, Faculdade de Farmácia, Hospital de S. João e Instituto Português de
Oncologia – Centro Regional do Norte), assim como da Fundação Calouste Gulbenkian, da Fundação de Serralves
e da Fundação Oriente.
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2. INVESTIGAÇÃO CIENTÍFICA
2a. Cancer Biology
2b. Cancer Genetics
2c. Carcinogenesis
2d. Genetics, Evolution and Pathology
2e. Population Genetics
2f. Tumour Evolution and Development
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CANCER BIOLOGY GROUP
Coordinator: Paula Soares, BSc, PhD.
Principal Investigators: Clara Sambade, MD, PhD; Helena Vasconcelos, BSc, PhD; José Manuel Lopes,
MD, PhD; Manuel Sobrinho-Simões, MD, PhD, (Scientific Consultant).
Post- Docs: Valdemar Máximo, BSc, PhD; Ana Preto, BSc, PhD; Ana Sofia Rocha, BSc, PhD.
PhD Students: Ana Carvalho*, Patrícia Castro*, Jorge Lima, Victor Trovisco.
MSc Students: Raquel Lima, Susana Ribeiro.
BI’s: Tália Feijão, Patrícia Pontes, Paula Rebocho, Lígia Gomes, Ricardo Marques, Ricardo Soares.
Clinicians: Manuel Cardoso de Oliveira, MD, PhD; António Taveira Gomes, MD, PhD, José Teixeira
Gomes, MD, PhD.
* - Concluded their thesis in 2005 (see below).
Objectives/Goals of the research activity
1. The main objective is to progress in the understanding of the etiopathogenesis of some types of
human cancer, with an emphasis on thyroid and neuroendocrine tumours. Within this frame a particular
attention is paid to: a) genetic alterations in tyrosine kinase receptors and signal transducing molecules
involved in the mitogen-activated protein kinases (MAPK) pathway; and b) mitochondrial alterations
secondary to mitochondrial DNA mutations/deletions or to mutations in nuclear genes encoding
mitochondrial enzymes.
2. Some members of the group are also involved in clinico-pathological studies in other types of human
tumours, and in projects aiming to validate and/or identify molecular targets for cancer treatment, namely
via the utilization of cell signalling inhibitors and the down regulation of gene expression with siRNAs.
Main research topics
Oncobiology of differentiated thyroid tumours
Radiation induced thyroid tumourigenesis
Oncobiology of familial forms of thyroid and neuroendocrine tumours.
Oncobiology of some haematological malignancies.
Validation of molecular targets (namely targets in cancer chemotherapy resistance)
Background and major achievements in 2005
In 2005 we have followed several strategies:
1. To start collecting and studying a large series of papillary and follicular carcinomas with detailed
clinico-pathologic data, appropriately sampled material (paraffin-embedded and, whenever
possible, also frozen material), long follow-up and reliable information on regional and distant
metastases, with three main objectives:
1a) To establish the relationship between the growth pattern of papillary thyroid carcinoma (PTC)
and the subtypes of BRAF mutation. In 2003, we have identified BRAFV600E mutation as a major
oncogenic event in PTC (in about 50% of the cases)(Soares et al, Oncogene, 2003). The
BRAFV600E mutation was specifically associated with the conventional type of PTC, as well as with
some variants of PTC displaying a prominent papillary growth pattern (Trovisco et al, J Pathol,
2004). In 2005 we detected a new type of BRAF mutation (BRAFVK6001E) in a solid variant of PTC
(Paper 9-Trovisco et al.) and summarized the available evidence on the peculiar genotypephenotype relationship between BRAF mutations and PTC histotypes (Paper 7– Sobrinho-Simoes
M) (see also below).
1b) To disclose the molecular characteristics of the follicular variant of PTC which is known to
behave differently from conventional PTC, namely regarding its tendency to give rise to lung and
bone metastases. After having shown in 2003 and 2004 that this variant displayed a different BRAF
mutation (BRAFK601E in about 7-9% of the cases and no BRAFV600E), we demonstrated, in 2005,
that it shared some of the molecular features of follicular carcinomas: frequent occurrence of
PAX8/PPARγ translocation and of N-RAS mutations (Paper 2 and 10 – Castro et al).
1c) To clarify the influence on the metastatic pattern of the tumours and on the patients’ prognosis of
BRAF mutations, RAS mutations and PAX8/PPARγ translocation. In our hands, and at variance with
the results of other groups, the presence of BRAF mutation does not seem to be linked with
increased clinical aggressiveness of the tumours (Paper 8 – Trovisco et al). We think we have
obtained enough evidence to claim that the on-going controversy on the prognostic meaning of
BRAF mutations in PTC can only be solved if one is able to study a large series of cases in which
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the influence of the age of the patients and the histotype of the tumours is controlled (Paper 8 –
Trovisco et al). In relation to this objective, and besides the above mentioned preliminary study
(Paper 9-Trovisco et al), we have also analysed, in collaboration with clinicians from H.S. João and
USP (Brazil) a cohort of PTC patients in an attempt to find if there is any particular type of BRAF
mutation associated with nodal metastization; our preliminary results do not confirm the advanced
higher propensity for metastization of BRAF mutated tumours (Trovisco et al, manuscript in
preparation).
In order to enlarge our series we have made protocols with the Cancer Institute of Porto, Hospital de
Matosinhos and University Hospitals of Santiago de Compostela, Oslo and S. Paulo (Brasil). We are,
at present, also negotiating similar protocols with the Cancer Institutes of Coimbra and Lisboa.
2. To progress in the understanding of the role played by BRAF mutations (a sort of cell biology
counterpart of the clinicopathological studies listed in Point 1), we have established an in vitro
system in which we can evaluate the biological role (proliferation, apoptosis, motility and
invasiveness) and the signalling pathways activated by the different BRAF mutations in thyroid
cancer derived cell lines, with and without BRAF mutation. Using this system we have been
studying the effects of kinase signalling inhibitors (BAY, Gleevec and PD) on the rate of
proliferation and apoptosis. In a parallel study we have been evaluating the effect of silencing of
BRAF gene by siRNA in the same thyroid cancer derived cell lines (Post- doc project of Ana
Preto). Finally, in a cell model, we have been comparing the transcription patterns of BRAF
mutants with those of RET/PTC transfectants; the results obtained to date show that STAT3
appears to be a key molecule activated by both oncogenic events ( PhD Project of Vitor
Trovisco). In a collateral approach we analysed, by immunohistochemistry, the association of
BRAF mutation and the expression of membrane receptors (p75/TRK and NGFR) in PTC and
found that there is a close relationship between polarized (apical) expression of PTC and BRAF
mutation in conventional PTC (Paper 11- Rocha et al). This cell biology approach will be
continued in 2006 by two post-docs (Ana Preto and Ana Sofia Rocha) using also melanoma cell
lines.
3. To identify mechanisms underlying the occurrence of aneuploidy in thyroid tumours.
The two major histotypes of differentiated thyroid carcinoma display different patterns of DNA
content: follicular carcinomas are clearly aneuploid whereas almost every papillary carcinoma is
diploid or quasi–diploid. We found an association between the presence of a polymorphism in H-Ras
and the occurrence of aneuploidy in thyroid tumours. We also detected a link between the presence
of H-RAS 81-C allele and significantly higher amounts of H-RAS p21 mRNA isoform. These findings
support the hypothesis that H-RAS 81 T-C may induce aneuploidy through the overexpression of
p21 isoform of H-RAS (Paper 12- Castro et al) This hypothesis will be tested by Patricia Castro in
her post-doc under the supervision of Eugénio Santos (University of Salamanca) and Paula Soares.
4. To progress in the understanding of the role played by mitochondrial alterations in
tumourigenesis.
It is not well understood the role of the mtDNA mutations/variants, nor the role of genetic alterations
in nuclear genes that codify for mitochondrial proteins, on the etiopathogenesis of several types of
tumors exhibiting mitochondrion rich neoplastic cells. This holds true for the so-called Hürthle cell
(oxyphilic, oncocytic) tumours of the thyroid, as well as to many types of mitochondrion-rich
neuroendocrine tumours, such as medullary carcinoma of the thyroid, paragangliomas and
pheochromocytomas. The succinate dehydrogenase (SDH) mitochondrial proteins (Complex I of the
mitochondrial respiratory chain and Krebs cycle) were implicated in familial forms of neuroendocrine
tumours (paraganglioma, pheocromocytoma), but their role in C-cell derived tumours was unknown.
We identified in 2003, in a familial case of C-cell hyperplasia without ret mutation, a new SDHD gene
variation that segregates with the disease (Lima et al, JCME, 2003). We are now estimating the
frequency of alterations in SDH genes in a series of medullary carcinomas without ret mutation, as
well as in numerous sporadic and familial paragangliomas and pheochromocitomas from Portugal
and Spain (the study of sporadic and familial paragangliomas from Spain is coordinated by Ginesa
Garcia-Rostan).
We have previously reported that alterations in mitochondrial genes can predispose to the
development of Hürthle cell tumours. Recently, a gene predisposing to thyroid tumors with cell
oxyphilia (TCO) was mapped to chromosome 19p13.2 in one family with Hürthle cell tumours and
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carcinomas. GRIM-19 (Gene associated with Retinoid-Interferon-Induced Mortality), a gene located
in the 19p13.2 region, has been found to fulfill two roles within the cell: i) It is a member of the
interferon-β and, a retinoic acid-induced pathway of cell death, and ii) It is part of the mitochondrial
Complex I assembly. It could therefore be involved both in cell and mitochondrial growth, two of the
characteristics features of Hürthle cell tumours. We found somatic and germline mutations in the
above mentioned gene (GRIM-19), in mitochondrion-rich tumours of the thyroid (Paper 4- Máximo et
al).Our findings reinforced the assumption that alterations in genes of the mitochondrial respiratory
chain (MRC) such as GRIM-19, as well as genes of MRC and Krebs cycle (SDHx), and of Krebs
cycle alone (Fumarate hydratase – FH) play a role in tumourigenesis (via the blockage of apoptosis
and/or the induction of a pseudo-hypoxic status). The relevance of our findings was highlighted by
the editorial comment published in the issue of Br J Cancer with our paper (Santoro et al, BJCancer,
2005). The study of other mitochondrial-related genes localized in some regions of the
chromosomes 17 and 19 (PRSS15, NDUFA7, TIMM44, TIMM22 and HCCS1) did not provide
evidence favouring the involvement of any of these genes in the etiopathogenesis of Hürthle cell
tumours (Maximo et al, manuscript in preparation).
Concerning the role of the mtDNA mutations/variants on the etiopathogenesis of mitochondrion-rich
(oncocytic, Hürthle cell) tumours we verified that mitochondrial D-loop instability is not a marker of
malignancy in thyroid tumours (Paper 5 - Máximo et al). (Hürthle cell tumours) we published a review
paper emphasising the practical aspects of our findings on thyroid (Paper 6 – Sobrinho-Simões et al)
and a letter to the editor on the Warthin’s tumour of the salivary gland that is also characterized by
oxyphilic cells (Paper 13 – Máximo V and Sobrinho-Simões M)
We had previously verified that irradiated tumours possess numerous alterations in mtDNA that are
present in the adjacent thyroid parenchyma but not in the peripheral blood of the patients (Lima J et al,
Carcinogenesis, 2004). After performing cloning analysis and large scale sequencing of mtDNA in paired
samples (blood, adjacent thyroid and tumour), we verified that the “mutated” sequences correspond to
different mitochondrial haplotypes. This led us to perform genotypic characterization in paired samples
(blood, adjacent thyroid and tumour) which showed that the samples did not match. Our results have
been confirmed by another group working on “Chernobyl” cases leading to the re-start of the study with
appropriate controls.
The studies on the role of mitochondrial alterations in tumourigenesis (PhD project of Jorge Lima) will
continue in 2006, using, besides the Chernobyl cases, the database of familial forms of thyroid tumours
(we have collected until now 17 families, with 154 affected and non-affected members), the familial and
sporadic cases of medullary carcinoma, pheocromocytomas and paragangliomas, and in the series of
Warthin’s tumours and renal oncocitomas diagnosed at the Hospital de S. João.
The possibility of studying a series of post-Chernobyl tumours led us to the development of a project that
aims to understand the effect of irradiation in mitochondrial DNA. To conclude this project we will
perform and characterize the presence of mt DNA large deletions and of mutations in genes of
Complexes I, II, III, IV and V genes (MRC) in these samples (Post-doc project of Valdemar Máximo and
PhD project of Jorge Lima).
In the field of radiation induced carcinogenesis we will initiate in 2006 a project (with financial support
from Fundação Calouste Gulbenkian) aiming to evaluate Head and Neck Tumours in a cohort of about
5000 individuals irradiated for the treatment of Tinea Capitis in the 1950’s.
Our Group will host the National Database of GastroIntestinal Stromal Tumours (GIST) and
NeuroEndocrine Tumours (NET), which have been designed as REGIST and REGENE respectively
(Responsible: José Manuel Lopes). The establishment and maintenance of the Database will be
supported by NOVARTIS ONCOLOGY.
Addendum:
Aim: Validation of molecular targets in cancer treatment or chemoresistance.
PI. Helena Vasconcelos
We verified that downregulation of multidrug resistance protein (P-gp) expression with siRNAs provided
their sensitisation of resistant chronic myeloid leukaemia (CML) cells to Imatinib, suggesting that P-gp
may be a good molecular target for an adjuvant therapy with Imatinib (Study financed by Novartis
Oncology Portugal).
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We are currently validating the relevance of XIAP (an antiapoptotic protein) in the resistance of acute
myelogenous leukaemia (AML) cells to chemotherapy (Study financed by the Portuguese Association
Against Leukemia – APCL).
We intend to continue the validation of new molecular targets for cancer treatment, focusing on antiapoptotic proteins and on signal transduction molecules, using both the RNAi and the kinase inhibitors
strategies.
Seminars, Scientific meetings:
1ª Reunião Internacional do Grupo de Patologia Endócrina do Hospital de S. João. “Recent advances in
the diagnosis and treatment of thyroid cancer.” IPATIMUP, March 10 and 11, 2005.
Reunião Anual Luso Espanhola (Regiões da Galiza, Astúrias, Castilla-Leon). “Thyroid and
neuroendocrine tumours”. IPATIMUP, November 18 and 19, 2005.
PhD Theses:
Ana Carvalho. Cellular and molecular analysis of surviving and the involvement of chromosomal
passengers in the multinucleation phenotype of Hodgkin’s disease. Supervisor: William Earnshaw
(Edimburgh), and co-supervisor: Clara Sambade. February 2005. Medical Faculty of Porto.
Papers: Wheatley SP, Carvalho A, et al. INCENP is required for proper targeting of Survivin to the
centromeres and the anaphase spindle during mitosis. Current Biology 11:886-90, 2001.
Gassmann R, Carvalho A, et al. Borealin: a novel chromosomal passenger required for stability of the
bipolar mitotic spindle. Journal of Cell Biology 19;166:179-791, 2004.
Carvalho A, et al.Survivin is required for stable checkpoint activation in taxol-treated HeLa cells. Journal
Cell Science 116:2987-2998, 2003.
Patrícia Castro. Molecular biology of the chromosomal instability phenotype of follicular thyroid tumours.
Supervisor: Raghnild Lothe (Oslo) and co-supervisor: Manuel Sobrinho-Simões. November 2005.
Medical Faculty of Porto.
Papers: Castro P, e tal. Fetal adenomas and minimally invasive follicular carcinomas of the thyroid
frequently display a triploid or near triploid DNA pattern.Virchows Archiv 438:336-42, 2001.
Castro P, et al. Pattern of chromosomal changes in follicular thyroid tumors with an emphasis on
fetal/embryonal adenomas. Journal of Pathologyl 206:305-311, 2005.
Castro P, et al. A subset of the follicular variant of papillary thyroid carcinoma harbors the PAX8PPARgamma translocation. International Journal Surgical Pathology 13:235-238, 2005.
Castro P, et al. PAX8-PPARgamma rearrangement is frequently detected in the follicular variant of
papillary thyroid carcinoma. Journal of Clinical Endocrinology and Metabolism 91:213-220, 2006.
Castro P, et al. H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of
the thyroid. Oncogene (in press).
Publications
A. Related with the main research topics
1- Castro P, Eknæs M, Teixeira MR, Danielsen H, Soares P, Lothe R, Sobrinho-Simões M. Pattern
of chromosomal changes in follicular thyroid tumors with an emphasis on fetal/embryonal
adenomas. Journal of Pathologyl 206:305-311, 2005.
2- Castro P, Roque L, Magalhaes J, Sobrinho-Simoes M. A subset of the follicular variant of
papillary thyroid carcinoma harbors the PAX8-PPARgamma translocation. International Journal
Surgical Pathology 13:235-238, 2005.
3- Lima J, Trovisco V, Soares P, Máximo V, Magalhães J, Salvatore G, Santoro M, Bogdanova T,
Tronko M, Abrosimov A, Jeremiah S, Thomas G, Williams D, Sobrinho-Simões M. Reply to: Low
prevalence of BRAF mutations in radiation-induced thyroid tumors in contrast to sporadic
papillary carcinomas. Cancer Letters 230:149-150, 2005.
4- Máximo V, Botelho T, Capela J, Soares P, Lima J, Taveira A, Amaro T, Barbosa AP, Preto A,
Harach HR, Williams D, Sobrinho-Simões M. Somatic and germline mutation in GRIM19, a dual
function gene involved in mitochondrial metabolism and cell death is linked to mitochondrion-rich
(Hürthle cell) tumours of the thyroid. British Journal of Cancer 92:1892-1898, 2005.
5- Máximo V, Lima J, Soares P, Botelho T, Gomes L, Sobrinho-Simoes M. Mitochondrial D-Loop
instability in thyroid tumours is not a marker of malignancy. Mitochondrion 5:333-340, 2005.
6- Sobrinho-Simões M, Máximo V, Vieira de Castro I, Fonseca E, Soares P, Garcia-Rostan G,
Cardoso de Oliveira M. Hürthle (oncocytic) cell tumors of thyroid: etiopathogenesis, diagnosis
and clinical significance. International Journal of Surgical Pathology 13:29-35, 2005.
7- Sobrinho-Simoes M. BRAF Mutations in thyroid carcinomas: phenotype-genotype correlations.
Advances in Anatomic Pathology 12:106-107, 2005.
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8- Trovisco V, Soares P, Preto A, Vieira de Castro I, Lima J, Castro P, Máximo V, Botelho T,
Moreira S, Meireles AM, Magalhães J, Abrosimov A, Cameselle-Teijeiro J, Sobrinho-Simões M.
Type and prevalence of BRAF mutations are closely associated to papillary thyroid carcinoma
histotype and patients’ age but not with tumour aggressiveness. Virchows Archiv 446:589-595,
2005.
9- Trovisco V, Soares P, Soares R, Magalhães J, Sá-Couto P, Sobrinho-Simões M.A new BRAF
gene mutation detected in a case of a solid variant of papillary thyroid carcinoma. Human
Pathology 36:694-697, 2005.
In Press:
10- Castro P, Rebocho AP, Soares RJ, Magalhaes J, Roque L, Trovisco V, Vieira de Castro I,
Cardoso-de-Oliveira M, Fonseca E, Soares P, Sobrinho-Simoes M. PAX8-PPARgamma
rearrangement is frequently detected in the follicular variant of papillary thyroid carcinoma.
Journal of Clinical Endocrinology and Metabolism 91:213-220, 2006.
11- Rocha AS, Risberg B, Magalhães J, Trovisco V, Vieira de Castro I, Lazarovici P, Soares P,
Davidson B, Sobrinho-Simões M. The p75 neurotrophin receptor is widely expressed in
conventional papillary thyroid carcinoma. Human Pathology (in press).
12- Castro P, Soares P, Gusmão L, Seruca R, Sobrinho-Simões M. H-RAS 81 polymorphism is
significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene (in press).
13- Máximo V, Sobrinho-Simões M. Warthin’s tumour. Letter to the Editor. Virchows Archiv (in
press).
B. Other topics
1. Basto D, Trovisco V, Lopes JM, Martins A, Pardal F, Soares P, Reis RM. Mutation analysis of BRAF gene in human gliomas. Acta Neuropathologica (Berl) 109:207-210, 2005.
2. Cameselle-Teijeiro J, Abdulkader I, Soares P, Alfonsín-Barreiro N, Moldes-Boullosa J, Manuel
Sobrinho-Simões. Cystic tumor of the atrioventricular node of the heart appears to be the heart
equivalent of the solid cell nests (ultimobranchial rests) of the thyroid. American Journal Clinical
Pathology 123:369-375, 2005.
3. Cameselle-Teijeiro J, Preto A, Soares P, Sobrinho-Simoes M. A stem cell role for thyroid solid
cell nests. Human Pathology 36:590-591, 2005.
4. Gouveia AM, Pimenta AP, Lopes JM, Capelinha AF, Ferreira SS, Valbuena C, Oliveira MC.
Esophageal GIST: therapeutic implications of an uncommon presentation of a rare tumor.
Diseases of the Esophagus18:70-73, 2005.
5. Lopes R, Castro I, Pontes P, Candeias J, Lemoine N, Sambade C. Expression profile of survivin
in acute leukemias: the importance of differential splicing. Leukemia 19:1284-1286, 2005.
6. Reis C, Carneiro E, Fonseca J, Pereira P, Vaz R, Pinto R, Capelinha AF, Lopes JM, Salgado
Epithelioid hemangioendothelioma and multiple thoraco-lumbar lateral meningoceles: two rare
pathological entities in a patient with NF-1. Neuroradiology 47:165-169, 2005.
7. Vasconcelos MH, Maia LF, Sousa C, Beleza SS and Guimaraes JE. Evidence for a specific
intracellular localization of an antisense oligonucleotide in K562 cells. Journal of
Pharmacological Sciences 99:105-108, 2005.
8. Sambade C, Berglund M, Lagercrantz S, Sällström J, Reis RM, Enblad G, Glimelius B,
Sundström C: U-2940: a human B-cell line derived from a diffuse large cell lymphoma sequential
to Hodgkin lymphoma. International Journal of Cancer 118 :555-563, 2006.
9. Lima RT, Martins LM, Guimarães JE, Sambade C and Vasconcelos MH. Chemosensitization
effects of XIAP downregulation in K562 leukemia cells. Journal of Chemotherapy (in press).
On going projects:
“Biological role of BRAF oncogene activaction in thyroid carcinogenesis. STI-571/gleeved as an inhibitor
agent.”
Principal Investigators: Raquel Seruca and Paula Soares
Duration:1/5/2004 to 1/6/2006;. Budget:78869 Euros(2005: 38752 E; 2006:117647 E)
Funding: NOVARTIS
“Teste para selecção in vitro de compostos com potencial actividade anti-oxidante, obtidos através de
plantas medicinais utilizadas tradicionalmente em Portugal.”
Principal Investigator: Valdemar Máximo
Duration:1/2/2005 to 1/2/2007;. Budget :48468 Euros(2005: 19387 E; 2006:19387 E)
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Funding: UNICER (beverage and food company)
“Mitochondrial DNA deletions and mutations in post-Chernobyl thyroid tumors and in the respective
normal thyroid parenchyma.”
Principal Investigators: M Sobrinho-Simões and Valdemar Máximo.
Duration:1/7/2004 to 1/7/2006; Budget: 46305 Euros (2004: 30105; 2005: 10000)
Funding:FCT
“Identification and characterisation of the mechanisms involved in the acquisition of motility and
invasiveness by papillary thyroid carcinoma cells.”
Principal Investigators: M Sobrinho-Simões and Ana Sofia Rocha.
Duration:1/1/2003 to 312/1/2005; Budget: 61798 Euros (2005:20287E; 2006: 20638E)
Funding:FCT
“Potential mechanisms of tolerance to wild-type p53 in human tumours using differentiated thyroid
carcinoma as a prototype.”
Principal Investigators: Paula Soares and Ana Preto.
Duration: 1/7/2003 to 1/7/2006. Budget: 67825 Euros (2005: 23150 E; 2006:22525 E)
Funding: FCT
“Role of raft domains in the modulation/alteration of the transducing pathways in normal thyroid and in
papillary thyroid carcinoma.”
Principal Investigator: Paula Soares
Duration:1/1/2004 to 1/1/2007. Budget: 72645 Euros (2005: 24219 E; 2006:24218 E)
Funding: FCT
“Biological role of BRAF oncogene activaction in thyroid carcinogenesis.”
Principal Investigators: Paula Soares
Duration: 1/9/2005 to 1/9/2008. Budget: 95250 Euros (2005: 30950 E; 2006:31900 E)
Funding: FCT
“Overcoming resistance to imatinib mesylate in leukemia cell line models by specific downregulation of
MDR1 with siRNAs”
Principal Investigators: J.E. Guimarães and M. Helena Vasconcelos Meehan
Duration: 15/2/2004 to 15/2/2006. Budget: 51000 Euros.
Funding: NOVARTIS
“Validação do papel do XIAP na resistência da leucemia mielógena aguda à quimioterapia: abrir
caminho para uma possível nova estratégia de tratamento
tendo o XIAP como alvo terapêutico”
Principal Investigators: M. Helena Vasconcelos Meehan
Duration: 1/6/2005 to 1/9/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia":
“Molecular dissection of the multinucleation phenotype of the neoplastic cells in Hodgkin´s lymphoma.”
Principal investigator: Clara Sambade
Duration: 1/6/2005 to 1/6/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia":
“Neuroendocrine tumors: Clinico-pathological and immunohistochemistry
identification of biological factors of aggressiveness”
Principal Investiga tor: José Manuel Lopes
Duration: 23/9/2004; to 23/9/2006. Budget: 53150 Euros.
Funding: NOVARTIS
characterization
11
and
Cancer Genetics Group
Coordinator: Raquel Seruca
Principal Investigators: Fátima Carneiro, Céu Figueiredo, José Carlos Machado, Carla Oliveira, Fernando Schmitt,
Gianpaolo Suriano.
Post-Doc students: Joana Paredes, Maria José Oliveira.
PhD students: André Albergaria, António Carlos Ferreira, Catarina Alves, Gonçalo Regalo, Joana Correia, Paulo
Canedo, Paulo Ferreira, Rachid Karam, Rita Mateus, Sérgia Velho, Sílvia Carvalho.
The research of our group focuses on the molecular genetics of three common types of epithelial cancer (gastric,
breast, and colorectal carcinoma). We aim at: 1) identifying individuals at risk for the various forms of these tumours;
2) identifying pathological features and molecular markers occurring in the setting of familial and sporadic
carcinoma; 3) identifying signalling pathways mediated by genetic and environmental factors in tumour development,
in order to find new molecular targets for therapeutic intervention. In this report we will describe, in a summarized
manner, the results obtained in 2005 according to the research goals planned in 2004.
1) Identifying individuals at risk of gastric cancer
We ascertained 18 new north-American hereditary diffuse gastric cancer (HDGC) families (14 accomplishing criteria
1 and 4 with criteria 2), 10 isolated early-onset DGC (<35 years), one family with DGC and lobular breast cancer
(LBC) and a patient with synchronous DGC and LBC for CDH1 germ line mutation screening. Thirty-six percent (5 of
14) of the HDGC families accomplishing criteria 1 and 2 of 10 early-onset DGC harboured CDH1 germ line
mutations. CDH1 germline mutations were also found in the patients with LBC. Our results indicate that CDH1
mutation screening should be offered not only to HDGC families but also to families with DGC and LBC and to
isolated cases of DGC in individuals younger than 35 years (Suriano G et al, 2005).
We screened a series of 16 new Portuguese families and found a CDH1 mutation (E757K) in one family with
complete criteria for HDGC. This mutation was later screened for in all first degree relatives alive and all of them,
except the mother, were carriers of the same CDH1 alteration. This alteration was absent in 100 individuals from a
Portuguese control population (data not published). We studied in vitro the pathogenic significance of this mutation
(E757K) and three other new germline missense mutation T118R, G239R, L214P found in New Zealand and in UK
families, using aggregation and invasion assays. All missense mutations were pathogenic in vitro (data not
published)
To disclose whether intragenic deletions or deletions encompassing the full CDH1 gene may be a mechanism
underlying a fraction of families with aggregation of gastric cancer, we collected a large panel (n=150) of families,
from different countries with low, moderate and high incidence of gastric cancer, which have proved to be negative
for germline CDH1 point mutations, as tested by different laboratories. No deletions have been found in these
families, either intragenic or encompassing the full CDH1 gene, excluding these type of CDH1 alterations as a
molecular mechanism underlying HDGC. From these 150 families, we have selected 41 families with available
biological material to screen for germline mutations in all exons and intron-exon boundaries of C-MET gene. We
found several new sequence variants. In none of the families we were able to detect segregation of the sequence
variant with the disease in the family. Based on the results we can role out C-MET germline mutations as major
players in familial gastric cancer (data not published).
We defined a new syndrome with clustering of diffuse gastric cancer and midline malformations harbouring a CDH1
splice-site germline mutation. This germline mutation (1137G>A) generates abnormal transcripts with PTCs in
codons 349 and 386 (Frebourg T et al, 2005). We analysed the allele-specific expression of CDH1 gene in normal
gastric mucosa from two asymptomatic mutation carriers (1137G>A) from this HDGC family and found that 85% of
transcripts were normal. We evaluated an asymptomatic 2061delTG mutation carrier from another HDGC family that
showed a 10-fold down regulation of the mutated CDH1 allele in comparison to the wild type allele. These results
prompted us to study the role of NMD in the regulation of CDH1 expression in HDGC mutation carriers.
We extended our study of identifying a specific genetic profile (of inflammation-related genetic polymorphisms)
associated with risk of development of Helicobacter pylori-associated gastric carcinoma, by analysing in a casecontrol study IL6, IL8, IL10, COX2, MIF, TNFA, CTL4 and IFNGR1 polymorphisms. Our results indicate that the
IFNGR1-56C/T polymorphism is yet another relevant host susceptibility factor for gastric carcinoma development.
The -56*T allele is associated with significantly increased expression of the IFNGR1 gene and may therefore play an
important role in modulating the host inflammatory response to Helicobacter pylori infection. We did not find any
significant association with any of the remaining polymorphisms included in the study.
1) Identifying individuals at risk of breast cancer
We extended the screening of BRCA1 and BRCA2 in families with aggregation of breast cancer and analyzed
negative families for other DNA repair genes (Rad51, XRCC1, XRCC3, XPD, TP53). We have carried out a casecontrol study in a Portuguese population. We analysed 633 DNA samples: 74 breast cancer patients with family
history (FH) of breast cancer, 219 breast cancer patients without FH and 340 healthy women, for several DNA
signalling/repair polymorphisms: XRCC1 Arg399Gln, XPD Lys751Gln, RAD51 G135C, XRCC3 Thr241Met, TP53
Arg72Pro and TP53 INS3, using PCR-RFLP technique. We observed a lower frequency of 399Gln genotypes in
breast cancer patients with negative FH (56.2%) comparing with control group (65.7%). These results suggest a
12
protective effect of 399Gln genotypes on breast cancer in women without FH (OR=0.66 95% CI 0.40-1.06; OR
adjusted for age by logistic regression). The analysis of XPD Lys751Gln polymorphism, as observed in previous
studies, did not show any statistical significant association between the 2 firsts groups and any of the genotypes
compared with healthy women. Concerning RAD51 G135C polymorphism, we did not find any statistical significant
association of sporadic breast cancer risk and this polymorphism. However, we observed an increased risk to breast
cancer in women carriers of GC135 or CC135 genotypes presenting a positive FH of breast cancer (OR=2.15 95%
CI 1.12-4.10). We analysed XRCC3 Thr241Met polymorphism and we did not find any statistically significant
difference in genotypes frequencies between breast cancer patients with and without FH and control group. We
found higher frequencies of INS3 A1A1 genotype in breast cancer patients with FH than in control group (18.4% and
4.7%, respectively). Our results showed correlation of TP53 INS3 polymorphism with increased breast cancer risk
(OR=4.54 95% CI 1.69 -12.5) in women with a positive FH of breast cancer. In relation to TP53 Arg72Pro
polymorphism, we did not find any statistical difference between the different groups.
2) Identifying pathological features and molecular markers occurring in the setting of familial and sporadic
cancer
Although about 20% of the HDGC families harbor germline mutations of the missense type, their clinical
management remains controversial. We analyzed the first two prophylactic gastrectomies performed on HDGCcarriers, of CDH1 germline mutations of the missense mutations (A634V, E757K), that choose to perform it after
genetic counseling. Both germline alterations impaired in vitro the ability of E-cadherin to mediate cell-to cell
adhesion and to suppress cell invasion, demonstrating their pathogenicity in vitro. Foci of early invasive diffuse
carcinoma (signet ring cell type) were found in specimens from both individuals submitted to prophylactic
gastrectomy. Furthermore, the distribution and size of the cancers in the gastrectomy specimens have clearly
indicated that standard endoscopic screening is unlikely to provide sufficiently sensitive clinical screening for at-risk
individuals. Our data indicates that prophylactic gastrectomy should be taken in consideration also to asymptomatic
carriers of germline missense mutations, when their pathogenic relevance is demonstrated in vitro (data not
published).
In familial breast cancer we identified a specific basal phenotype (ER and HER-2 negativity and overexpression of
P-cadherin, CK5, and p63) that can be used as a valuable tool for identifying familiar cases within breast carcinomas
(Matos et al., 2005). We analysed 168 invasive breast carcinomas classified into four different subtypes: luminal A
and B, HER2 and basal-type. Basal-type tumors were ER and HER2 negative and represented 7.6% of our series;
HER2-overexpressing tumors did not express ER and represented 17.7%; luminal-type tumors expressed ER and
represented 72.8% of this series (luminal A 56.3%, luminal B 16.5%). Moreover, we characterized each subtype
based on P-cadherin (P-CD), p63, cytokeratin (CK) 5, BCL2, and Ki67 expression. Basal-type tumors were mostly
grade III, more frequently P-CD-, p63-, and CK5-positive, and had a high proliferation rate. With this study, we show
that P-CD, p63, and CK5 are important molecular markers that can be used to distinguish a basal phenotype. In
addition, we also demonstrate the usefulness of TMAs in breast carcinoma immunoprofiling.
This study still validated the frequent presence of a basal phenotype in a series of familiar breast carcinoma. We
would like to validate in breast carcinomas harboring germline mutations of BRCA genes in order to support these
pathological/molecular markers as reliable, fast, and low cost strategy for the identification of individuals with
germline mutations in BRCA genes.
Comparative genomic hybridization (CGH) has been the technique of choice over the last 10 years for mapping DNA
copy number changes in human tumors. We reviewed the literature to demonstrate how CGH has contributed to the
comprehension of molecular aspects of breast tumorigenesis (Reis-Filho et al., 2005). At least two distinct molecular
pathways of breast cancer have been characterized, that show a strong correlation with histological grade. It seems
that grade I invasive ductal carcinomas (IDCs) arise from well-differentiated ductal carcinoma in situ (DCIS),
whereas grade III IDCs come from poorly differentiated DCIS. In addition, dedifferentiation from a low- to a highgrade breast cancer has proven an unlikely phenomenon. CGH has been instrumental in dissecting distinct
molecular pathways toward breast malignancy and in establishing a direct relationship between genotype and
clinical pathological features.
In colorectal cancer, we verified that BRAFV600E mutation is never found in HNPCC families with hMSH6 germline
mutations, in contrast to its presence in 40% of MSI sporadic carcinomas. Our data allows the use of this molecular
marker (BRAFV600E mutation) in the genetic testing of HNPCC with germline mutations of MSH6 as it was verified
for hMSH2, hMLH1. (Domingo E et al, 2005). Furthermore, we analysed a panel of early-onset colorectal carcinoma
patients for BRAF mutations and found no V600E mutations in this setting. According, these patients will be included
in the screening for germline mutations of MMR genes hMSH2, hMLH1 and hMSH6 (data not published).
MMR defects lead to an increase rate of mutations, giving rise to instability at microsatellite sequences (MSI) and an
increased frequency of missense mutations. Recently, we systematically analysed alterations in KRAS, BRAF and
RASSF1A, in a panel of 31 colorectal and 25 gastric MSI sporadic carcinomas and 20 MSI HNPCC in order to
define the frequency and the pattern of genetic/epigenetic alterations in these distinct subsets of MSI tumours. Thirty
six percent of MSI sporadic colorectal carcinomas had RASSF1A methylation and activating mutations at KRAS
and/or BRAF. In contrast, only 10% and 8% of HNPCC and sporadic gastric carcinomas, respectively, had
concomitant KRAS mutations and RASSF1A methylation. We demonstrate that MSI sporadic colorectal carcinomas
accumulated significantly more epi/genetic alterations in RASSF1A, KRAS/BRAF than MSI sporadic gastric or
13
HNPCC carcinomas. (Oliveira C et al, 2005). Further, we have determined the frequency of PIK3CA mutations in 47
gastric and 103 colorectal carcinomas characterised for MSI status and analysed the association between PIK3CA
mutations and KRAS or BRAF mutations. PIK3CA mutations in exons 9 and 20 were present in 13.6% and 10.6% of
colorectal and gastric carcinomas, respectively. No differences in frequency and type of PIK3CA mutations were
found between MSI and microsatellite stable (MSS) colorectal carcinomas. All gastric carcinomas with PIK3CA
mutations were MSI. In colorectal carcinoma, PIK3CA mutations occur, preferentially, together with activating KRASBRAF mutations while in gastric carcinomas PIK3CA mutations tend to occur as isolated events (Velho S et al,
2005).
Overall, the results obtained are important since they are likely to have therapeutic implications in the near future, by
the use of specific kinase inhibitors alone or in conjunction with demethylating agents in the specific settings of
gastrointestinal tumours.
Furthermore, we aim at verifying the frequency of KRAS and BRAF mutations in MSS colorectal carcinomas. We
screened 250 MSS primary CRC and 45 lymph node metastases for both genes and studied their pathologic
features. We demonstrated that KRAS and BRAF are alternative events in early MSS CRC, as it was previously
verified for MSI carcinomas. KRAS mutations alone increase along colorectal carcinoma progression. The same is
not verified for BRAF. Concomitant mutations at KRAS and BRAF increase during MSS CRC metastization
(submitted for publication). Our results prompted us to verify in colorectal tumours if the activation of both genes is
likely to harbour a synergistic effect in tumour invasion.
3) Identification of signaling pathways mediated by genetic and environmental factors in the distinct cancer
models, and target-specific therapeutic drugs
Signaling pathways mediated by H. pylori- We examined the relevance of H. pylori cag pathogenicity island
(cagPAI), CagA, and VacA in cell invasion and the mechanisms underlying this process. We demonstrated, in AGC
cells, that H. pylori strains with an intact cagPAI stimulate gastric epithelial cell invasion, through a c-Met dependent
signalling pathway and by increasing MMP-2 and MMP-9 activity. VacA does not contribute to epithelial cell invasion
(submitted for publication).
Chronic infection with H. pylori results in a series of alterations on the gastric mucosa. H. pylori virulence factors
constitute an important source of variation in the clinical outcome of the infection in adults, but there is limited
knowledge in paediatric age. We evaluated the role of H. pylori and its virulence genotypes in clinical, endoscopic,
and histological features of a Portuguese paediatric population (n=125). The prevalence of H. pylori in this
population was 50%, 17% of which infected with multiple strains. H. pylori infection was associated with endoscopic
antral nodularity and with the histological features of gastritis and activity, but not with clinical symptoms of
abdominal pain or dyspepsia. No associations were found between H. pylori genotypes and clinical symptoms,
endoscopic appearance or histology. Infection with multiple strains was more frequent in dyspeptic (58%) than in
non-dyspeptic (18%) children, and also more frequent in nodular (100%) than in normal mucosa (52%). In children,
virulence genotypes can not be used to evaluate disease severity. The role of multiple infections needs further
clarification (data not published).
Signaling pathways mediated by Slug and E12/E47 - It’s been shown that Slug and E12/E47, two transcription
factors involved in embryonic development, are responsible for the repression of the E-cadherin gene expression,
through the binding to the E-box regulatory regions in the E-cadherin promoter. In our study we try to understand
whether these two genes have a role during downregulation of E-cadherin in gastric cancer. We have analysed the
expression profiles of Slug and E12/E47 in a series of 59 primary gastric carcinomas and correlated this with Ecadherin expression. We have found that Slug overexpression in tumour when compared to matched normal
mucosa was significantly correlated with downregulation of E-cadherin expression (p<0.001). Overexpression of
Slug could be correlated with cases showing distant metastases (p=0.008) and when it was accompanied with E-cad
downregulation it could be associated to more advanced cases (stage III-IV) (p=0.043) and the presence of distant
metastases (p=0.008). No correlation could be established between E12/E47 upregulation and E-cadherin
downregulation (data not published). In order to establish an in vitro model that will allow us to better understand a
putative role of Slug in cancer progression, we are currently transfecting cell lines and studying the consequences of
Slug ectopic expression regarding E-cadherin expression and the alterations in the phenotype and behavior of the
cells (invasion, adhesion, motility).
Signaling pathways mediated by E-cadherin- We have previously shown that two germline missense mutations of Ecadherin (T340A and V832M) found in HDGC patients affect in vitro the ability of E- cadherin to mediate cell-to-cell
adhesion, motility and invasion. However the in vitro assays used till now did not address the biological role
mediated by the extracellular matrix (ECM) in the motility behavior of cancer cells harboring these defects of Ecadherin. To this end, cell lines stably expressing the E-cadherin mutants T340A and V832M as well as the wild-type
protein were seeded on a layer of collagen type I and their motility behavior studied by time-lapse microscopy.
T340A expressing cells showed a 20% increased cell migration when compared to wild-type E-cadherin expressing
cells, accompanied by evident cytoskeletal rearrangements. In contrast, cells expressing the V832M mutation
behaved in a similar manner as the wild-type E-cadherin expressing cells. In line with these observations, an
increase in the phosphorylation level of βintegrin was observed for cells expressing the V832M mutation, while
14
T340A expressing cells show reduced levels of βintegrin activity when compared to cells expressing the wild-type
protein. These results demonstrate that E-cadherin mutations can influence the motility behaviour of tumour cells,
by perturbing their interaction with the ECM. We compared the expression profile, by cDNA array analysis, of two
non-expressing human E-cadherin cell lines (MDA-MB 231 and MDA-MB 435) infected with mock, wild-type Ecadherin and E-cadherin missense mutations T340A and V832M. We verified overexpression of Rho in T340A cell
line and overexpression of NFkB in V832M cell line. In mutated cell lines we verified overexpression of MMP’s and
Bcl2. The later results prompted us to study the apoptotic level of these mutated cells. Using the same subset of
mutations, we were recently able to demonstrate that in vitro loss of functional E-cadherin renders cells more
resistant to apoptotic stimuli (Ferreira et al. 2005). We also showed the existence of a possible interplay between Ecadherin and the antiapoptotic bcl-2, bringing new insights into the understanding of the tumorigenic process
dependent of E-cadherin deregulation. As well worthy of note, the apoptotic agent taxol was used in this study to
induce cell death. Taxol is a chemical widely used in the treatment of advanced cancers including epithelial tumors
resulting from E-cadherin loss; our results question the effectiveness of this treatment in this type of tumors and calls
for the need of further research on the subject.
Signaling pathways mediated by IL-6R- Recent studies suggest that the interleukin-6R/GP130 receptor signalling
pathway, where the transcription factor C/EBPbeta plays a key role, is likely to be involved in gastric carcinoma. In
this study, expression of C/EBPbeta was analysed in a series of 90 gastric carcinomas. CEBPB gene mutations
were screened for in a series of 35 primary gastric carcinomas. The functional activity of C/EBPbeta was analysed in
gastric carcinoma cell lines. In normal gastric mucosa, C/EBPbeta expression was restricted to cells with a
proliferative phenotype. In intestinal metaplasia, dysplasia, and gastric carcinoma of the intestinal and atypical
subtypes, C/EBPbeta was overexpressed (P=0.0005, for the association with histological type). C/EBPbeta and
Ki67, a marker of cell proliferation, were also co-expressed in primary gastric carcinoma. In one gastric carcinoma
we found a mutation of the CEBPB gene. Using gastric carcinoma cell lines we showed that transfected C/EBPbeta
is able to regulate the expression of endogenous cyclooxygenase-2 (COX-2). Back in the series of metaplasia,
dysplasia and gastric carcinoma lesions we observed a very high degree of overlap between expression of C/EBPbeta and COX-2. In gastric carcinoma cell lines we have also shown that transfected C/EBP-beta is able to modulate
proliferation in these cells. These results suggest that C/EBPbeta may both be a marker of neoplastic transformation
and play an active role in gastric carcinogenesis through its ability to up-regulate COX-2 expression.
3d) Signaling pathways mediated by P-cadherin - We found that hypomethylation of P-cadherin promoter is
associated to P-cadherin expression in breast invasive carcinomas (Paredes et al., 2005). P-cadherin expression
showed a strong correlation with high histologic grade, increased proliferation, c-erbB-2 and p53 expression, lack of
estrogen receptor, and poor patient survival. This overexpression can be regulated by gene promoter methylation
because the 5-Aza-2’-deoxycytidine treatment of MCF-7/AZ cells increased P-cadherin mRNA and protein levels.
Additionally, we found that 71% of P-cadherin-negative cases showed promoter methylation, whereas 65% of
positive ones were unmethylated (P = 0.005). The normal P-cadherin-negative breast epithelial cells showed
consistent CDH3 promoter methylation. Functionally, we verified that breast cancer cells stably expressing Pcadherin (by retroviral transduction) are invasive in collagen and Matrigel matrixes and we identified that this
functional effect requires the juxtamembrane domain of its cytoplasmic tail. Based on this previous data, we did not
know if the induction of cell invasiveness by P-cadherin expression was a consequence of increased migration
and/or due to other factors, such as the upregulation of metalloproteinases. In order to analyse cell migration, we
performed a wound-healing assay for MCF-7/AZ cells retrovirally transduced with P-cadherin. P-cadherin
transduced cells migrated significantly faster than vector-transduced cells. We still have found that these cells
express higher levels of MMP-1 and MMP-2, and these are more active than the ones expressed in the control cells.
3e) Known target-specific therapeutic drugs in breast cancer
We have been focused on PDGFR-α/c-KIT (Imatinib) , EGFR (IRESSA) andHER2/neu (Herceptin),
We investigate the potential significance of PDGFR-α expression in invasive mammary carcinomas (Carvalho et al.,
2005). We used immunohistochemistry to detect PDGFR-α overexpression on a series of 181 formalin-fixed paraffin
embedded invasive ductal breast carcinomas and in two breast cancer cell lines: MCF-7 and HS578T. PDGFR-α
expression was observed in 39.2% of the breast carcinomas and showed an association with lymph node
metastasis (P = 0.0079), HER-2 expression (P = 0.0265) and Bcl2 expression (P = 0.0121). A correlation was also
found with the expression of platelet-derived growth factor A (PDGF-A; P= 0.0194). The two cell lines tested did not
express PDGFR-α. Screening for mutations revealed alterations in the PDGFR-α gene at the following locations:
2500A->G, 2529T->A and 2472C->T in exon 18 and 1701G->A in exon 12. We also found an intronic insertion
IVS17-50insA at exon 18 in all sequenced cases. None of these genetic alterations was correlated with PDGFR-α
expression. The cell lines did not reveal any alterations in the PDGFR-α gene sequence. As a conclusion we
observed that PDGFR-α is expressed in invasive breast carcinomas and is associated with biological
aggressiveness. The genetic alterations described were not correlated with protein expression, but other
mechanisms such as gene amplification or constitutive activation of a signalling pathway inducing this receptor could
still sustain PDGFR-α as a potential therapeutic target.
Further, we studied a series of metaplastic breast carcinomas for EGFR and HER2 expression and amplification
(Reis-Filho et al., 2005). Metaplastic breast carcinomas constitute a heterogeneous group of neoplasms, accounting
for less than 1% of all invasive mammary carcinomas. HER 2 and EGFR have attracted much attention in the
15
medical literature over the past few years owing to the fact that humanized monoclonal antibodies against HER2 and
therapies directed against the extracellular ligand-binding domain or the intracellular tyrosine kinase domain of
EGFR have proven successful in treating certain types of human cancer. We investigated whether HER2 and EGFR
overexpression was present and evaluated gene amplification in a series of metaplastic breast carcinomas. Twentyfive metaplastic breast carcinomas were immunohistochemically analyzed using a monoclonal antibody (31G7) for
EGFR and two antibodies for HER2 (Herceptest and CB11) and scored using the Herceptest scoring system. Gene
amplification was evaluated by chromogenic in situ hybridization using Zymed Spot-Light EGFR and HER2
amplification probe. Nineteen (76%) metaplastic breast carcinomas showed EGFR ovexpression, and EGFR
amplification was detected in seven cases (37%): three carcinomas with squamous differentiation and four spindle
cell carcinomas. One case exhibited HER2 overexpression of grade 2+, but HER2 gene amplification was not
detected. In conclusion we observed that metaplastic breast carcinomas frequently overexpressed EGFR, which
was associated with EGFR gene amplification in one-third of cases. Our findings suggest that some patients with
metaplastic breast carcinomas might benefit from novel therapies targeting EGFR.
We have still evaluated the status of HER2 in tissue microarrays (TMAs) of invasive breast cancer, previously
evaluated by fluorescence in situ hybridization method in conventional sections. We performed IHC, with the mouse
monoclonal antibody CB11 and the rabbit monoclonal antibody SP3, and CISH in 10 TMA blocks with 190 formalinfixed paraffin-embedded invasive breast carcinomas. In parallel, HER2 activation status was assessed by IHC using
a phosphorylated HER2 antibody (PN2A). The correlation between the two antibodies, SP3 and CB11, was
statistically significant (p<0,0001) with an agreement rate of 97,7%. We found expression of the phosphorylated
HER2 in 1,6% of the cases, all with gene amplification. The two antibodies revealed specificity of 98% and sensitivity
between 59% (CB11) and 56% (SP3), using FISH as the gold standard method. The agreement between FISH and
CISH was excellent (kappa test= 0,91), with high positive and negative predictive values (92% and 98%,
respectively). Our results show that both SP3 and CISH can be used as methods to evaluate HER2 status, in
alternative to CB11 and FISH. However, further studies are required to confirm these results.
Research plans for 2006
The group aims to follow the same line of research, to establish new international collaborations and to win national
and international projects. We aim to submit one patent in 2006 for evaluation. We aim to publish between 15-25
manuscripts in peer-review International Journals with IF above 3 and 2 manuscripts above 6 IF.
We aim at evaluating if metaplastic breast carcinomas share molecular and pathological features previously found in
basal like breast carcinomas in order to be able to offer a precise prognosis and correct therapeutics in this type of
breast cancer.
We aim to profile by CGH array distinct groups of breast carcinoma to identify chromosomal regions that can
harbour genes that can be used as molecular markers for diagnosis, prognosis or therapeutic evaluation in breast
cancer.
We aim at further studying the signalling pathways mediated by de novo expression of P-cadherin and find the
pivotal proteins for cell invasion in breast cancer.
We aim at characterizing the expression profile (cDNA microarray) and the protein activation status (kinase and
phosphatase screenings) of cell lines stably expressing either wild-type E-cadherin or distinct HDGC-associated Ecadherin germline missense mutations. For this study, we will select mutations that were shown to have distinct
cellular behaviours (i.e motility) in vitro.
We aim at identifying second-hit mechanisms leading to E-cadherin dowregulation in sporadic and hereditary forms
of diffuse gastric carcinoma and to identify epi/genetic mechanisms leading to E-cadherin silencing in the germline of
HDGC patients.
We aim at investigating how H. pylori strains with different virulence mediate host cell effects and interfere with
signalling pathways relevant to gastric carcinogenesis.
We aim to unravel the signalling pathways that lead to overexpression of CEBP in gastric carcinoma and studying its
cellular effects.
We aim at identifying the cellular effects mediated by BRAF activating in colon cancer cell lines and to verify its
timing of occurrence using colorectal lesion in different stages of malignancy. Further we aim to unravel the
molecular mechanism underlying the association between hMLH1 promoter methylation methylation and BRAF
activating mutation in colorectal MSI carcinoma.
Financial support of the Cancer Genetics Group
The group got for 2005, after deduction of 20% overheads, grants and missions for lab running costs- 224.974.91
Euros.
16
Apart from the projects listed/numbered below the group has won two re-equipment Projects:
Project from Fundação para a Ciência e a Tecnologia (REEQ/218/2001) PI: Céu Figueiredo (125.000.00 Euros) and
other from Fundação Calouste Gulbenkian (3563274-S) -PI: Céu Figueiredo (69.000.00 Euros).
PI: F Schmitt
1.
2002-2006 - “DNA repair gene polymorphisms in breast cancer patients from Portuguese origin”, FLAD
(172/2002), 50.000,00€.
2.
2003-2006 - “Myoepithelial differentiation in breast carcinomas: pathological characterization and clinical
implications”, FCT (POCTI/CBO/45157/2002), 54.000,00€.
3.
2004-2005 - “Study of the molecular pattern of the p63 gene in human breast tumours”, DRCT Governo
Regional dos Açores (76/2004), 16.775,00€.
4.
2005-2008 - “P-cadherin in Breast Cancer: what regulates its aberrant expression and how it can induce
invasion of neoplastic cells?”, FCT (POCTI/BIA-BCM/59252/2004), 90.000,00€.
FSchmitt got for 2005 for lab running costs- 16.146.16€.
PI: C Figueiredo
1.
2005- 2008- “Effects of Helicobacter pylori on gastric epithelial cells”. FCT (POCI/SAU-IMI/56681/2004),
100.000,00€
2.
2005-2007- “Gene-environment interactions in human carcinogenesis with emphasis on gastric cancer”
FCT (CONC-REEQ/218/2001), 125.000,00€
3.
2003-2005- “Avaliação das consequências da infecção pelo Vírus do Papiloma Humano (HPV) e seus
diferentes genótipos no desenvolvimento de carcinoma do colo do útero numa população portuguesa”.
Fundação Calouste Gulbenkian (3563274-S), 69.900,00€
4.
2005-2007- “Avaliação dos riscos do consumo de sal e da infecção por Helicobacter pylori no
desenvolvimento de carcinoma gástrico e das suas lesões precursoras em Portugal” Agência
Financiadora: Agência Portuguesa de Segurança Alimentar, 186.446,00€
CFigueiredo got for 2005 for lab running costs- 22.886.00€.
PI: F Carneiro
1.
2002-2005- “Application of microarray CGH for the study of chromosomal instability at different stages of
gastric carcinogenesis” FCT (POCTI/CBO/41179/2001), 101.110,00€
2.
2005-2008-“Functional analysis of the E-cadherin repressors ZEB1, Slug and E12/E47 in gastric
carcinogenesis” FCT (POCI/SAU-OBS/57275/2004), 96.800,00€
FCarneiro got for 2005 for lab running costs- 14.860.80€.
PI : JC Machado
1. 2003 -2005” Helicobacter pylori virulence and interleukin-1 polymorphisms: interplay in gastric
carcinogenesis.” FCT(POCTI/CBO/41550/2001), 93.615,00€
2.
3.
2004-2006- “Identification of genetic markers for risk of development of gastric carcinoma in the
Portuguese population”. Programa Operacional de Saúde / SAUDE XXI (FEDER), Ministério da Saúde,
APIFARMA (SIFEC nº 15-01-01-FDR-00158), 300.000,00€
2005-2008- “Teste de susceptibilidade genética para determinação de risco de desenvolvimento de
Doença de Crohn na população Portuguesa” - Programa IDEIA, Agência de Inovação (TSG-CROHN),
200.000,00€
JCMachado got for 2005 for lab running costs- 67.774.81€.
PI: G Suriano
1.
2005-2008-“E-cadherin germline missense mutations and hereditary diffuse gastric cancer: a model for the
identification of the E-cadherin-dependent signaling pathways pivotal for cell invasion”. FCT (POCTI/SAUOBS/57670/2004), 43.590,00€
2.
2005-2007-“Study of pathological phenotypic heterogeneity using the ornithine transcarbamylase (OTC) as
model”. FCT (POCI/CVT/58082/2004), 63.760, 00€
17
3.
2005-2008-“Mechanisms of cell invasion: possible application to oncobiology”. Programa IDEIA, Agência de
Inovação (INV-ONC-DPN), 294.899,00€
GSuriano got for 2005 for lab running costs- 28.824.84€.
PI: C Oliveira
1. 2005-2008- “Identification of molecular mechanisms underlying gastric cancer in positive and negative
families for germline CDH1 mutations”. FCT (POCTI/SAU-OBS/58111/2004). 91.180,00€
COliveira got for 2005 for lab running costs- 14.950.00€.
PI: R Seruca
1.
2002-2005-“Cadherin germline mutations and hereditary diffuse gastric cancer: functional biochemical and
molecular characterization”. FCT (POCTI/CBO/40820/2001), 89.275,00€
2.
2004-2006-“Biological role of Braf activation in colon carcinogenesis. STI-571/Gleevec as therapeutic
agent?” Novartis-Portugal, 56.399,00€
3.
2005-2008- “BRAF and KRAS mutations in mismatch repair deficiency colon cancer.New prognostic and
therapeutic tool?”.FCT (POCI/SAU-OBS/56921/2004), 78.690,00€
RSeruca got for 2005 for lab running costs- 58.372.33€.
1.
PI: J Paredes
1.
2006-2007 – “Cell signalling mediated by P-cadherin expression in neoplastic invasion modulation using in
vitro assays”, Prémio Gulbenkian de Estímulo à Investigação (FCG 55/05), 10.000€.
Supervision of PHD and Master thesis:
1.
F Schmitt- Carla Costa – PhD thesis - “Essential role for angiogenic factors and bone marrow-derived
endothelial/hematopoietic precursor cells in the growth of solid tumours”, Medical Faculty of Porto
University, 2005.
2.
F Schmitt- Manuela Lacerda – PhD thesis - "Breast cancer progression study using as model in situ
carcinoma and invasive carcinoma with predominant in situ component", Medical Faculty of Porto
University, 2005.
3.
F Schmitt- Inês Matos França de Carvalho – Master thesis - “PDGFR alpha expression in invasive breast
carcinomas”, Molecular Genetics Master Course of Minho University, 2005.
4.
F Schmitt- Maria João Coelho Machado – Master thesis - “ Oestrogen and TGF beta transduction pathway
in angiogenesis”, Molecular Master Course of Aveiro University, 2005.
5.
F Schmitt- André João Almeida Marques Soares de Albergaria – Master thesis - “ Methylation pattern of
CDH3 gene in human mammary tumours”, Molecular Oncology Master Course of Medical Faculty, Porto
University, 2005.
6.
F Schmitt- Sílvia Teresa Valmor da Silva Pinto de Carvalho – Master thesis - “EGFR evaluation in invasive
breast carcinomas: correlation between immunohistochemistry and chromogenic in situ hybridization”,
Molecular Oncology Master Course of Medical Faculty, Porto University, 2005.
7.
F Schmitt- Sara Alexandra Vinhas Ricardo – Master thesis - “HER2 evaluation in tissue microarrays of
invasive breast carcinomas by immunohistochemistry and chromogenic in situ hybridization”, Molecular
Oncology Master Course of Medical Faculty, Porto University, 2005.
Published abstracts
1.
Albergaria A, Paredes J, Oliveira J, Ricardo S, Leitão D, Milanezi F, Schmitt F, Jerónimo C. Aberrant Pcadherin expression is an indicator of clinical outcome in invasive breast carcinomas and is associated with
CDH3 promoter methylation status. FEBS Journal 272: 474, 2005
2.
Carvalho I, Milanezi F, Martins A, Reis RM, Schmitt F. Overexpression of platelet-derived growth factor
receptor alpha in breast cancer is associated with tumour progression. Breast Cancer Research 7: R788R795, 2005.
3.
Figueira P, Vieira A, Saraiva C, Carneiro F, Tavarela-Veloso F: Limitações no diagnóstico imagiológico de
lesões hepáticas focais. Jornal Português de Gastrenterologia 12: 87, 2005.
18
4.
Figueiredo C, Maximo V, Soares P, Sousa S, Carneiro F, Seruca R, Sobrinho-Simoes M. MtDNA
alterations in Helicobacter pylori chronic gastritis and gastric carcinoma. Helicobacter 10: 473-4.
5.
Macedo G, Lopes S, Pereira P, Castro I, Maia JC, Fonseca E, Carneiro F, Tavarela-Veloso F: Complicação
tardia de transplante hepático num doente de Wilson. Jornal Português de Gastrenterologia 12: 26, 2005.
6.
Macedo G, Sousa Machado A, Lopes S, Araújo F, Carneiro F, Tavarela-Veloso F: Caracterização
histológica e imunocitoquímica numa população com hepatite B crónica e AgHBc negativo. Jornal
Português de Gastrenterologia 12: 90, 2005
7.
Oliveira MJ, Costa AC, Henriques L, Thomas RJ, Atherton J, Machado JC, Mareel M, Leroy A, Figueiredo
C. Helicobacter pylori stimulates cancer cell invasion: molecular mechanisms involved. Helicobacter 10:
475.
8.
Paredes J, Albergaria A, Ribeiro AS, Milanezi F, Schmitt F. P-cadherin expression is involved in migration
induction of MCF-7/AZ breast cancer cells. FEBS Journal 272: 75, 2005)
9.
Regalo G, Canedo P, Campos ML, Loncar MB, Figueiredo C, Carneiro F, Machado JC. C/EBP-beta is
expressed in gastric carcinoma and leads to overexpression of cyclooxygenase-2. Helicobacter 10: 506,
2005.
10. Rocha GA, Silva JP, Guerra JB, Rocha AM, Bittencourt P, Cabral MM, Nogueira AMM, Figueiredo C,
Queiroz D. Helicobacter pylori babA2 strains as a risk factor for duodenal ulcer in childhood.
Gastroenterology 128: A592.
Papers published in 2005:
1.
Carneiro F, Sobrinho-Simões M: Hereditary diffuse gastric cancer. Lessons from histopathology. Advances
in Anatomic Pathology 12:151-152, 2005.
2.
Domingo E, Niessen RC, Oliveira C, Alhopuro P, Moutinho C, Espín E, Armengol M, Sijmons RH,
Kleibeuker JH, Seruca R, Aaltonen LA, Imai K, Yamamoto H, Schwartz SJr, Hofstra R MW: BRAF-V600E
in not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2
genes. Oncogene 24:3995-3998, 2005.
3.
Duarte IS, Santos AM, Sousa H, Catarino R, Pinto D, Matos A, Pereira D, Moutinho J, Canedo P, Machado
JC, Medeiros R. G-308A TNF-alpha polymorphism is associated with an increased risk of Invasive cervical
cancer. Biochemical Biophysics Research Communications 334: 588-592, 2005.
4.
Dufloth RM, Carvalho S, Heinrich JK, Shinzato JY, Santos C, Zeferino LC, Schmitt F. Analysis of BRCA1
and BRCA2 mutations in Brazilian breast cancer patients with positive family history. Sao Paulo Med J 123:
192-197, 2005.
5.
Dufloth RM, Costa S, Schmitt F, Zeferino LC. DNA repair gene polymorphisms and susceptibility to familial
breast cancer in a group of patients from Campinas, Brazil. Genetics and Molecular Research 4: 771-782,
2005.
6.
Ferreira AC, Almeida S, Tavares M, Canedo P, Pereira F, Regalo G, Figueiredo C, Trindade E, Seruca R,
Carneiro F, Amil J, Machado JC, Tavarela-Veloso F. NOD2/CARD15 and TNFA, but not IL1B and IL1RN,
are associated with Crohn’s disease. Inflammatory Bowel Disease 11:331-339, 2005.
7.
Ferreira P, Oliveira MJ, Beraldi E, Mateus AR, Nakajima T, Gleave M, Yokota J, Carneiro F, Huntsman D,
Seruca R, Suriano G: Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol
in vitro. Experimental Cell Research 310:99-104, 2005.
In this work we demonstrate that loss of functional E-cadherin not only increases cell invasion but
also render cells more resistant to pro-apoptotic agents, such as taxol.
8.
Figueiredo C, Machado JC, Yamaoka Y. Pathogenesis of Helicobacter pylori infection. Helicobacter 10:.1420, 2005.
9.
Frebourg T, Oliveira C, Hochain P, Karam R, Manouvrier S, Graziadio C, Vekemans M, Hartmann A, BaertDesurmont S, Alexandre C, Lejeune Dumoulin S, Marroni C, Martin C, Castedo S, Lovett M, Winston J,
Machado JC, Attie T, Jabs EW, Cai J, Pellerin P, Triboulet JP, Scotte M, Le Pessot F, Hedouin A, Carneiro
F, Blayau M, Seruca R: Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse
gastric cancer. Journal of Medical Genetics 2005 Apr 14; [Epub ahead of print].
10. Gonzalez CA, Pera G, Agudo A, Bueno-de-Mesquita HB, Ceroti M, Boeing H, Schulz M, Del Giudice G,
Plebani M, Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S, Berglund G, Siman H, Hallmans G,
Stenling R, Martinez C, Dorronsoro M, Barricarte A, Navarro C, Quiros JR, Allen N, Key TJ, Bingham S, Day
NE, Linseisen J, Nagel G, Overvad K, Jensen MK, Olsen A, Tjonneland A, Buchner FL, Peeters PH,
19
Numans ME, Clavel-Chapelon F, Boutron-Ruault MC, Roukos D, Trichopoulou A, Psaltopoulou T, Lund E,
Casagrande C, Slimani N, Jenab M, Riboli E. Fruit and vegetable intake and the risk of stomach and
oesophagus adenocarcinoma in the European Prospective Investigation into Cancer and Nutrition (EPICEURGAST). International Journal of Cancer. 2005 Dec 27; [Epub ahead of print].
11. Gorringe KL, Chin SF, Pharoah P, Staines JM, Oliveira C, Edwards PA, Caldas C: Evidence that both
genetic instability and selection contribute to the accumulation of chromosome alterations in cancer.
Carcinogenesis 26:923-930, 2005.
12. Granja NM, Begnami M, Bertolan J, Longatto-Filho A, Schmitt FC. Desmoplastic small round cell tumour:
cytological and immunocytochemical features. Cytojournal 2:6, 2005.
13. Granja NM, Ricardo SA, Longatto A, Alves VA, Bedrossian CW, Wiley E, Schmitt FC. Potential use of loss
of heterozygosity in pleural effusions of breast cancer metastases using the microsatellite marker of the
16q22.1 region of the CDH1 gene. Anal Quant Cytol Histol 27: 61-66, 2005.
14. Karam R, Oliveira C, Seruca R, Carneiro F: Cancer Gástrico Hereditário. In: Atualização em Cancer Gástrico.
Linhares E, Lourenço L, Sano T (eds). Editora Tecmedd, São Paulo, 2005.
15. Longatto-Filho A, Albergaria A, Paredes J, Moreira MA, Milanezi F, Schmitt FC. P-cadherin expression in
glandular lesions of the uterine cervix detected by liquid-based cytology. Cytopathology 16: 88-93, 2005.
16. Longatto-Filho A, Martins A, Costa SMA, Schmitt FC. VEGFR-3 expression in breast cancer tissue is not
restricted to lymphatic vessels. Pathology Research Practice 201: 93-99, 2005.
17. Lynch HT, Grady W, Suriano G, Huntsman D. Gastric Cancer: New Genetic Developments. Journal Surgical
Oncology, 90:114-133, 2005.
18. Magro F, Pereira P, Carneiro F, Lopes JM, Teixeira A, Sarmento C, Lima J, Saraiva A, Teixeira A,
Tavarela-Veloso F: Colon stenosis in a patient with ulcerative colitis as a manifestation of mixed Mullerian
tumor of the peritoneum. Scandinavian Journal of Gastroenterology 40: 1251-1254, 2005.
19. Magro F, Pereira P, Carneiro F, Tavarela-Veloso F: Reactive hepatitis in a patient with Crhon’s disease
successfully treated with infliximab: does tumor necrosis factor alpha play a role in reactive hepatitis?
Inflammatory Bowel Disease 11: 88-90, 2005.
20. Magro F, Pereira P, Carneiro F, Tavarela-Veloso F: Unusual presentation of tuberculosis after infliximab
therapy. Inflammatory Bowel Disease 11: 82-84, 2005.
21. Maia L, Dinis J, Cravo M, Claro I, Baltazar C, Fonseca I, Veloso T, Capelinha AF, Carneiro F, Nobre-Leitão
C: Who takes the lead in the development of ulcerative colitis-associated colorectal cancers (UCACRCs):
mutator, suppressor or methylator pathway? Cancer Genetics and Cytogenetics 162: 68-73, 2005.
22. Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F. P63, Cytokeratin 5 and P-Cadherin: three
molecular markers to distinguish basal phenotype in breast carcinomas. Virchows Archives 447: 688-694,
2005.
23. Mergulhao P, Magro F, Pereira P, Correia R, Lopes JM, Magalhaes J, Dias JM, Carneiro F, Tavarela-Veloso
F: Gingival hyperplasia as a first manifestation of Crohn's disease. Digestive Diseases and Sciences
50:1946-1949, 2005.
24. Oliveira C, Moreira H, Seruca R, Cardoso de Oliveira M, Carneiro F: Role of pathology in the identification
of Hereditary Diffuse Gastric Cancer: Report of a Portuguese family. Virchows Archives 446:181-184, 2005.
In this work we describe the first HDGC Portuguese family with E-cadherin germline mutations.
25. Oliveira C, Velho S, Domingo E, Preto A, Hofstra RMW, Hamelin R, Yamamoto H, Seruca R, Schwartz Jr
S: Concomintant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI
sporadic colorectal cancer. Oncogene 24:7630-7634, 2005.
26. Oliveira MS, Fraga AG, Torrado E, Castro AG, Pereira JP, Longatto-Filho A, Milanezi F, Schmitt FC,
Wayne MM, Portaels F, Silva MT, Pedrosa J. Infection with Mycobacterium ulcerans induces persistent
inflammatory responses in mice. Infection and Immunity 73: 6299-6310, 2005.
27. Paredes J, Albergaria A, Oliveira JT, Jerónimo C, Milanezi F, Schmitt FC. P-cadherin overexpression is an
indicator of clinical outcome in invasive breast carcinomas and is associated with CDH3 promoter
hypomethylation. Clinical Cancer Research 11: 5869-5877, 2005.
28. Regalo G, Wright NA, Machado JC. Trefoil factors: from ulceration to neoplasia. Cell Molecular Life
Science 62: 2910-2915, 2005.
In this work we discuss the role of trefoil peptides in the development of gastric tumours.
20
29. Reis R, Martins A, Ribeiro SA, Basto D, Longatto-Filho A, Schmitt FC, Lopes J. Molecular characterization
of PDGFR-alpha/PDGF-A and c-KIT/SCF in gliosarcomas. Cell Oncology 27: 319-326, 2005.
30. Reis-Filho JS, Milanezi F, Carvalho S, Simpson PT, Steele D, Savage K, Lambros MBK, Pereira E,
Nesland J, Lakhani SR, Schmitt FC. Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene
amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysis.
Breast Cancer Research 7: R1028-R1035, 2005.
31. Reis-Filho JS, Schmitt FC. Fluorescence in situ hybridization, comparative genomic hybridization, and other
molecular biology techniques in the analysis of effusions. Diagnostic Cytopathol 33: 294-299, 2005.
32. Reis-Filho JS, Simpson PT, Jones C, Steele D, Mackay A, Iravani M, Fenwick K, Valgeirsson H, Lambros
M, Ashworth A, Palacios J, Schmitt F, Lakhani S. Pleomorphic lobular carcinoma of the breast: role of
comprehensive molecular pathology in chararacterization of an entity. Journal of Pathology 207: 1-13,
2005.
33. Reis-Filho JS, Simpson PT, Galea T, Lakhani SR. The molecular genetics of breast cancer: The
contribution of comparative genomic hybridisation. Pathology Research Practice 201: 713-725, 2005.
34. Suriano G, Vrcelj N, Senz J, Ferreira P, Masoudi H, Cox K, Nabais S, Lopes C, Machado JC, Seruca R,
Carneiro F, Huntsman DG: beta-Catenin (CTNNB1) gene amplification: A new mechanism of protein
overexpression in cancer. Genes Chromosomes and Cancer 42:238-246, 2005.
In this work we describe a new mechanism of beta-catenin activation, a signalling molecule pivotal
for colon cancer development, which is also involved in the progression of a small percentage of
gastric carcinomas.
35. Suriano G, Yew S, Ferreira P, Senz J, Kaurah P, Ford JM, Longacre TA, Norton JA, Chun N, Young S, Oliveira
MJ, MacGillivray B, Rao A, Sears D, Jackson CE, Boyd J, Yee C, Deters C, Pai GS, Hammond LS, McGivern
BJ, Medgyesy D, Sartz D, Arun B, Oelschlager BK, Upton MP, Neufeld-Kaiser W, Silva OE, Donenberg TR,
Kooby DA, Sharma S, Jonsson BA, Gronberg H, Gallinger S, Seruca R, Lynch H, Huntsman DG The
characterization of a recurrent germline mutation of the E-cadherin gene: implications for genetic testing and
clinical management. Clinical Cancer Research, 2005, 11: 5401-9
36. Van Marck V, Stove C, Van Den Bossche K, Stove V, Paredes J, Vander Haeghen Y, Bracke M. Pcadherin promotes cell-cell adhesion and counteracts invasion in human melanoma. Cancer Research 65:
8774-83, 2005.
37. Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S Jr, Duval A, Carneiro F, Machado JC,
Hamelin R, Raquel Seruca R: The prevalence of PIK3CA mutations in gastric and colon cancer. European
Journal of Cancer 41:1649-1654, 2005.
In this work we describe for the first time PIK3Ca mutations in gastric tumours. These mutations are
likely to represent a new therapeutic target for gastrointestinal malignancies.
38. Willems S, Carneiro F, Geboes K: Gastric carcinoma with osteoclast-like giant cells and lymphoepithelioma-like
carcinoma of the stomach: two of a kind? Histopathology 47: 331-333, 2005.
21
Carcinogenesis
Group Leader: Leonor David, MD PhD.
Staff members: Fátima Gartner, PhD; Celso Reis, PhD; Raquel Almeida, PhD; Luis Filipe
Santos Silva, PhD; Ana Carvalho, post-Doc; Nuno Marcos, PhD student; Salomé Pinho, PhD
student; Paula Paulo, BI; Ana Magalhães, BI; Natália Costa, BI; Rita Barros, BI; Maria Manuel
Azevedo, BI; Joana Gomes, BI; Nuno Mendes, technician.
Objectives/Goals of the research activity - The main objective of the group is to identify
alterations of mucins and mucin glycosylation, associated with gastric carcinoma and
precancerous lesions, that may be relevant for the development of diagnostic and therapeutic
strategies. We are also engaged in understanding the molecular mechanisms involved in the
development of such alterations, including the identification of transcription factors responsible
for cancer/pre-cancer transdifferentiation.
Background and major achievements during 2005 – We demonstrated that the T
(Thomsen-Friedenreich) antigen expression is associated, in vivo and in vitro, with large alleles
of MUC1 mucin (large VNTR domain) (Paper 1). For the first time, we established a link
between protein size variation and glycosylation, which may be relevant to clarify previous
epidemiological evidence from the group showing associations of MUC1 VNTR with risk for
development of intestinal metaplasia and gastric carcinoma. Our group has previously shown
that individuals with small MUC1 mucin (small VNTR domain) were at increased risk for
development of gastric carcinoma and intestinal metaplasia both in Portugal and in Colombia.
To dissect the relevance of VNTR size we constructed a gastric carcinoma cell line model
(GP202) transfected with FLAG-MUC1 constructs with 0, 3, 9 and 42 TRs. In the transfected
clones expression of the cancer-associated glycan structure designated Thomsen-Friedenreich
antigen was observed in the clones with 9 and 42 repeats but not in the 0 and 3 TRs
transfected clones. To assess the consistency of the in vitro observations we analyzed a series
of gastric cancer patients typed by Southern blotting for the MUC1 VNTR and showed that the
same association holds in vivo – cancers from the patients with large VNTR domains have a
significantly higher frequency of Thomsen-Friedenreich antigen expression. The group has now
established a SiRNA approach to silence endogenous MUC1. The strategy aims at generating
a gastric carcinoma cell line knocked-down for MUC1 which will serve two future developments
of our work: 1. To compare biologic properties (adhesion, motility) of the cancer cells with and
without MUC1 and; 2. To generate transfected clones with different VNTRs and silenced
endogenous MUC1 to proceed in the evaluation of the role of mucin size/glycosylation in cell
biology and in adhesion of Helicobacter pylori to the engineered cells (Projects 1 and 2).
The group collaborated with international networks for the development of MUC1 mucin
cancer-associated glycopeptides. The MUC1 mucin represents a target antigen for cancer
immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas.
We performed chemoenzymatic synthesis of MUC1 Tandem Repeat glycopeptides, with
cancer-associated O-glycosylation, using a panel of recombinant human glycosyltransferases.
MUC1 glycopeptides with different densities of Tn and STn glycoforms were developed and
used as immunogens to evaluate in pre-clinical studies in animal models, and to define an
optimal vaccine design. The studies showed that the glycopeptides with complete O-glycan
occupancy (five sites per repeat) elicited the strongest antibody response suggesting that this
glycopeptide holds promise as a cancer vaccine. The results also suggest the glycopeptides
can be used for immunotherapy in cancer patients (Papers 2,3,4) (Project 3).
We have also established stable transfectants of gastric cell lines expressing the
sialyltranferase ST6GalNAc-I. This enzyme is responsible for the biosynthesis of the cancerassociated carbohydrate antigen Sialyl-Tn. In order to characterize the functional role of the
Sialyl-Tn antigen in gastric carcinoma cells we have performed in vitro studies to evaluate the
22
phenotypic behavior of the gastric cell transfectants. Preliminary studies shows major
phenotypic alterations of the Sialyl-Tn expressing transfectants, including decreased cell-cell
adhesion, increased motility and invasion. Immunoprecipitation assays indicate that the MUC1
mucin is one of the carriers of the Sialyl-Tn antigen. Further studies of co-localization of MUC1
and Sialyl-Tn are going to be performed by proximity ligation assays (Project 4).
The group is also addressing the role of carbohydrate antigens in the adhesion of Helicobacter
pylori using a Glyco-gene Chip array. The attachment of Helicobacter pylori to gastric cells is a
multistep process mediated by the interaction of bacterial adhesins to epithelial cell surface
glycans. HP binding/interaction with host cells is known to alter the expression of host genes,
leading to increased expression of pro-inflammatory molecules and modification of
carbohydrate structures that further contribute to gastric colonization. Unraveling the structures
responsible for this clearance and understanding the molecular mechanisms which lead to their
expression would greatly contribute to understand how HP infects the gastric epithelia and may
be a valuable help to design novel therapeutic strategies to combat HP infection. To that end
we have performed co-culture of human gastric cell lines with high and low pathogenicity
strains of HP. We have extracted RNA from the gastric cell line, prepared cDNA and used it for
Microarray hybridization analysis using the Glyco-gene Chip array from the Consortium for
Functional Glycomics. The Glyco-gene Chip array contains probe sets to monitor the
expression of 1,814 human transcripts and has been developed using Affymetrix technology.
The following classes of genes are represented on the GLYCOv2 Gene Chip:
glycosyltransferases;
glycan-binding
proteins;
sulfotransferases;
C-type
lectins,
neuraminidases, nucleotide Sugar synthesis and transporter proteins, N-glycan biosynthesis
related genes, Mucins, Proteoglycans, Growth factors, Cytokines, Chemokines, Conserved
oligomeric Golgi complex genes. A full list of the genes monitored by the Glyco-gene Chip
array
is
available
at
the
site:http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml
(Project 5).
We substantially progressed in the clarification of genotype/phenotype associations in the
Secretor and Lewis systems in an attempt to clarify their relevance for gastrointestinal
infectious diseases – Helicobacter pylori and Norwalk virus – and for the generation of cancer
associated Lewis structures. Hypomethylation of the FUT3 gene promoter was identified as a
key factor for the aberrant expression of Lewisa in cancer (Paper 5). Phenotypes for Secretor
and Lewis status were defined in a large cohort of individuals with FUT2 and FUT3 gene
polymorphisms previously characterized by the group, and their relevance for Helicobacter
pylori and Norwalk virus infection is currently being analyzed. Preliminary analysis of the data
shows that the phenotype/genotype characteristics of the two systems is not relevant for
Helicobacter pylori infection. The amount and completeness of data we have collected will help
to clarify a longstanding controversy in the literature about the putative relevance of Secretor
and Lewis status on Helicobacter pylori infection. On the other hand, our data indicate that
Secretor status, together with ABO histo-blood group phenotype for some viral strains, is
determinant for adhesion of viruses of the calicivirus family. In 2006 we will further clarify, in
collaboration with the group of Prof.Jorge Rocha from IPATIMUP, the paradoxical observation
that a new FUT2 mutation characterized by our group shows an almost absence of
heterozygotes in Portuguese and Danish populations (Projects 6,7, 8 and 9).
The group identified a new transcription factor – OCT-1 – that binds CDX2 gene promoter and
can play a role in initiating intestinal transdifferentiation of the gastric mucosa (Paper 6). We
have previously shown that CDX2 is critical for MUC2 gene transactivation, reinforcing the role
of CDX2 in intestinal differentiation/transdifferentiation. AP2 methylation-dependent binding to
MUC2 promoter was also characterized by our group (Paper 7). We have shown, by ChIP
assays, that CDX2 binds to its own promoter in several locations and are currently clarifying
the relevance of the different binding sites by site-directed mutagenesis of CDX2 promoter
constructs. The main objective of the group at this stage is to identify initiators of aberrant
23
CDX2 expression in gastric cells. OCT-1 is a candidate initiator despite that by itself it does not
transactivate CDX2 (see first period of this paragraph). We have excluded the direct relevance
of Helicobacter pylori in transactivation of CDX2 using two different approaches – co-cultures
of Helicobacter pylori with gastric cells and transfection studies with expression vectors for the
GagA gene. Two approaches are currently being explored: 1. The first hypothesis (bottom-up
strategy) is based on CDX2 promoter analysis with identification of Smad4 putative binding
sites, which led us to demonstrate CDX2 transactivation by Smad4, a transcription factor
involved in the transcription of both TGF-beta and BMPs target genes. Cdx2 regulation by
TGF-Beta/BMPs pathways is currently under investigation; 2. The second hypothesis (topdown strategy) is exploring cytokines that might be capable of transactivating CDX2, by using a
stable transfection of a CDX2 promoter/luciferase reporter.
The group was involved during 2005 in a large study of follow-up of a cohort of healthy
volunteers from Estaleiros Navais de Viana do Castelo, which represented a lot of effort for the
group: collection of blood-samples for genetic studies, saliva for secretor status, endoscopies
and biopsies, food questionnaires, etc. This study will allow to settle the relevance of geneenvironment interactions identified in previous studies and also to explore new candidate
targets for individual susceptibility for acquisition of Helicobacter pylori infection and for the
development of pre-neoplastic lesions of the stomach. A parallel study was launched in
Mozambique during 2005, which will give us access to a population that, despite having a high
rate of Helicobacter pylori infection, has a low rate of intestinal metaplasia and gastric
carcinoma. We expect that the combined analysis of both studies will clarify the real impact of
genetic and environmental factors in gastric carcinogenesis. We also expect to identify the
parameters (genetic and environmental) that should be included in the protocols for treatment
and follow-up of the patients infected with Helicobacter pylori. The study implies a close
collaboration with the Department of Pathology of Hospital Central de Maputo in Mozambique,
with the Departments of Gastroenterology of Hospital S.João in Porto and Hospital Central de
Maputo in Mozambique, with the Medical staff of Estaleiros Navais de Viana do Castelo and
with the Department of Epidemiology of the Medical Faculty of Porto (Projects 8 and 9).
The group was also involved in setting up, together with a biotechnology company from
Portugal – ATGC, and a company from Denmark - Dako, a kit for the diagnosis of intestinal
metaplasia based on the identification of alterations of the mucin expression profile using
monoclonal antibodies previously produced by the group.
The group “incorporated” Prof. Fátima Gartner, Salomé Pinho and Joana Gomes, that have
been working on breast cancer models in dogs. We are developing joint projects incorporating
the main expertise of the group into the dog breast cancer model. MUC1 represents a
promising marker in breast cancer, and tumour-associated carbohydrate antigens are
predictors of the clinical course and prognosis in mammary tumours. These molecules,
especially MUC1, have been the focus of attention for immunotherapeutic applications and
cellular and humoral immune responses to MUC1 have been documented in benign and
malignant breast tumours. We aim to establish dog mammary tumour cell lines in vitro and in
vivo, in athymic nude mice, with aberrant expression of cancer-associated carbohydrates.
Finally, we intend to develop animal models to test the immunogenicity of cancer cell lines with
different tumour-associated carbohydrate antigen profiles and test their capacity to generate
efficient immune responses (Projects 11 and 12).
Apart from the mainstream of the group objectives we collaborated in several publications,
mainly clinico-pathologic descriptions (see “Other” publications)
Work plan for 2006
Our work plan for 2006 aims to: 1. Generate a gastric carcinoma cell line silenced for MUC1
expression using SiRNA, which will be used to assess the adhesion and motility of the silenced
24
clones and the role of MUC1 in adhesion of Helicobacter pylori to the engineered cells; 2.
Identify the protein carrier(s) of the S-Tn carbohydrate in gastric cells and to identify and
characterize the alterations in glycosyltransferases expression induced by co-culture of gastric
cells with Helicobacter pylori (Glyco-gene Chip array); 3. Clarify the role of Secretor and Lewis
phenotypes/genotypes in Helicobacter pylori and Norwalk virus infections and to clarify the
“exclusion” of heterozygotes for a new mutation identified by our group in the Secretor (FUT2)
gene; 4. Identify the transcription factors/pathways involved in initiating gastric intestinal
metaplasia through activation of CDX2 intestinal homebox gene; 5. Characterize the
mucin/carbohydrate profile of breast cancers from dogs and generate cell lines with different
capacities to stimulate immune responses. The group will continue the involvement on the
epidemiology projects in Viana do Castelo and Mozambique and will be involved in setting up
the MALDI-TOF facility.
Financing/Projects
Apart from the projects listed/numbered below the group has won a Re-equipment Project:
“Mass spectrometry (MALDI-TOF) for protein characterization and proteomics in the north of
Portugal”. Re-equipment Project from Fundação para a Ciência e a Tecnologia
(REEQ/899/SAU/2005) PI: Celso Reis. Total budget for 2005-2006: 536.690€ (2005 – 50.000€;
2006 – 486.690€).
The group has also won Royalties from monoclonal antibodies sells: 2005 – 3.946€
• MUC5AC (MAB2011, Clone CLH2). CHEMICON INTERNATIONAL INC. 28835
Single oak drive, Temecula, CA 92590; California, USA.
• MUC5AC (NCL-MUC5). NOVOCASTRA LABORATORIES Ltd. Balliol Business
Park West, Benton Lane, Newcastle upon Tyne; NE12 8EW, United Kingdom.
• MUC6 (NCL-MUC6). NOVOCASTRA LABORATORIES Ltd. Balliol Business
Park West, Benton Lane, Newcastle upon Tyne; NE12 8EW, United Kingdom.
The group got 290.347€ funding during 2005 and has approved funding for 2006 (301.356€).
The funding for 2005, after deduction of 20% overheads (58.070€), and salaries (60.595€: one
technician – 13.700€/year; one pos-doc – 13.455€/9 months; an informatic technician at the
Epidemiology Department in the Medical Faculty - 8.940€/year; a student who performed
dietary questionnaires in Mozambique - 2.500€/year and 22.000€ for the 6BIs/variable
periods), included 171.682 € for Lab running costs.
1.
“Clarification of the biological role of MUC1 mucin variability in gastric carcinogenesis”.
Fundação para a Ciência e a Tecnologia (POCTI/CBO/44812/2002). PI: Luís Filipe
santos Silva. Total budget for 2003-2006 - 72.000€ (2005-24.000€; 2006-24.000€).
2.
“Clarification of the relevance of MUC1 mucin polymorphism in Helicobacter pylori
infection”. Fundação para a Ciência e a Tecnologia (POCI/SAU-IMI/56895/2004). PI:
Luís Filipe santos Silva. Total budget for 2005-2008 - 100.000€ (2005-33.000€; 200633.000€).
3. “Molecular mechanisms of biosynthesis of cancer associated mucin carbohydrate
antigens in intestinal metaplasia”. Fundação para a Ciência e a Tecnologia
(POCTI/CBO/44598/2002). PI: Celso Reis. Total budget for 2003-2006 - 66.600€ (2005
– 22.200€; 2006 – 22.200€).
4. “Molecular mechanisms of biosynthesis of cancer associated mucin carbohydrate
antigens in gastric carcinoma”. Association for International Cancer Research (AICR
grant Ref: 05-088). PI: Celso Reis. Total budget for 2005 – 2008 - 112.320€ (2005 –
37.440€; 2006 -37.440€).
25
5. “Identification of Glycosylation-associated genes induced by H. pylori in gastric cells: a
glycomic approach”. Fundação para a Ciência e a Tecnologia. POCI/SAUOBS/56686/2004 PI: Celso Reis. Total budget for 2005-2007: 55.000€. (2005 –
27.500€; 2006 – 27.500€).
6. “Genes associated to intestinal metaplasia of the gastric mucosa (MUC2 mucin and
fucosyltransferase FUT3): transcriptional regulation and relevance for Helicobacter
pylori adhesion” (Project POCI/SAU-OBS/55549/2004). PI: Leonor David. Total budget
for 2005-2008: 96.680€ (2006 – 33.000€).
7.
“Regulation of aberrant MUC2 mucin gene expression in intestinal metaplasia,
mucinous gastric carcinomas and gastric carcinoma cell lines: role of methylation and
putative transcription factors”. Fundação para a Ciência e a Tecnologia (Project POCTI/
CBO/39075/2001). PI: Leonor David. Total budget for 2002-2005: 83.000€ (2005 27.600€)
8. “Characterization of gastric lesions associated with Helicobacter pylori infection in
workers of a shipyard from Viana do Castelo (ENVC), three years after an initial
screening for the identification of risk factors from the host and from Helicobacter pylori
associated to the development of gastric disease, namely peptic ulcer and cancer”.
Fundação Calouste Gulbenkian (Project FC-54918). PI: Leonor David. Total budget for
2003-2005: 120.000€ (2005 - 40.000€).
9. “Lesions of the gastric mucosa in Mozambique and Portugal: the “African enigma”.
Study of biologic factors and lifestyle that contribute to understand the differences in
incidence of gastric carcinoma in two countries with a high prevalence of Helicobacter
pylori infection: Mozambique and Portugal”. Fundação Calouste Gulbenkian (Project
FC-68697). PI: Leonor David. Total budget for 2005-2007: 100.000€ (2005 - 33.000€;
2006 - 33.000€).
10. “Identification of signalling pathways involved in Cdx2 regulation in two human models
of altered intestinal differentiation: intestinal metaplasia and juvenile polyposis”.
Fundação para a Ciência e a Tecnologia (Project POCTI/SAU-OBS/55840/2004). PI:
Raquel Almeida. Total budget for 2005-2008: 98.500€ (2005 – 16.416€; 2006 –
32.833€).
11. “Biological characterization of canine mammary mixed tumours: histogenesis, tumour
progression and genetic alterations”. Fundação para a Ciência e a Tecnologia
(POCI/CVT/57795/2004). PI: Fátima Gartner. Total budget for 2005-2008: 92.225€
(2005 – 15.370€; 2006 – 30.741€).
12. “Cat mammary tumours-Pathologic, Molecular and Cytogenetic approaches”.
Fundação para a Ciência e a Tecnologia (POCI/CVT/62940/2004). PI: Fátima Gartner.
Total budget for 2005-2008: 82.927€ (2005 – 13.821€; 2006 – 27.642€).
Main international collaborations
•
Eppley Institute, University of Omaha – Michel Anthony Hollingsworth. This
collaboration was fundamental for the development of the FLAG-MUC1 clones and for
the present development of the work consisting in the implementation of the SiRNA for
silencing of endogenous MUC1 gene expression.
26
•
Faculty of Health Sciences of the University of Copenhagen – Ulla Mandel and Henrik
Clausen. This collaboration has been fundamental for characterization of carbohydrate
antigens and glycosyltransferases using unique monoclonal antibodies.
•
INSERM, Nantes – Jacques Le Pendu. This collaboration has been critical for the
prosecution of the study of Secretor and Lewis phenotypes/genotypes, due to the
unique expertise of Jacques Le Pendu in such a complex field.
•
INSERM, Lille – Isabelle Van Seuningen. This collaboration has been fundamental for
the establishment of promoter studies, namely transient transfection assays with
luciferase reporters and electrophoretic mobility shift assays (EMSA).
•
IMIM, Barcelona – Francisco Real and Carme de Bolos. This collaboration has allowed
completion of in situ hybridization techniques for mucin gene expression.
•
IMIM, Barcelona – Antonio Garcia Herreros. This collaboration was essential to set up
chromatin immunoprecipitation assays (ChIp).
•
Universitite des Sciences et Technologies de Lille – Anne Harduin-Lepers. This
collaboration has been useful in the cloning and recombinant expression of
silayltransferases.
•
University of Uppsala - Ola Soderberg. Will help us to establish proximity ligation
assays for the visualization and confirmation of protein-glycan interactions.
PhD Thesis
In 2005 three PhD students of the group defended their thesis at the Medical Faculty of Porto.
•
Carla Carrilho – A Pathologist from Mozambique, identified for the first time the HPV
types associated to cervix carcinomas from Mozambique and concluded that multiple
infections by several viral types are common (HPVs 16,18,31,33,35,45 e 58). These
results suggest the need for generating multivalent prophylactic vaccines to prevent this
neoplasia in Africa. Carla also identified markers useful for diagnostic purposes at initial
phases of the carcinogenesis process (P53, Ki67, simple mucin-type carbohydrates and
keratins). Title of the Thesis: “Cervix cancer in Mozambique. Role of human
papillomavirus (HPV) in the etiopathogenesis of cervix cancer and evaluation of the
usefulness of some markers in the diagnosis”.
•
Jacinta Serpa – A Biologist from Azores, explored the interface between humans and
many microorganisms mediated by two systems called Lewis and Secretor. The two
enzymatic systems determine the addition of fucoses to proteins, mainly mucins, and
glycolipids, and have a bearing on our susceptibility to get infections. Her work explored
the genetic polymorphisms responsible for inter-individual variability. She identified
known polymorphisms and, in the case of FUT2 gene, responsible for the secretor
status, two new gene variants were identified and characterized. She also
demonstrated that FUT3 gene promoter methylation modulates Lewis antigen, namely
cancer-associated silyl-Lewisa antigen, expression. Her work substantially contributed
to clarify genotype/phenotype associations in these systems and to evaluate the
relevance of susceptibility for bacterial (eg. Helicobacter pylori) and viral infections. Title
27
of the Thesis: “Secretor and Lewis phenotype/genotype and their relevance for
Helicobacter pylori infection”
•
Patrícia Mesquita – A Biochemist from Lisbon, clarified the mechanisms underlying the
aberrant expression of MUC2 mucin, normally expressed in the intestine, in gastric
intestinal metaplasia and 30% of gastric carcinomas. She identified the transcription
factor (CDX2) that is essential to trigger aberrant expression of the MUC2 mucin in
gastric cells and mapped the promoter sites used by CDX2. She further demonstrated
that site-specific hypomethylation of MUC2 promoter is essential for gene expression.
Her work contributed to understand regulatory mechanisms involved in generating
intestinal metaplasia that in many cases precedes gastric carcinoma. Title of the Thesis:
“Regulation of MUC2 mucin gene expression in gastric carcinoma and gastric
carcinoma cell lines – role of methylation and putative transcription factors”.
Selected publications:
1. Santos-Silva F, Fonseca A, Caffrey T, Carvalho F, Mesquita P, Reis C, Almeida R, David L,
Hollingsworth MA: Thomsen-Friedenreich antigen expression in gastric carcinomas is
associated with MUC1 mucin VNTR polymorphism. Glycobiology 15:511-517, 2005.
2. Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V,
Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, Burchell J, Clausen H:
Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer
specific anti-MUC1 antibody responses and override tolerance. Glycobiology 16(2):96107, 2006; 2005 Oct 5; [Epub ahead of print]
3. Kagan E, Ragupathi G, Yi SS, Reis CA, Gildersleeve J, Kahne D, Clausen H, Danishefsky
SJ, Livingston PO: Comparison of antigen constructs and carrier molecules for
augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn.
Cancer Immunology, Immunotherapy 54: 424-430, 2005.
4. Slovin SF, Ragupathi G, Fernandez C, Jefferson MP, Diani M, Wilton AS, Powell S,
Spassova M, Reis C, Clausen H, Danishefsky S, Livingston P, Scher HI: A bivalent conjugate
vaccine in the treatment of biochemically relapsed prostate cancer: a study of
glycosylated MUC-2-KLH and Globo H-KLH conjugate vaccines given with the new semisynthetic saponin immunological adjuvant GPI-0100 OR QS-21. Vaccine 23:3114-122,
2005.
5.Serpa J, Mesquita P, Mendes N, Oliveira C, Almeida R, Santos-Silva F, Reis CA, Le Pendu
J, David L: Expression of Lea in gastric cancer cell lines depends on FUT3 expression
regulated by promoter methylation. Cancer Letters (in press).
6.Almeida R, Almeida J, Shoshkes M, Mendes N, Mesquita P, Silva E, Van seuningen I, Reis
CA, Santos-Silva F, David L: OCT-1 is over-expressed in intestinal metaplasia and
intestinal gastric carcinomas and bind to, but does not transactivate, CDX2 in gastric
cells. Journal of Pathology 207: 396-401, 2005.
7. Mesquita P, Almeida R, Lunet N, Reis CA, Santos-Silva F, Serpa J, Van Seuningen I, Barros
H, David L: Metaplasia – a transdifferentiation process that facilitates cancer
development. The model of gastric intestinal metaplasia. Critical Reviews in Oncogenesis
(in press).
28
“Other” publications:
1. Marques T, David L, Reis CA, Nogueira A: Topographic expression of MUC5AC and
MUC6 in the gastric mucosa infected by Helicobacter pylori and in associated diseases.
Pathology Research and Practice 201: 665-672, 2005.
2. Carrilho C, Cirnes L, Alberto M, Buane L, Mendes N, David L: Distribution of HPV
infection and tumor markers in cervical intraepithelial neoplasia from cone biopsies of
Mozambican women. Journal of Clinical Pathology 58: 61-68, 2005.
3. Silva LF, Ribeiro D, David L, Felino A: Oral osteossarcoma: case report. Oral Oncology
41: 195-197, 2005.
4. Matos A, Faustino A, Lopes C, Ruttenam G, Gärtner F: Use of cytokeratin staining for the
detection of lymph node micrometastasis in canine mammary malignant tumours.
Veterinary Record (in press).
5. Matos A, Duarte S, Lopes C, Lopes JM, Gärtner F: Canine splenic hamartoma.: A case
report. Veterinary Record (in press)
29
GENETICS, EVOLUTION AND PATHOLOGY
Group Leader: Jorge Rocha, BSc, PhD
Staff members: Susana Seixas, BSc, post-Doc; Sandra Beleza, BSc, post-Doc (since May 2005);
Margarida Coelho, BSc, BI; João Oliveira, BSc, BI (since October 2005); Susana Pereira, undergraduate
student (Biochemistry).
Research interests and goals: The major goal of our research is to characterize the evolutionary forces
that shaped the current patterns of human genetic diversity and their implications in health and disease.
Our research focus both on the study of specific populations and on the analysis of the evolutionary
history of particular genes. At the population level, we are particularly interested in the study of African
populations and human groups derived from recent historical events where admixture is an important
component of population structure. At the gene level, we are especially interested in the detection of
genetic signatures of natural selection as a way to uncover functional relevance of genetic variation and
understand genetic adaptation to environmental changes.
Major activities in 2005: We have concluded a study on the evolutionary history of human lactase
persistence (lactose tolerance) based on the analysis of haplotype diversity around the lactase gene
(LCT) in 794 chromosomes from five ethnically diverse populations with different genetic backgrounds
and subsistence patterns (Portugal, Italy, Fulbe from Cameroon, São Tomé and Mozambique). Lactose
intolerance (lactase restriction), the primitive condition in all mammals, limits the use of fresh milk in
adults due to the decline in lactase activity after the weaning phase. The prevalence of this trait in
humans is highly variable and is negatively correlated with milk drinking habits of different human
populations. A major hypothesis- the Gene Culture Coevolution Hypothesis- explains the distribution of
lactose tolerance as the result of the recent selection pressure caused by the advantages of milk drinking,
but this hypothesis had been mostly based on geographic correlations. By using microsatellite markers
linked to a candidate mutation for lactose tolerance in a putative LCT promoter, we provided formal
genetic evidence that the mutation affording tolerance to lactose arose about 12 000 years ago in Eurasia
and underwent a rapid increase in frequency due to natural selection (Paper 1). This work was the main
subject of the Master Thesis of Margarida Coelho (see below) and is part of a project that aims at
understanding how major changes in subsistence patterns provided selective pressures leading to human
genetic adaptation (Project 1).
Still in the scope of our project on human genetic adaptation, we started to characterize the
intrahaplotypic diversity of a panel of hemoglobin β*S (HBB*S) mutants, collected in various ethnic
groups from different African populations (São Tomé, Angola, Mozambique, Cameroon). Spreading of
malaria in Africa is presently thought to be due to the adoption of agricultural practices, which created
the optimal conditions for the breeding of Anopheles vectors and the diffusion of plasmodial parasites.
However, population genetics support to the link between malaria and agriculture is still mostly based on
geographic correlations. We reasoned that a major line of evidence supporting the hypothesis that only
with the origins of agriculture in Africa did malaria have a significant selective impact, could be based
on the demonstration of the temporal closeness between the introduction of agriculture and the
emergence of malaria-protective mutations in human populations. As the HBB*S mutation is one of the
most important protective genetic factors against Plasmodium falciparum, we are presently
characterizing a battery of highly polymorphic microsatellites located at different distances from the
HBB locus in order to test major hypotheses about the evolutionary history of the HBB*S haplotypes
and its implications for the link between malaria and agriculture in Africa. This work is being done in
collaboration by João Oliveira and Margarida Coelho.
We concluded a resequencing study across 19 kb in a Serine Protease Inhibitor (SERPIN) gene subcluster located in chromosome 14q32.1, which includes the genes for α1-antitrypsin (PI), corticosteroid
binding globulin (CBG) and a α1-antitrypsin-like sequence (PIL), which is generally though to be a
pseudogene. SERPINs are a superfamily of homologous proteins that probably arose by gene duplication
30
and divergent evolution. Most SERPINs act as regulators of serine proteases in a number of fundamental
biological processes, like blood coagulation, fertilization, complement activation, fibrinolysis, tissue
repair, inflammation and tumor suppression. PI, the archetypical SERPIN, has a wide spectrum of
normal isoforms, and several deficiency-causing alleles. PI deficiency, one of the most frequent
hereditary diseases in populations of European origin, is associated to early-onset pulmonary emphysema
and different forms of liver disease. By surveying functional and non-functional regions within the
SERPIN subcluster in two ethnically distinct samples from Europe and Africa, we aimed to disclose
unknown genetic polymorphisms and provide a deeper insight into the evolutionary history of this gene
family and its implications in health and disease. We found a strong signal of natural selection in the
African population characterized by the high frequency of a relatively recent derived haplotype, carrying
a common 2 kb deletion in the PIL gene and displaying a considerable extended homozygosity across the
surveyed region. This signal of natural selection was confirmed by both formal tests of neutrality and
empirical analyzes based on HapMap interrogation. Furthermore, in contrast to previous suggestions that
PIL is a pseudogene, we found that a non-deletion PIL allele is associated with gene expression in testis
and leukocytes. In collaboration with Gianpaolo Suriano from the Cancer Genetics Group at IPATIMUP,
we developed a three dimensional model of the corresponding protein showing that it may be a new
secretory SERPIN with altered substrate specificity. Our finding that the non-deleted form of PIL is
indeed an active gene expressed in different human tissues, together with the observation that PIL is
deleted in chimpanzee, but intact in other Catarhine Primates, further suggests that natural selection is
favoring the pseudogenization of PIL. This work provides an illustration of how functionally relevant
genetic variation may be identified by studying natural selection. Moreover, this is one of the first
evidences that pseudogenization is not always a neutral process and may be actively favored by
directional selection. The work, presently in preparation for submission, has been developed by Susana
Seixas, as part of her post-Doc, with co-supervision of Prof. Anna Di Rienzo (Department of Human
Genetics of the University of Chicago). In 2006 we expect to further investigate the function of the PIL
gene by isolating the encoded protein through in vitro expression, followed by binding and function
assays to identify substrates and characterize inhibitory activity.
We concluded a fine scale characterization of the population structure of the Island of São Tomé, Gulf of
Guinea (Project 2). Our interest in São Tomé stems from the perception that human populations derived
from the Atlantic slaving process are natural laboratories that provide unique opportunities for integrative
studies aiming at the identification of key evolutionary determinants of current patterns of human cultural
and biological variation. Due to its limited geographic dimension (~850 km2), small population size (~150
000) and relatively recent complex peopling process, São Tomé may be considered an excellent model to
assess the microevolutionary impact and bio-cultural implications of the slave trade. In this study, we
attempted to describe and interpret the genetic structure of São Tomé without relying on predefined ethnic,
anatomical or geographical categories. To this end, we inverted the sequence by which the relations between
genetic and cultural variation are usually investigated by first sorting individuals into genetic clusters based
solely on multilocus microsatellite genotypes and then comparing the distribution of additional
phylogeographic informative markers (mt-DNA, Y-chromosome haplotypes, β-globin and Duffy blood
group loci) across the inferred genetic clusters. Using data from only 15 randomly selected microsatellite
loci, typed in 394 unrelated individuals from 14 localities, we coupled a transect sampling strategy with a
Bayesian clustering approach and found that São Tomé is far from being a single panmitic unit, despite the
maximum distance between any two sampled sites being less than 50 km. This uneven distribution was
found to be clearly more related to language than to geographic distance and was best captured by two
clusters. One of the clusters predominates in villages where the Angolar creole is the major autochthonous
language, the other cluster is linguistically much more heterogeneous and includes Forro speakers and
descendants of contracted laborers that originated from Mozambique, Angola and, especially since mid 20th
century, from Cape-Verde. By using different types of markers to analyze genetic variation across inferred
clusters, we found that the Angolar cluster, in spite of retaining the signature of genetic contributions from
both West Africa and Central-West Africa, carries a clear imprint of genetic drift. In contrast, the nonAngolar cluster remained open to more diverse genetic contributions, including higher levels of admixture
with European settlers. Comparison of these results with the available historical and linguistic data indicated
that our data are compatible with a long held popular belief that the Angolar group was founded by the
survivors of the wreckage of a slave ship, occurring just off the southeast shore of São Tomé. However, the
31
observed genetic patterns are also compatible with an alternative fission model in which genetic divergence
was caused by a kin-structured split in an ancestral population previously formed by the amalgamation of
diverse African contributions. These observations demonstrate that neither genetic microdifferentiation is
confined to archaic human societies nor homogenization is the only expected outcome of modern periods of
population expansion. Our focus in the small-scale study of genetic patterning in São Tomé provides an
illustration of how small discontinuous jumps lead to formation of clusters that may latter become important
aspects of human genetic variation in a more global scale. The work, presently prepared for submission, has
been substantially developed by Margarida Coelho, in collaboration with Cíntia Alves and Prof. António
Amorim from the Population Genetics group at IPATIMUP. We have also had the collaboration of Prof.
Giovanni Destro-Bisol (University of Rome1), Prof. Donata Luiselli (University of Bologne) and Dr. Tjerk
Hagemeijer, a linguist from the University of Lisbon.
During the typing of autosomal microsatellite loci in São Tomé, Cíntia Alves found that 2% of males had an
apparent X chromosome Amelogenin locus drop-out due to a failure in PCR amplification caused by a
mutation in a primer binding site (Paper 2). X chromosome Amelogenin had been previously reported in
Europeans, but at a much lower 0.3% frequency. With a 2% frequency in African males it is expected that
the frequency of female carriers and homozygotes will be 3.92% and 0.04%, respectively. This may have a
non-trivial impact in pre-natal diagnosis of certain XY chromosome abnormalities, like XXY, using
quantitative assays. These results emphasize the need for caution when applying solely amelogenin for sex
determination.
Still in the scope of our project on the Anthropogenetics of São Tomé (Project 2) and its implications in
trans-oceanic migrations launched by the Portuguese maritime trade, we proceeded with our cooperation
with Prof. Sérgio Pena (Federal University of Minas Gerais, Brazil) and collaborated in a study of Y
chromosome diversity in Brazil (Paper 3). Using slow evolving Y chromosome polymorphisms, we had
previously shown that, contrary to mt-DNA sequence variation, Y chromosomes of “white” Brazilians have
their immediate geographical origin exclusively in Europe, with low frequency of sub-Saharan African
chromosomes and virtual absence of Amerindian contribution. We had also found no differences between
Brazilians and Portuguese and even among Brazilians from distinct regions of Brazil. In order to test if the
lack of differentiation was a sex-biased and not a marker-biased phenomenon, we studied faster evolving Y
chromosome markers in samples from Brazil and Portugal. The population structure revealed by this work
confirmed that there are indeed no differences between Brazil and Portugal and no population
differentiation within four major geographical regions of Brazil. Nevertheless the fast evolving markers did
uncover a higher within population diversity in Brazil than in Portugal, which could be explained by the
input of diverse European Y chromosomes carried by several migration waves to Brazil. The data highlight
the significance of typing and combining markers evolving at distinct mutational paces to usefully assess the
levels of diversity in populations derived from distinct geographical origins, like Brazil.
As part of our activities in supporting the training of undergraduate students, we hosted in our group
Susana Pereira who is now a Biochemist from the University of Algarve. Susana has concluded a
preliminary work on DNA sequence variation in the human Matrix Gla Protein (MGP) in European and
African populations. Matrix Gla protein is a vitamin K-dependent protein accumulates in the
extracellular matrix of bone and cartilage and is an important inhibitor of cartilage and vessel
calcification. Mutations leading to loss of MGP function are responsible for Keutel syndrome, an
autosomal recessive disorder. The study involved the complete direct ressequencing of the whole MGP
gene in population samples from Europe and Africa and the characterization of major patterns of linkage
disequilibrium in the populations studied. By applying neutrality tests, Susana demonstrated that the
levels of polymorphism in MGP are lower than expected on the basis of interspecific divergence under
neutrality, suggesting the MGP locus may be under strong negative selection. This undergraduate work
was developed in collaboration with Prof. Leonor Cancela from the University of Algarve.
The group was also actively involved in field work in Africa during 2005. In June 2005 we made a field trip
to Mozambique (Jorge Rocha and Margarida Coelho) in order to collect buccal swab samples and establish
contacts with official and traditional authorities from the provinces of Cabo-Delgado, Nampula, Zambézia,
32
Sofala, Manica and Maputo. This work was done in collaboration with Prof. António Prista (Pedagogic
University of Mozambique), as part of a broader project aimed to characterize the human biological
diversity in Mozambique, which involves a multidisciplinary team with interests in biomedical research. A
pilot study within this project has received funding from local Mozambican agency, which allowed
intensive sampling of a rural village (Calanga) located in Maputo province. This involved not only the
collection of buccal swabs and blood samples but also the evaluation of several phenotypes related to
physical fitness, cardiovascular risk, and parasite load. In December 2005, we made a field trip to the
Province of Namibe, in Angola (Jorge Rocha and Sandra Beleza). We collected buccal swabs from different
ethno-linguistic groups, including the semi-nomadic Herero/Kuvale herders in the Namibe desert. This work
was done in collaboration with the local health authorities and the provincial government of Namibe and
allowed the establishment of cooperation for characterizing the epidemiology of hereditary anemias in that
province of Angola. Both field collection efforts will be invaluable for studying the evolutionary history of
adaptative mutations (see above) and for carrying out further studies on human migrations in Africa, to be
developed in the PhD thesis of Margarida Coelho (see below).
In May 2005, we submitted to FCT one PhD and one post-Doc proposal for Margarida Coelho and
Sandra Beleza, respectively. Both projects were approved for funding. The work plan of Margarida
Coelho included two major parts, both dealing with the analysis of African populations and the
combined use of SNPs and STRs to characterize autosomal haplotype diversity. The first part is linked to
our adaptation project (see above) and will be focused on the interpretation of the detailed
phylogeography of mutations from four loci that might have been targets for natural selection in response
to malaria: haemoglobin S, α-thalasemia, glucose-6-phosphate dehydrogenase and duffy blood group O.
In the second part we will use several autosomal SNP-STRs to characterize genetic diversity in São
Tomé (West Africa), Angola and Mozambique in order to characterize the signatures of major
population movements within Africa, especially the Bantu expansion. This project will be developed in
co-supervision with Prof. Joanna Mountain (Laboratory of Anthropological Genetics, Standford
University). The project of Sandra Beleza aims at characterizing the admixture structure of Cape Verde
and its implications for understanding the biology of three complex traits: skin and eye pigmentation,
obesity and hypertension. This project builds upon the concept of admixture mapping and the notion that
recently admixed populations are especially well suited for studying the biology of complex traits with
different frequencies in previously isolated human groups. The project will involve: a) the
characterization of the individual admixture structure of Cape-Verde using ancestral informative markers
(AIMs); b) the assessment of associations between individual ancestry and complex traits related to skin
and eye pigmentation, obesity and hypertension; c) testing the effect of specific candidate genes on the
studied complex traits by using the admixture mapping; d) Identifying genes involved in skin
pigmentation by admixture mapping; e) analyzing the evolutionary history of candidate genes that may
influence skin pigmentation using re-sequencing approaches. The project will be co-supervised by Prof.
Esteban Parra (Department of Anthropology, University of Toronto) and will have the collaboration of
Prof. Mark Shriver (Department of Anthropology, Penn State University). In Cape Verde, the project
will be hosted by the local University and the Ministries of Education and Health.
Work plan for 2006: Our work plan for 2006 aims to: a) proceed with our analysis of the SERPIN
cluster, according to the post-doctoral plan of Susana Seixas, and obtain financial support to carry out the
functional characterization of PIL gene through isolation of the new SERPIN protein, in collaboration
with Gianpaolo Suriano from the Cancer Genetics Group at IPATIMUP; b) proceed with the PhD plan of
Margarida Coelho and data collection for Project 1; c) start a collaboration with Prof. Leonor David from
the Carcinogenesis Group in IPATIMUP aiming at the characterization of the patterns of variation in the
FUT2 gene involved in susceptibility to Helicobacter pylori and Norwalk virus infection; d) begin field
work in Cape-Verde according to the aims of Sandra Beleza’s post-doctoral plan.
Financing/Projects: The group got 39636 € funding during 2005: 5852 Є corresponding to 3 months
since the beginning of Project 1, 29 300 € from Project 2 and 4484 € from our diagnosis of α1-antrypsin
deficiency cases. For 2006 we expect a total budget of 38 000 €, from Project 2 and α1-antrypsin
deficiency diagnosis.
33
1. “Bio-cultural adaptation: human evolutionary responses to major changes in subsistence
economy” (FCT-project; POCTI/BIA-BDE/56654/2004). PI: Jorge Rocha. Total budget for
2005-2008 = 78 000 €.
2. “Anthropogenetics of São Tomé e Príncipe: a case study on human microevolution” (FCTproject; POCTI/42510/ANT/2001). PI: Jorge Rocha. Total budget for 2003-2006 = 90 000 €.
Main international collaborations:
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Pedagogic University of Mozambique: Prof. António Prista.
Department of Human Genetics, University of Chicago: Prof Anna Di Rienzo.
Department of Anthropology, University of Rome1: Prof. Giovanni Destro-Bisol.
Department of Anthropology, University of Bologne: Profs. Davide Pettener and Donata
Luiselli.
Department of Biochemistry, University of Minas Gerais: Prof. Sérgio Pena.
Laboratory of Anthropological Genetics, Standford University: Prof. Joanna Mountain.
Department of Anthropology, University of Toronto: Prof Esteban Parra.
Department of Anthropology, Penn State University: Prof. Mark Shriver.
Theses:
Master thesis:
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Human lactase persistence: evaluation of concordance between the breath hydrogen test and
molecular genotyping; analysis of the evolutionary history using a microsatellite approach.
Margarida Coelho
Graduation thesis:
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Characterization of the levels and patterns of genetic diversity in the MGP gene: a resequencing
study in African and European populations. Susana Pereira
Publications:
1-Coelho M, Luiselli M, Bertorelle G, Lopes AI, Seixas S: Microsatellite variation and evolution of
human lactase persistence. Human Genetics 117: 329-339, 2005.
2-Alves C, Coelho M, Rocha J, Amorim A: The Amelogenin locus displays high frequency of X
homologue failure in São Tomé Island (West Africa). Progress in Forensic Genetics 11 (in press).
3-Carvalho-Silva DR, Tarazona-Santos E, Rocha J, Pena SDJ, Santos FR: Y chromsome diversity in
Brazilians: switching perpspectives from slow to fast evolving markers. Genetica 126: 1-10, 2005.
34
POPULATION GENETICS GROUP
Group Leader: António Amorim, PhD
Staff members: Maria João Prata, PhD; Leonor Gusmão, PhD; Luísa Pereira, PhD; Luísa Azevedo, PhD (post-Doc);
Ana Almeida, PhD student; Alexandra Lopes, PhD student; Barbara van Asch, PhD student; Elisabete Oliveira, PhD
student; Filipe Pereira, PhD student; Iva Gomes, PhD student; Sandra Martins, PhD student; Rita Quental, PhD
student; Sofia Quental, PhD student; Cíntia Alves, technician.
Objectives/Goals of the research activity
The group aims at understanding the origin and evolution of (mainly) human genetic diversity and their
consequences and applications, both normal and pathological (using autosomal, X and Y linked, as well as mtDNA
markers).
This requires the development of descriptive and analytical formal tools and techniques adequate to specific
genomic segments, in order to achieve the genetic characterisation of normal populations, their origins, phylogeny
and evolution, and disease susceptibility profiles.
The applications in which we concentrate our efforts are molecular diagnostics and forensics, but a new line of
research involving the history, conservation and management of domesticates and laboratory animals as well as
food quality assessment is now established.
Background and major achievements during 2005
The issue of the genetic matching quality of samples in case/control researches was addressed in fertility studies
[16] and the problems related to genetically profiling heterogeneous populations was demonstrated in the context
of North African populations [21, 29].
Substantial contributions to the origin and history of various human populations were achieved, as in the case of
Europeans [3], Africans [11], and in particular Ashkenazi [31], Iberians [18] and Azoreans [24, 35]. Furthermore,
genetic profiling of various populations worldwide with relevance for anthropological and forensic studies was
carried out [1, 4, 5, 7, 9, 10, 14, 15, 21, 25, 27, 30, 33].
ProtocadherinX (PCDH11X or PCDHX) is a recently described gene expressed in brain. In humans, PCDH11X has a
homologue on the Y chromosome (the X-linked gene was transposed to the Y chromosome after the humanchimpanzee lineages split) and is predicted to escape from X-inactivation. We presented evidence providing a strong
indication that PCDH11X indeed escapes inactivation in humans and, furthermore, that women present an up to 2fold excess in the abundance of PCDH11X transcripts, a difference that can be related to sexually dimorphic traits in
the human brain [32].
Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate metabolism and in DNA
methylation and synthesis. We demonstrated that the previously reported role of MTHFR*677T and MTHFR*1298C
polymorphisms in ALL susceptibility could not be discerned in North Portugal, so that if existent, it seems to be
influenced by population-specific gene-environmental interactions [20].
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare severe neurometabolic disorder determined by increased levels of
L-2-hydroxyglutaric acid in body fluids. We identified seven novel mutations in the L-2-HGA gene, but for three
families, no pathogenic mutations were found, which suggests either alterations in regulatory regions of the gene or
of its intervening sequences, compound heterozygosity for large genomic deletion or further genetic heterogeneity
[22].
Mitochondrial DNA single macrodeletions are frequently associated with myopathies; we demonstrated that there
is no matrilineal related increased risk for the occurrence of this type of mutations [23].
Molecular tools suitable for diagnosis of strains in candidiasis [19] were developed and tested.
We mapped the genetic landscape of the maternal lineages of the Portuguese autochthonous breeds of
domesticates (dog [13] and goat [12]), placing them in the context of worldwide genetic diversity and launching the
bases for adequate conservation policies, and forensic or food quality assessment.
Among the forensically relevant research results (a) we demonstrated the presence of mixed up benign and
neoplastic tissue sections from two individuals on the same prostatic biopsy slide, a technique that can be used in
alleged cases of medical malpractice [2], (b) we forecasted the problems raised by the introduction of SNPs in
parentage testing by performing simulation studies, proving that the replacement of the now current markers
(STRs) would result in a worrying number of difficult cases [8], and (c) we participated in the Commission of the
International Society of Forensic Genetics (ISFG) for the update of the recommendations on the use of Y-STRs in
forensic analysis [34].
An international collaborative study on the mutation rates of human Y-specific STRs was coordinated by our
group, revealing that repeat gains were more frequent than losses, longer alleles were more mutable, and that
mutation rate increased with the father's age [28].
WORK PLAN FOR 2006
35
We intend,
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to develop and validate a PCR multiplex for X-chromosome STR typing
to continue the studies of normal and pathological variation associated with mtDNA
to extend the genetic profiling of Portuguese domesticates, including sheep and pig
to explore the genetic traceability of processed animal products (cheese and smoked meat)
to access the impact of cis-acting variation on differences in expression levels between individuals, in
humans
to clarify the origin and dispersion of Machado Joseph Disease and the evolutionary dynamics of the locus
(both normal and expanded alleles)
to study susceptibility factors and pharmacogenetics of pediatric acute lymphoblastic leukemia
to continue the anthropological and forensic studies of human populations, with particular emphasis on
Portugal and Timor
PAPERS
1. CHERNI L, LOUESLATI YAACOUBI B, PEREIRA L, ALVES C, KHODJET EL KILL H, BEN AMMAR EL GAAIED A,
AMORIM A (2005) Data for 15 autosomal STR markers (Powerplex 16 System) from two Tunisian populations:
Kesra (Berber) and Zriba (Arab). Forensic Sci Int 147(1):101-6.
2. ALONSO A, ALVES C, SUAREZ-MIER MP, ALBARRAN C, PEREIRA L, FERNANDEZ DE SIMON L, MARTIN P,
GARCIA O, GUSMAO L, SANCHO M, AMORIM A (2005) Mitochondrial DNA haplotyping revealed the presence of
mixed up benign and neoplastic tissue sections from two individuals on the same prostatic biopsy slide. J Clin
Pathol 58(1): 83-6.
3. PEREIRA L, RICHARDS M, GOIOS A, ALONSO A, ALBARRAN C, GARCIA O, BEHAR DM, GOLGE M, HATINA J, ALGAZALI L, BRADLEY DG, MACAULAY V, AMORIM A (2005) High-resolution mtDNA evidence for the late-glacial
resettlement of Europe from an Iberian refugium. Genome Res. 15: 19-24.
4. ALVES C, GUSMAO L, LOPEZ-PARRA AM, MESA MS, AMORIM A, ARROYO-PARDO E (2005) STR allelic
frequencies for an African population sample (Equatorial Guinea) using AmpFlSTR Identifiler and Powerplex 16
kits. Forensic Sci Int. 148:239-42.
5. DE SOUZA GOES AC, DE CARVALHO EF, GOMES I, DA SILVA DA, GIL EH, AMORIM A, GUSMAO L (2005)
Population and mutation analysis of 17 Y-STR loci from Rio de Janeiro (Brazil). Int J Legal Med. 119(2):70-6.
6. DIEDERICHE M, MARTIN P, AMORIM A, CORTE-REAL F, GUSMAO L (2005) A case of double alleles at three YSTR loci: forensic implications. Int J Legal Med. 119(4):223-5.
7. ARROYO-PARDO E, GUSMAO L, LOPEZ-PARRA AM, BAEZA C, MESA MS, AMORIM A (2005) Genetic variability of
16 Y-chromosome STRs in a sample from Equatorial Guinea (Central Africa). Forensic Sci Int. 149:109-13.
8. AMORIM A, PEREIRA L (2005) Pros and cons in the use of SNPs in forensic kinship investigation: a comparative
analysis with STRs. Forensic Sci Int. 150:17-21.
9. MARTÍNEZ B, CARABALLO L, GUSMÃO L, AMORIM A, CARRACEDO A (2005) Autosomic STR population data in
two Caribbean samples from Colômbia. Forensic Sci Int. 152: 79-81
10. CHERNI L, PEREIRA L, GOIOS A, YACOUBI LOUESLATI B, KHODJET EL KHIL H, GOMES I, GUSMÃO L, ALVES C,
SLAMA A, AMORIM A, BENAMMAR ELGAAIED A (2005) Y-chromosomal STR haplotypes in three ethnic groups and
one cosmopolitan population from Tunísia. Forensic Sci Int. 152: 95-99
11. BELEZA S, GUSMAO L, AMORIM A, CARRACEDO A, SALAS A (2005) The genetic legacy of western Bantu
migrations. Hum Genet. 117: 366-375
12. PEREIRA F, PEREIRA L, VAN ASCH B, BRADLEY DG, AMORIM A (2005) The mtDNA catalogue of all Portuguese
autochthonous goat (Capra hircus) breeds: high diversity of female lineages at the western fringe of European
distribution. Mol Ecol 14: 2313-8.
13. VAN ASCH B, PEREIRA L, PEREIRA F, SANTA-RITA P, LIMA M, AMORIM A (2005) MtDNA diversity among four
Portuguese autochthonous dog breeds: a fine-scale characterisation. BMC Genet. 6:37.
14. FRIGI S, PEREIRA F, PEREIRA L, YACOUBI B, GUSMAO L, ALVES C, KHIL HK, CHERNI L, AMORIM A, GAAIED
AE (2005) Data for Y-chromosome haplotypes defined by 17 STRs (AmpFLSTR((R)) Yfilertrade mark) in two
Tunisian Berber communities. Forensic Sci Int. 2005 Jul 6; [Epub ahead of print]
15. MARTINEZ B, CARABALLO L, BARON F, GUSMAO L, AMORIM A, CARRACEDO A (2005) Analysis of STR loci in
Cartagena, a Caribbean city of Colombia. Forensic Sci Int. 2005 Jul 14; [Epub ahead of print]
16. PEREIRA L, GONCALVES J, GOIOS A, ROCHA T, AMORIM A (2005) Human mtDNA haplogroups and reduced
male fertility: real association or hidden population substructuring. Int J Androl. 28:241-7.
17. PEREIRA L, RICHARDS M, GOIOS A, ALONSO A, ALBARRAN C, GARCIA O, BEHAR DM, GOLGE M, HATINA J, ALGAZALI L, BRADLEY DG, MACAULAY V, AMORIM A. Evaluating the forensic informativeness of mtDNA haplogroup
H sub-typing on a Eurasian scale. Forensic Sci Int. 2005 Jul 30; [Epub ahead of print]
18. PEREIRA L, CUNHA C, ALVES C, AMORIM A (2005) African Female Heritage in Iberia: A Reassessment of
mtDNA Lineage Distribution in Present Times. Hum Biol 77: 213–229
36
19. SAMPAIO P, GUSMAO L, CORREIA A, ALVES C, RODRIGUES AG, PINA-VAZ C, AMORIM A, PAIS C (2005) New
Microsatellite Multiplex PCR for Candida albicans Strain Typing Reveals Microevolutionary Changes.J Clin Microbiol.
43 :3869-76
20. OLIVEIRA E, ALVES S, QUENTAL S, FERREIRA F, NORTON L, COSTA V, AMORIM A, PRATA MJ (2005) The
MTHFR C677T and A1298C Polymorphisms and Susceptibility to Childhood Acute Lymphoblastic Leukemia in
Portugal. J Pediatr Hematol Oncol. 27: 425-429
21. CHERNI L, LOUESLATI BY, PEREIRA L, ENNAFAA H, AMORIM A, EL GAAIED AB (2005) Female gene pools of
Berber and Arab neighboring communities in central Tunisia: microstructure of mtDNA variation in North Africa.
Hum Biol. 77: 61-70
22. VILARINHO L, CARDOSO ML, GASPAR P, BARBOT C, AZEVEDO L, DIOGO L, SANTOS M, CARRILHO I, FINEZA I,
KOK F, CHORAO R, ALEGRIA P, MARTINS E, TEIXEIRA J, CABRAL FERNANDES H, VERHOEVEN NM, SALOMONS
GS, SANTORELLI FM, CABRAL P, AMORIM A, JAKOBS C (2005) Novel L2HGDH mutations in 21 patients with L-2hydroxyglutaric aciduria of Portuguese origin. Hum Mutat. 26(4): 395-6.
23. GOIOS A, NOGUEIRA C, PEREIRA C, VILARINHO L, AMORIM A, PEREIRA L (2005) mtDNA single macrodeletions
associated with myopathies: Absence of haplogroup-related increased risk. J Inherit Metab Dis 28(5): 769-78.
24. FERNANDO O, MOTA P, LIMA M, SILVA C, MONTIEL R, AMORIM A, PRATA MJ (2005) Peopling of the Azores
Islands (Portugal): data from the Y chromosome. Hum Biol 77:189-99.
25. SOUTO L, ALVES C, GUSMAO L, FERREIRA E, AMORIM A, CORTE-REAL F, VIEIRA DN (2005) Population data on
15 autosomal STRs in a sample from East Timor. Forensic Sci Int. 155(1):77-80.
26. CRESPILLO M, PAREDES MR, PRIETO L, MONTESINO M, SALAS A, ALBARRAN C, V AI, AMORIN A, BERNIELLLEE G, BREHM A, CARRIL JC, CORACH D, CUEVAS N, DI LONARDO AM, DOUTREMEPUICH C, ESPINHEIRA RM,
ESPINOZA M, GOMEZ F, GONZALEZ A, HERNANDEZ A, HIDALGO M, JIMENEZ M, LEITE FP, LOPEZ AM, LOPEZSOTO M, LORENTE JA, PAGANO S, PALACIO AM, PESTANO JJ, PINHEIRO MF, RAIMONDI E, RAMON MM, TOVAR
F, VIDAL-RIOJA L, VIDE MC, WHITTLE MR, YUNIS JJ, GARCIA-HIRSCHFEL J (2005) Results of the 2003-2004 GEPISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain.
Forensic Sci Int. [Epub ahead of print]
27. BUILES JJ, BRAVO ML, GOMEZ C, ESPINAL C, AGUIRRE D, GOMEZ A, RODRIGUEZ J, CASTANEDA P, MONTOYA
A, MORENO M, AMORIM A, GUSMAO L (2005) Y-chromosome STRs in an Antioquian (Colombia) population
sample. Forensic Sci Int. 2005 Nov 7; [Epub ahead of print]
28. GUSMÃO L, SANCHEZ-DIZ P, CALAFELL F, MARTIN P, ALONSO CA, ALVAREZ-FERNANDEZ F, ALVES C, BORJASFAJARDO L, BOZZO WR, BRAVO ML, BUILES JJ, CAPILLA J, CARVALHO M, CASTILLO C, CATANESI CI, CORACH D,
DI LONARDO AM, ESPINHEIRA R, FAGUNDES DE CARVALHO E, FARFAN MJ, FIGUEIREDO HP, GOMES I, LOJO
MM, MARINO M, PINHEIRO MF, PONTES ML, PRIETO V, RAMOS-LUIS E, RIANCHO JA, SOUZA GOES AC, SANTAPA
OA, SUMITA DR, VALLEJO G, VIDAL RIOJA L, VIDE MC, VIEIRA DA SILVA CI, WHITTLE MR, ZABALA W,
ZARRABEITIA MT, ALONSO A, CARRACEDO A, AMORIM A (2005) Mutation rates at Y chromosome specific
microsatellites. Hum Mutat 26: 520-8.
29. LOUESLATI BY, CHERNI L, KHODJET-ELKHIL H, ENNAFAA H, PEREIRA L, AMORIM A, BEN AYED F, BEN AMMAR
ELGAAIED A (2006) Islands inside an island: reproductive isolates on Jerba island. Am J Hum Biol 18:149-53.
30. SOUTO L, GUSMÃO L, FERREIRA E, AMORIM A, CÔRTE-REAL F, VIEIRA DN (2006) Y-chromosome STR
haplotypes in East Timor: Forensic evaluation and population data. Forensic Sci Int 156: 261-265.
31. BEHAR DM, METSPALU E, KIVISILD T, ACHILLI A, HADID Y, TZUR S, PEREIRA L, AMORIM A, QUINTANAMURCI L, MAJAMAA K, HERRNSTADT C, HOWELL N, BALANOVSKY O, KUTUEV I, PSHENICHNOV A, GURWITZ D,
BONNE-TAMIR B, TORRONI A, VILLEMS R, SKORECKI K (2006) The Matrilineal Ancestry of Ashkenazi Jewry:
Portrait of a Recent Founder Event. Am J Hum Genet 78(3) [Epub ahead of print]
32. LOPES AM, ROSS N, CLOSE J, DAGNALL A, AMORIM A, CROW TJ (2006) Inactivation status of PCDH11X: sexual
dimorphisms in gene expression levels in brain. Hum Genet. [Epub ahead of print]
33. BUILES JJ, MARTINEZ B, GOMEZ A, CARABALLO L, ESPINAL C, AGUIRRE D, MONTOYA A, MORENO M, AMORIM
A, GUSMAO L, BRAVO ML Y chromosome STR haplotypes in the Caribbean city of Cartagena (Colombia). Forensic
Sci Int. 2006 Jan 30; [Epub ahead of print]
34. GUSMÃO L, BUTLER JM, CARRACEDO A, GILL P, KAYSER M, MAYR WR, MORLING N, PRINZ M, ROEWER L,
TYLER-SMITH C, SCHNEIDER PM. DNA Commission of the International Society of Forensic Genetics (ISFG): an
update of the recommendations on the use of Y-STRs in forensic analysis. Int J Legal Med. 2005 Aug 26;:1-10
[Epub ahead of print]
35. MONTIEL R, BETTENCOURT C, SILVA C, SANTOS C, PRATA MJ, LIMA M (2005) Analysis of Y-chromosome
variability and its comparison with mtDNA variability reveals different demographic histories between islands in the
Azores Archipelago (Portugal). Ann Hum Genet.69:135-144
36. Beleza S, Gusmão L, Lopes A, Alves A, Gomes I, Giouzeli M, Calafell F, Carracedo A, Amorim A. Microphylogeographic and demographic history of Portuguese male lineages. Annals Hum Genet (in press).
Book Chapters
1. AMORIM A (2005) Perspectivas de aplicação histórica dos marcadores genéticos uni- e biparentais. Alguns
exemplos do Norte de Portugal no contexto ibérico. In CARRACEDO A, PEREIRA G (coord.) Xenética e Historia no
Noroeste Peninsular. Unha perspectiva interdisciplinaria. Consello da Cultura Galega, Santiago de Compostela.
37
2. GUSMÃO L (2005) Linhagens masculinas em Portugal. In CARRACEDO A, PEREIRA G (coord.) Xenética e
Historia no Noroeste Peninsular. Unha perspectiva interdisciplinaria. Consello da Cultura Galega, Santiago de
Compostela.
3. BELEZA S (2005) A homogeneidade e a heterogeneidade das linhagens masculinas do Noroeste da Península
Ibérica. In CARRACEDO A, PEREIRA G (coord.) Xenética e Historia no Noroeste Peninsular. Unha perspectiva
interdisciplinaria. Consello da Cultura Galega, Santiago de Compostela.
4. GUSMÃO L, ALVES C (2005) Y chromosome STR typing. In Forensic DNA Typing Protocols, v.297 (Carracedo A.,
ed.), Humana Press Inc., Totowa, New Jersey, pp. 67-81.
Prizes
Best poster “XLI Conferências de Genética Instituto Genética Médica Jacinto de Magalhães”
OLIVEIRA E, ALVES S, QUENTAL S, FERREIRA F, NORTON L, COSTA V, AMORIM A, PRATA MJ (2005) Implicações
na terapia da leucemia linfoblástica aguda infantil do polimorfismo genético da tiopurina S-metiltransferase.
PhDs
Finished
Beleza S. Phylogenetic and demographic history of two human populations revealed by the analysis of two nonrecombining segments of the genome: Y-chromosome and mitochondrial DNA (U Santiago de Compostela;
supervisor: L Gusmão).
Lopes AM (submitted) Gene evolution and regulation within the Xq21.3/Yp11.2 hominid-specific homology
block. (U Porto; supervisor: A Amorim)
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Ongoing
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Pereira F “Development of uniparentally transmitted genetic markers for the characterization of male and
female gene pools of Portuguese small ruminants autochthonous breeds” Faculty of Sciences, University of
Porto, IPATIMUP, and Department of Genetics, Smurfit Institute, Trinity College, Dublin 2, Ireland. Fundação
para a Ciência e Tecnologia (SFRH/BD/19585/2004). Since October 2004
Oliveira E “Study of Genetic Polimorphisms in Pediatric Acute Lymphoblastic Leukemia - Pharmacogenetic Role
in the Treatment and Relationship with susceptibility to the leukemogenic process” Faculty of Sciences,
University of Porto, IPATIMUP, and School of Medicine , Washington University in St. Louis. Fundação para a
Ciência e Tecnologia (SFRH/BD/17124/2004). Since November 2004.
Goios A “Dynamics of the mitochondrial genome – the moving boundary between normal and pathogenic
diversity” Faculty of Sciences, University of Porto, IPATIMUP and Department of Statistics, University of
Glasgow. Fundação para a Ciência e Tecnologia (SFRH/BD/16518/2004). Since November 2004.
Gomes I “X chromosome markers: genetic characterization, population analysis and forensic applications”
University Santiago de Compostela, IPATIMUP and CEGEN (National Genotyping Center of Spain). Fundação
para a Ciência e Tecnologia (SFRH/BD/21647/2005). Since November 2004.
Martins S “Epidemiologia genética da doença de Machado-Joseph. Estudo evolutivo e comparação com outras
ataxias, Faculty of Sciences, University of Porto, IPATIMUP and Univ. Pompeu Fabra, Barcelona
(SFRH/BD/8880/2002)
Quental R “Congenital Disorders of Glycosylation (CDG): Enzymatic, Biochemical and Molecular Features”
Faculty of Sciences, University of Porto, IPATIMUP and Department of Human Genetics, Catholic University of
Leuven, Belgium. Fundação para a Ciência e Tecnologia (SFRH/BD/23657/2005). Since February 2005.
Quental S “Functional, Expression and Structural investigation of the mutational spectrum of Portuguese Maple
Syrup Urine Disease patients” Faculty of Sciences, University of Porto, IPATIMUP and Department of Human
Genetics, School of Medicine, Emory University, Atlanta, USA. Fundação para a Ciência e Tecnologia
(SFRH/BD/22685/2005). Since January 2005.
NATIONAL AND INTERNATIONAL COOPERATIONS
The
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group is currently engaged in various collaborative projects with
Smurfit Institute, Trinity College, Dublin (genetics of domesticates), Dan Bradley
Univ Pompeu Fabra (population genetics modelling), Francesc Callafel
"Hôpital Nôtre Dame”, Montreal, Canada(Machado-Joseph disease), Guy Rouleau
Univ. Leeds (mtDNA), Martin Richards
Dep. Statistics, Univ. Glasgow (mtDNA), Vincent Macaulay
Inst. Med. Legal, Univ. Santiago Compostela (forensic genetics), Angel Carracedo
38
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Univ. Oxford (gene function evolution), Rosalind Harding
Univ. of Pennsylvania (gene expression), Vivian Cheung
Technion and Rambam Medical Center, Haifa, Israel (jewish populations) Doron Behar
Instituto de Genética Médica Jacinto de Magalhães (human genetic diseases), Laura Vilarinho
Instituto Nacional de Medicina Legal (forensic genetics and databases), Francisco Corte-Real
Faculdade de Medicina, UP (Aspergillus genotyping), Cidália Pina-Vaz
Visiting researchers at IPATIMUP
Sabeh Frigi, Tunis University, March
Paula Sanchez-Diz, University of Santiago de Compostela, Spain, February
Ana María López Parra, Complutense University of Madrid, Spain, February
Silvia Jimenez, University of Cartagena, Colombia, March
Beatriz Martinez, University of Cartagena, Colombia, March
Ulises Toscanini, Fundación Favaloro, Argentina, April
Dayse Aparecida da Silva, University of Rio de Janeiro, Brazil, April
Visits / Courses Abroad
PEREIRA L: Extant mtDNA diversities in humans and domesticates. Institut Jacques Monod, Paris, France. 24/06.
PEREIRA L: Choosing the adequate marker for population structure detection. Research Center for Medicine,
Children’s National Medical Center, Washington DC, USA. 26/10.
Organization of Scientific Meetings
• 21st Congress of the International Society for Forensic Genetics, 13-17 September
• X Jornadas de Genética Forense, Grupo Espanhol e Português da ISFG, 12-13 September
• Portugaliae Genetica 8th. edition (One evolution, four modes of inheritance), 17-19 March 2005
1.
National Projects
POCTI/MGI/45076/2002 “Tratamento e susceptibilidade na leucemia linfoblástica aguda infantil: influência de factores
genéticos em enzimas destoxificadoras”
01-05-2003 - 01-05-2006
PI: MJ Prata
total budget: €56200
POCTI/ANT/45139/2002 – “Reavaliação da diversidade mitocondrial europeia: distribuições das linhagens femininas no
presente e no passado”.
01-05-2003 - 01-05-2006
PI: L Pereira
total budget: €70000
POCI/ANT/57037/2004 – “Estudo evolutivo e epidemiológico de focos de anemias hereditárias em Portugal”.
01-10-2005 - 01-10-2008
PI: MJ Prata
total budget: €30000
POCI/AFR/62242/2004 – “Etnias e genética na fronteira da rota de migração Bantu: o caso dos Karamojong”.
Ethnicity and genetics in the fringe of Bantu migration route: The Karamojong case
01-01-2005 - 01-01-2008
PI: L Gusmão
total budget:
€30000
2.
International Projects
Factores de riesgo en asma: genes relacionados con la remodelación bronquial. Instituto de Investigaciones
Inmunológicas, Universidad de Cartagena, Colombia.
3. International Jurys
A Amorim: PhD jury, U Santiago de Compostela (Sandra Beleza)
39
Title of the group activities
1. Tumor evolution and development
Group Leader
Luís Teixeira da Costa, PhD, Researcher at IPATIMUP
Staff members
Ângela Costa, BSc – BI
Marta Novais, BSc – BI (through September 2005)
Joana Macedo, MD – MSc Student (part-time)
Isabel Castro, BSc – BI (from November 2005)
Catarina Osório, BSc – BI (from November 2005)
Elisabete Figueiredo, BSc – BI (from November 2005)
Objectives/Goals of the research activity
Our main long term goal is to understand the molecular mechanisms underlying tumor
evolution. Rather than focusing on an organ or tissue-specific type of tumor, we will pursue this
goal by using different models to try to answer specific fundamental questions within that broad
goal. At this early stage, this was translated into focusing on problems with which we had
previous experience and felt we have the technical and financial means to tackle: a) the
regulation of TCF4, which is a key player in intestinal tumorigenesis; b) the role played by 53
mutations in chromosomal instability in tumors. A second goal, derived from IPATIMUP’s profile
and objectives, is to directly put our knowledge of and technical expertise in cancer molecular
genetics to the service of patients.
Background and major achievements during 2005
2005 was another transition year for the lab. Setting up continued to take a significant portion
of our time, for multiple reasons: a) Need to enlarge our “technical base”; b) Major changes in
lab composition; c) Major alterations in IPATIMUP’s infra-structure; d) Addition of a new line of
research.
Our first FCT-financed project, “Identification of TCF4-interacting proteins”, was almost
completed (but see below) during 2005. Our interest in TCF4 stems from the PI’s previous
experience in colorectal cancer, a tumor type initiated, in an overwhelming majority of cases,
by inactivating mutations of the tumor suppressor gene APC or “activating” mutations of βcatenin. Both result in an abnormal increase in the cellular levels of a dimeric transcription
factor including by β-catenin and members of the TCF-LEF transcription factor family, in
particular TCF4. TCF4 has also been shown to be a key player in the differentiation of mouse
gut epithelial cells, a role consistent with the fact that β-catenin is an effector of the "WntWingless" signaling pathway, whose importance in a number of developmental processes in
multiple animal species is well established.
Our approach to identify new TCF4 partners involves the use of a yeast two-hybrid system.
The initial yeast stage of the project (primary and secondary screens, identification and
individual re-testing of candidates) was carried out in 2004. In 2005, we proceeded to test the
candidates in mammalian cells by co-immunoprecipitation/western blot and a mammalian twohybrid system. These tests led us to concentrate on two candidates: Grg5 and “clone 149”, a
previously uncharacterized gene. We have demonstrated that the 149-TCF4 interaction
extends to other family members (e.g. TCF1), and are now studying the gene’s expression
patterns in both human tumor cell lines (so far we found it to be expressed in colorectal, breast
and thyroid cell lines lines) and normal (mouse) tissues, as well as the protein’s intracellular
distribution. As for the TCF4-Grg5 interaction, we have mapped TCF4’s Grg5 binding domain
40
to a 131 aminoacid fragment by deletion analysis. Interestingly, the same fragment could
account for TCF4’s binding to 149. Subsequent analysis with smaller fragments suggests that
the binding domains do not completely overlap (see Worplan for 2006). We went on to
generate 24 different single-aminoacid mutants within this 131aa fragment, to try to identify
critical residues, and have preliminary results suggesting that at least 4 of those mutants have
a significant impact on Grg5 binding. Partial results have been reported to FCT and presented
at the annual meeting of SPGH (Portuguese society for human genetics). Additionally, we have
continued to work on improvements of the two-hybrid system that should facilitate both the
elimination of false-positives in yeast and testing of bonafide candidates in mammalian cells,
thus improving the system’s efficiency. Some of the vectors we generated in the process have
already been used, for instance in the generation of the TCF4 mutants.
Our second FCT-funded project, “Analysis of the role of p53 in cancer's chromosomal
instability”, required the generation of knock-out and knock-in human tumor cell lines. Given the
difficulties we’ve had with our tissue culture facilities during 2005, we decided to delay the most
cell culture intensive part of the work, and therefore concentrated instead on the reagents and
methods to carry it out. A major part of our efforts was directed at a system to improve the
efficiency of gene-targeting by knock-out vectors. Such a system would greatly reduce both the
cost and time (and hence the risks of contaminations) of generating knock-outs and knock-ins.
However, the changes in lab members, as well as recent advances in high-efficiency somaticcell knock-out technology, eventually leds us to drop our own attempts.
In 2005, we added a new line of research – on using molecular genetics analysis in lung
cancer management. We started with a pilot project in which we attempted to detect p53
mutations in different clinical samples, including biopsies, bronco-alveolar lavages (BAL),
bronchial brushings (BB) and sputum. All four biopsies analyzed were found to have p53
mutations: of those, one could also be detected in BAL and another in BB. These preliminary
results contributed to an MSc thesis in Molecular Oncology and Medicine submitted to the
University of Porto. They also constitued the starting point for a grant proposal in which we
proposed to use molecular genetic data obtained from clinical samples to help in diagnosis,
prognosis and clinical management of lung cancer patients.
Work plan for 2006
2. TCF4 interactors.
We will follow up on the current work on the genes we have been focusing on. More
specifically, we will: a) complete the analysis of the effect of TCF4 mutations on TCF4-Grg5
binding and extend it to TCF4-149 interactions; b) perform similar mapping analyses on
Grg5 and 149; c) attempt to generate mutants impaired only in their ability to bind one
partner (e.g. a mutant TCF4 able to bind DNA and β –catenin, but not Groucho family
members), which would constitute good reagents to test hypotheses concerning the role of
the different proteins and interactions in carcinogenesis; d) complete the study on
expression patterns and intracellular distribution of 149; e) test the effect of Grg5 and 149 in
TCF4/ β-catenin signalling in mammalian cells.
3. p53 and instability
We will start the tissue culture-intensive part of the project, attempting to perform cell
fusions between different colorectal tumor cell lines, and generating a conditional p53
knock-in in one of them.
4. Lung cancer
41
We will start the project on clinical use genetic alterations in lung cancer. Most of the work
in this first year will focus on sample gathering and setting up methods for detecting specific
mutations present only in a small percentage of DNA molecules from a specific sample.
Financing/Projects
1. “Analysis of the role of p53 in cancer's chromosomal instability”, Fundação para a Ciência e
a Tecnologia (POCTI/MGI/48201/2002), 53 490€.
2. “Utilização Clínica dos Alvos Genéticos do Tabaco” (Clinical Use of Tobacco’s Molecular
Targets), Fundação Calouste Gulbenkian, 35 300€.
42
3. EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA
Relatório de actividades da Unidade de Educação Contínua e Difusão Científica - 2005
A UNIDADE DE EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA do IPATIMUP (UECDC-IPATIMUP)
desenvolveu durante o ano de 2005 um conjunto de iniciativas em diversos domínios da promoção do pensamento
e cultura científica:
A- Programas e Projectos
1-Programa “Ciência Viva em Férias”
A UECDC-IPATIMUP promoveu durante as seis quinzenas das Férias de Verão o programa “Ciência Viva em
Férias” para 22 alunos do Ensino Secundário, que participaram em estágios de iniciação científica, acompanhando
activamente e com tarefas distribuídas, o trabalho dos investigadores do IPATIMUP.
2- Programa “Viver uma Escola Diferente”
A UECDC-IPATIMUP promoveu durante o ano de 2005 a IPATIMUP sessões mensais com professores e alunos de
Escolas do primeiro ciclo de ensino básico de forma a promover o ensino lúdico e experimental das ciências
permitindo o primeiro contacto com as técnicas e princípios básicos da ciência. Neste âmbito a UECDC-IPATIMUP
estabeleceu um protocolo de cooperação com o Centro de Formação de Professores da escola secundária António
Nobre. Programa apoiado por protocolo com o Pelouro da Educação da Câmara Municipal do Porto.
3- Programa “Autolaboratório – Da Célula ao ADN”
Iniciativa de promoção do ensino experimental das ciências na sala de aula. O “Autolaboratório” visitou 36 escolas
da região Norte do País, em carro próprio adaptado a laboratório ambulante.
4- Projecto “Promoção e Internacionalização das Ciências da Vida no Norte de Portugal”
A UECDC-IPATIMUP concluiu em 2005 a execução do Projecto “Promoção e Internacionalização das Ciências da
Vida no Norte de Portugal”. O projecto que tem por objectivos, a promoção e internacionalização das Ciências da
Vida na Região do Norte, reforçou durante 2005 a cooperação inter-institucional, a informação e divulgação das
Ciências da Vida no universo científico e na sociedade civil.
5- Projecto “A Magia da Ciência”
A UECDC-IPATIMUP deu inicio ao projecto “A magia da Ciência – POCTI/DIV/2005/00061” financiado pela FCT –
Programa POCTI do III Quadro comunitário de apoio - Medida 3.1. - “Atribuição de Financiamento para projectos de
divulgação da cultura científica e tecnológica”. Projecto que tem por objectivo a produção e divulgação de
conteúdos científicos e educativos de natureza alargada que promovam junto da comunidade escolar e da
população em geral a vertente experimental da actividade científica.
6- Projecto “Autolaboratório”
A UECDC do IPATIMUP preparou e submeteu com sucesso a candidatura do projecto “AUTOLABORÁTORIO” ao
concurso
de
“Apoio
a
Iniciativas
de
Promoção
da
Cultura
Científica e Tecnológica” promovido pela Agência Nacional para a Cultura Científica e Tecnológica - Ciência Viva.
Projecto “Autolaboratório” dará continuidade ao programa iniciado em 2003.
7- Projecto “Despertar para a Ciência”
A UECDC-IPATIMUP em articulação com a Câmara Municipal da Trofa, preparou e submeteu em Dezembro de
2005, uma candidatura ao concurso “Apoio a Iniciativas de Promoção da Cultura Científica e Tecnológica”
promovido pela Agência Nacional para a Cultura Científica. Aguarda decisão. O projecto “Despertar para a Ciência”
tem como objectivo implantar nos diversos níveis de ensino da comunidade educativa da Trofa, um modelo
pedagógico baseado na prática sustentada e consequente de uma ciência dinâmica e vivida que contribuirá
decisivamente para uma qualificação do capital humano e progresso do concelho da Trofa.
B- Conferências, Colóquios e Palestras
“Curso livre sobre - Medicina Molecular e Cancro”
A UECDC-IPATIMUP em colaboração com a Fundação Calouste Gulbenkian, Fundação de Serralves e Associação
de Estudantes da Faculdade de Medicina da Universidade do Porto, organizou o “Curso Livre sobre Medicina
Molecular e Cancro” constituído por 5 sessões (20h) que decorreram durante o mês de Outubro de 2005 nos
auditórios do IPATIMUP, Museu de Serralves e Faculdade de Medicina do Porto.
“Colóquios sobre - A Medicina Preventiva Do Cancro”
A UECDC-IPATIMUP em colaboração com a Fundação Calouste Gulbenkian e a Fundação de Serralves organizou,
em 5 e 12 de Abril, na Fundação Calouste Gulbenkian e em 12 e 19 de Outubro na
Fundação de Serralves, o segundo Ciclo de Colóquios (2005) sobre Medicina e Cancro, este ano subordinado ao
tema “Procurando vencer o cancro: A hora dos tratamentos “biológicos”.
43
“IX Conferencia do Equinócio – Poíèsis criação e poesia”
A UECDC-IPATIMUP organizou a IX conferência do Equinócio intitulada “Poiésis – criação e poesia”, realizada a 11
de Outubro de 2005, com a coordenação da Prof. Manuel António Pina e os conferencistas João Lobo Antunes,
Luís Quintais e Pedro Guedes de Oliveira.
"A failure to communicate - Workshop for scientists and journalists" e "Workshop for scientist media skills"
A UECDC-IPATIMUP organizou, nos dias 11 e 12 de Julho de 2005, uma formação na área da comunicação e
ciência ("A failure to communicate - Workshop for scientists and journalists" e "Workshop for scientist media skills"),
orientada pelo Prof. Tom Linden da University of North Carolina.
Palestras
A UECDC-IPATIMUP promoveu durante o ano de 2005, a realização de palestras sobre temas como a Biologia, a
Genética, o Cancro, em várias escolas secundárias (Esc. Sec. Monte da Ola – Viana – Março e Novembro de 2005;
Esc. Sec. Rio Tinto – Porto – Outubro de 2005; Escola da Ponte – Vila das Aves – Novembro de 2005; Esc. Sec.
Rodrigues de Freitas – Porto – Novembro de 2005; Esc. Sec. Gondomar – Porto – Novembro de 2005).
C- Exposições
“3ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto”
A UECDC-IPATIMUP participou na “3ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto”, promovida
pela Universidade do Porto, que decorreu de 21 a 24 de Abril de 2005, no Pavilhão Rosa Mota – Porto. O stand fez
a divulgação das diversas vertentes de actividade do IPATIMUP (Investigação, Formação, Diagnóstico e
Divulgação), proporcionando ainda um conjunto de experiências sobre os temas a célula e o ADN.
“1ª Feira da Saúde do Porto”
A UECDC-IPATIMUP participou na “1ª Feira da Saúde do Porto”, promovida pela Câmara Municipal do Porto, de 22
a 23 de Março de 2005, no Pavilhão Rosa Mota – Porto. O stand fez a divulgação das diversas vertentes de
actividade do IPATIMUP (Investigação, Formação, Diagnóstico e Divulgação), proporcionando ainda um conjunto
de experiências sobre os temas a célula e o ADN.
“Semana da Ciência e da Tecnologia”
A UECDC-IPATIMUP organizou na “Semana da Ciência e da Tecnologia”, de 21 a 25 de Novembro de 2005, uma
iniciativa de visitas para alunos que frequentaram o “Ciência Viva em Férias” intitulada -“ Trás um amigo também”.
Nesta iniciativa o IPATIMUP esteve de “portas abertas” ao regresso dos alunos do programa “Ciência Viva nas
Férias”, possibilitando que os mesmos trouxessem novos colegas/amigos para contactarem com a realidade da
investigação científica.
“Laboratório de Ciências no Museu dos Transportes e Comunicações”
A UECDC-IPATIMUP continuou a prestar apoio científico ao laboratório de ciência que integra a exposição
permanente “Comunicação do Conhecimento e da Imaginação” do Museu dos Transportes e Comunicações do
Porto.
D- Participação em iniciativas de Divulgação Científica
A UECDC-IPATIMUP participou no “Encontro Nacional de Visualização Científica 2005”, organizado a 17 de
Setembro de 2005 no Centro Multimeios de Espinho, pela Universidade do Porto e Fundação Navegar.
A UECDC-IPATIMUP participou
no curso "Application and
Development of
Instructional
Software in Biosciences" do “International Postgraduation Program” organizado de 25 a 29 de Julho de 2005, pela
Escola de Ciências da Saúde da Universidade do Minho.
A UECDC-IPATIMUP participou na conferência “Communicating European Research 2005” realizada em
Bruxelas a 14 e 15 de Novembro de 2005, promovida pela “European Commission - Directorate-General for
Research Information and Communication Unit”.
44
5. SERVIÇO À COMUNIDADE
5a. UPS
5b. UPSi
5c. UPSs
45
Unidade de Prestação de Serviços (UPS)
Relatório de Actividades 2005
Introdução
Em Março de 2005 a Unidade de Prestação de Serviços atingiu o objectivo a que se propôs desde 2002, ou seja, obter a
acreditação pelo Colégio Americano de Patologistas (CAP). Durante este ano nosso principal objectivo foi o de manter o
Sistema de Gestão de Qualidade definido desde 2003, de acordo com as normas propostas pelo CAP e realizar a nossa primeira
auditoria interna antes da segunda inspecção pelos inspectores da CAP prevista para 2007. Apesar de todo o esforço realizado e
o facto de que os exames de biologia molecular passaram para outra Unidade de Prestação de Serviços do IPATIMUP, tivemos
um aumento do número de exames (mais 1036 exames do que 2004). Como vem sucedendo há vários anos, continuámos a
actuar como um centro de formação profissional pós-graduado, tendo recebido em 2005, 5 patologistas e 3 técnicos de
diferentes países.
1.
Recrutamento de pessoal:
•
Em Março de 2005 contratamos, em regime de contrato sem termo, a administrativa Cecília Seabra.
2. Aquisição de Equipamento e Testes de Proficiência::
Com a finalidade de equipar o laboratório de rotina diagnostica, no âmbito do Programa de Acreditação da Unidade via CAP
com o Apoio do Programa Saúde XXI, foram realizados os seguintes gastos com equipamentos e testes de proficiência do CAP
Aparelho/Empresa – Modelo
1 ecógrafo Toshiba, modelo Famio e acessórios *
1 Centrífuga eléctrica de bancada **
Testes de Proficiência do Colégio Americano de Patologistas
**
Preço c/ IVA
24.395,00 Euros
3.062,51 Euros
7.156,75 Euros
(*) O Projecto Saúde XXI comparticipa com 75% do total.
(**) Adquirido integralmente com recursos da Unidade.
3. Estágios, visitas de curta duração e treino de internos:
4.
Estágios
Nome
Amanda Oliveira Arruda, Técnica Superior,
UNICAMP, São Paulo, Brasil
Ana Paula Beltrame Farina, Interna de Anatomia
Patológica, Universidade Federal de Santa
Catarina, Santa Catarina, Brasil
Ana Sofia Fortunato Santos, Técnica de
Anatomia Patológica,
Hospital
Fernando
Fonseca, Lisboa, Portugal
Carla Firmo, Técnica de Anatomia Patológica,
Hospital Egas Moniz, Lisboa, Portugal
Carlos Henrique Aguiar Botelho, Especialista em
Anatomia Patológica, Universidade de Brasília,
Brasília, Brasil
Juliano Carvalho Freitas, Interno em Anatomia
Patológica, Universidade Federal da Bahia,
Bahia, Brasil
Maísa Momesso Quintal, Interna em Anatomia
Patológica, UNICAMP, São Paulo, Brasil
Período
14/07/2005 a
03/08/2005
01/06/2005 a
29/07/2005
Tipo de Estágio
Biologia Molecular
Situação Actual
Concluído
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Técnica de
Hibridização in situ
Concluído
16/05/2005 a
20/05/2005
11/07/2006 a
25/07/2006
Técnica de
Hibridização in situ
Imunocitoquímica
Concluído
02/05/2005 a
02/07/2005
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Concluído
Rafael Sarlo Vilela, Interno em Anatomia
Patológica, Hospital do Câncer, São Paulo, Brasil
12/09/2005 a
19/11/2005
26/09/2005 a
14/10/2005
01/10/2005 a
23/12/2005
Concluído
Concluído
Concluído
Concluído
46
4. Publicações com material da U.P.S.
Longatto-Filho A, Albergaria A, Paredes J, Moreira MA, Milanezi F, Schmitt FC. P-cadherin expression in glandular lesions of
the uterine cervix detected by liquid-based cytology. Cytopathology 16: 88-93, 2005.
Longatto-Filho A, Martins A, Costa SMA, Schmitt FC. VEGFR-3 expression in breast cancer tissue is not restricted to
lymphatic vessels. Pathology Research and Practice 201: 93-99, 2005.
Granja NM, Ricardo SA, Longatto A, Alves VA, Bedrossian CW, Wiley E, Schmitt FC. Potential use of loss of heterozygosity
in pleural effusions of breast cancer metastases using the microsatellite marker of the 16q22.1 region of the CDH1 gene.
Analytical Quantitative Cytology and Histology 27: 61-66, 2005.
Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F. p63, cytokeratin 5, and P-cadherin: three molecular markers to
distinguish basal phenotype in breast carcinomas. Virchows Arch 447: 688-694, 2005.
Carvalho I, Milanezi F, Martins A, Reis RM, Schmitt F. Overexpression of platelet-derived growth factor receptor alpha in
breast cancer is associated with tumour progression. Breast Cancer Res 7: R788-R795, 2005.
Paredes J, Albergaria A, Oliveira JT, Jerónimo C, Milanezi F, Schmitt FC. P-cadherin overexpression is an indicator of clinical
outcome in invasive breast carcinomas and is associated with CDH3 promoter hypomethylation. Clin Cancer Res 11: 58695877, 2005.
Reis R, Martins A, Ribeiro SA, Basto D, Longatto-Filho A, Schmitt FC, Lopes J. Molecular characterization of PDGFRalpha/PDGF-A and c-KIT/SCF in gliosarcomas. Cell Oncol 27: 319-326, 2005.
5.
Exames realizados na U.P.S.
Nº total de exames: 13.770
Captura Híbrida: 46
Citologias: 9.001 (6.709 - Projecto de Santo Tirso)
Citologias Aspirativas: 1.883
Histológicos: 1.745 (510 Autópsias)
Histoquímicos: 109
Imuno-histoquímicos: 168
Hibridização in situ: (Projecto ROCHE): 337
Imunofluorescência Directa: 9
Relatório Complementar: 14
Exames de proficiência da CAP
CAP-FISH: 2
CAP-MK: 8
CAP-NGC: 20
CAP-PAPM: 20
CAP-PIP: 50
Genética Molecular e Citogenética (Até Maio 2005)
Diagnóstico de Sindrome de Prader-Willi: 23
Diagnóstico de Síndrome de Angelman: 12
Diagnóstico de Síndrome de X-frágil: 1
Estudo molecular de delecção 1p: 1
Estudo molecular de translocações: 1
Fenotipagem de α-1 antitripsina: 62
Pesquisa de Instabilidade de Microssatélites: 13
Pesquisa de Mutação no RET: 10
Pesquisa de Mutações nos genes BRCA1 e BRCA2: 12
Pesquisa de Mutações do gene OTC: 12
Pesquisa de Mutações do gene E-caderina: 6
Pesquisa de Mutações UBE3A: 7
Citometria
Citometria de Fluxo: 1
Citometria de Imagem: 0
47
Casos em consulta*: 197
•
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Os casos em consulta foram oriundos das seguintes instituições:
A. Galvão Teles - Núcleo de Endocrinologia, Diabetes e Metabolismo – Lisboa - Portugal
Afaf M. Elhag - The Laboratory Mafrag Hospital - Abu Dhabi - U.A.E.
Afonso Fernandes - Hospital Santa Maria – Lisboa - Portugal
Agostinho Sanches - Hospital Senhora de Oliveira Guimarães - Portugal
Alberto Veiga Barreiro - Complexo Hospitalario Xeral-Calde - Galicia - Espanha
Albino Oliveira - Lab. Anatomia Patológica Dr. Albino Oliveira, Lda. - Portugal
Ana Paula Martins - Hospital Santa Cruz, S.A.- Portugal
Andreja Zidar - Institute of Oncology – Ljubljana - Eslovénia
Angela Logullo - S. Paulo - Brasil
B. Iglesias - Centro Médico POVIS - Vigo (Pontevedra) - Espanha
Beatriz Elizaguirre - Hospital de Galdakao - Espanha
Carla Carrilho – Hospital Central de Maputo - Moçambique
Carlos Alvarez Alvarez - Complejo Hospitalario Pontevedra-Hospital Motecelo - Espanha
Carlos Caldas - University of Cambridge - Famil. Gast.Cancer Study – Reino Unido
Carlos Prada Puentes - North Manchester Gen. Hospital – Reino Unido
Chrisoula D. Scopa - University of Patras - School of Medicine - Patras - Grécia
Christian Gulmann - Beaumont Hospital - Irlanda
Christine Dellau Vieira - Hospital Distrital de Santarém, EPE - Portugal
Christophe Duc - Institut Central des Hôpitaux Valaisans - Sion - Suiça
Christophe Girardet - Institut Central des Hôpitaux Valaisans – Sion - Suiça
Conchita de Miguel - Hospital Virgem Del Camino - Espanha
Décio Fausto Gorini - Histolab -Lab. Anatomia Patológica e Citopatologia – Brasília - Brasil
Élbio C. de Paula – GOIAS - Brasil
Fátima Magalhães – Unidade Local de Saúde Matosinhos – Portugal
Fernando Bal Nieves - Complexo Hospitalario Xeral-Calde - Galicia - Espanha
Fernando Miziara - Sector Hosp. Loc. Sul - Brasilia - Brasil
Francesc Felipo - Hospital M. Servet - Espanha
Francisco Garcia Herreros - Valencia - Espanha
Françoise Brachmanski – Lausanne - Suiça
Gearóid Ó Laoi - Mercy University Hospital - Irlanda
Geneviève Belleannee - CHU - Hôpitaux de Bordeaux - França
Gerrit A. Meijer - VU University Medical Center - Holanda
H. Kokka - A. Fleming General Hospital - Athens - Grécia
H. Van Dijck - Laboratorium Voor Pathologische Ontleedkunde - Bélgica
Halil Dinçer Azizlerli - Mediteks Saglik Hizm. Tibbi Malz.San. Ve Dísticas - Turquia
Hélène Trouette - CHU - Hôpitaux de Bordeaux - França
Inês Vieira de Castro - Brasil
Isabel Amendoeira – Hospital S. João - Portugal
João Carlos Coelho Filho - Fundação José Silveira - Salvador-Bahia - Brasil
Joaquim Rodrigues - Hospital Senhora de Oliveira – Guimarães - Portugal
José Cameselle Teijeiro - Hospital Clínico Universitário - Espanha
José Maria Rivera Pomar - Hospital de Cruces - Espanha
Juan M. Mosquera - Complexo Hospitalario Juan Canalejo - A Coruña - Espanha
Júlia Diego - Hospital Donostia - Espanha
K. Sikand - Christie Hospital – Reino Unido
Kay Washington - Vanderbilt University Medical Center – Nashville - EUA
Krystyna Kotanska-Groholt - The Norwegian Radium Hospital – Oslo - Noruega
L. Van Kerckvoorde - H. Hartkliniek - Bélgica
Lia Menasce - Christie Hospital – Reino Unido
Linda Giannikaki - University Hospital of Crete - Crete - Grécia
Lucea Menéndez - Hospital de Jarrio - Espanha
Lucilia Monteiro - Hospital de Egas Moniz, S.A. - Portugal
Luisa Cristina - Hospital Santa Maria – Lisboa - Portugal
M. Akif Demir - Celal Bayar University - School of Medicine – Manisa - Turquia
M. Angeles Peteiro Cancelo - Hospital Povisa - Espanha
M. Luz Carpintero Saiz - Complejo Hospitalario Pontevedra-Hospital Motecelo - Espanha
Manuel F. Fresno - Hospital Central de Asturias - Espanha
Manuela Maia – Hospital de Egas Moniz, S.A., - Portugal
48
Dr.ª
Prof.
Dr.
Dr.
Profª
Dr.
Dr.
Drª
Dr.
Dr.
Dr.
Dr.ª
Dr.ª
Drª
Drª
Dr.ª
Dr.
Dr.
Dr.ª
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.ª
Dr.
Profª.
Dr.ª
Dr.ª
Dr.
Prof.
Dr
Marcela Duran - Centro Hospitalar Cova da Beira - Portugal
Marcello Franco - UNIFESP/EPM - São Paulo - Brasil
Marcus Aurelho de Lima – Uberaba - Brasil
Maria José Brito - Hospital Garcia da Orta, S.A. Almada - Portugal
Mario Luna - The University of Texas, MD Anderson Cancer Center – Houston EUA
Mary Toner - University Teaching Hospit. Trinity College Dublin - Irlanda
Mônica Blaya de Azevedo - Laboratório de Patologia- ANATPAT – Porto Alegre - Brasil
Müller-Hermelink - Inst. Universität Würzburg – Würzbur - Alemanha
Noureddine Bouzourene - Laboratoire Argot Lab – Lausanne - Suiça
Noureddine Bouzourene - Laboratoire Argot Lab – Lausanne - Suiça
Palmira Malo - Hospital de Zumárraga - Espanha
Paola Souza - Souza Anatomia Patológica - Brasil
Patrícia Sabino de Matos - FCM/ Unicamp – Campinas - Brasil
Paula Guerra - Hospital SAMS - Portugal
Pilar San Miguel - Hospital Povisa - Espanha
Rogério de Almeida Ribeiro – Brasília - Brasil
Ronald de Krijger - Erasmus MC - Holanda
Rosete Nogueira – Centro Hospitalar Vila Nova de Gaia - Portugal
Rui Carrapato - Hospital S. Sebastião, S.A.-Santa Maria da Feira - Portugal
Sanz-Anquela - Hospital Principe de Astúrias – Madrid - Espanha
Sergio Gonzalez - Universidad Catolica de Chile - Espanha
Serpil Dizbay Sak - Ankara University Faculty of Medicine - Turquia
Snjezana Frkovic-Grazio - Institute of Oncology – Ljubljana - Eslovénia
Sofia Loureiro Santos - Hospital Garcia da Orta, S.A. Almada - Portugal
Steven Mackll - York Hospitals – Reino Unido
Suna Erkilic - Gaziantep University (School of Medicine) - Turkey
Thais Mavad - Universidade de São Paulo - Faculdade de Medicina - São Paulo -Brasil
Umit Ince - Oruc Patoloji ve Sitoloji Lab. - Turquia
Valdeci Ferreira - Fortaleza – Ceará - Brasil
6.
Controle de Qualidade
•
•
Data de Instituição
19-01-1998
Membros
•
Prof. Fernando Schmitt
•
Drª Fernanda Milanezi
•
D. Susana Silva
•
•
Nº de Casos Revistos em 2005 – 311 casos de patologia cirúrgica e citopatologia (não ginecológica)
Nº de Casos Revistos em 2005 de citologias ginecológicas: 2.123
•
Principais Conclusões no Final do 8º Ano:
Este foi o primeiro ano em que todo o Sistema de Controle de Qualidade foi realizado através do sistema informático Sislab,
cujos resultados estão publicados no Manual da Qualidade da UPS-2005. Os principais achados comparativamente a 2004
foram:
Total nº de casos
Casos Revistos
2004
12.755
302
2005
13.573
311
Tipo de Exames
Citologia aspirativa por agulha fina
Citologia não ginecológica
Histológico de biópsias
Histológico de Peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
2004
121
6
27
68
4
11
3
3
2005
164
6
24
91
2
11
9
3
49
Citologia ginecológica a)
Imunofluorescência
54a)
N/A
1
a)
Desde Abril de 2004 os casos de citologia ginecológica foram avaliados separadamente de acordo com o procedimento
PR.MED.01
Valores de discordância:
Critério
Identificação do espécime, arquivo e macroscopia
Diagnóstico
Codificação
2004
2.1%
0.4%b)
0.9%
2005
2.6%
0.6%c)
0.6%
“Turn-around-time”
Citologia aspirativa por agulha fina
Citologia não ginecológica
Histológico de biópsias
Histológico de Peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
Citologia ginecológica a)
a)
2004
2.8 dias
4.5 dias
4.9 dias
9.5 dias
3.2 dias
7.2 dias
6.9 dias
0.3 dias
14.7 dias
2005
1.8 dias
4.7 dias
2.4 dias
3.2 dias
5.0 dias
5.2 dias
1.9 dias
N/A
2.4 dias
(Janeiro a Março)
Na análise do controle de qualidade de citologia ginecológica, os principais resultados foram os seguintes:
Total de casos
Casos revistos
Concordância
patologistas
citotécnico
Discordância
patologista
citotécnico
entre
e
entre
e
2004
5920
1404
2005
8952
2123
2004
1301
(92.6%)
2005
1962
(92.4%)
103 (7.4%)
161
(7.5%)
O número total de citologias consideradas não satisfatórias para a análise foi de 3.5% (4.1% em 2004), o que representa uma
diminuição conforme um dos objectivos propostos no último ano.
Ascus foi diagnosticado em 1.9% o que representa igualmente uma redução em relação a 2004 (2.5%), bem como a relação
ASCUS/Lesão foi de 4.1.
Para além da participação com aprovação em todos os testes de proficiência da CAP, continuamos a participar de forma positiva
nos programas de controlo de qualidade externo das técnicas de imuno-histoquímica do UK-NEQAS e do Programa de
Qualidade da Sociedade Brasileira de Patologia (PIQ). Todas estas actividades estão registadas de acordo com o Procedimento
Regulamentador PR MED-05 – Programas Externos de Educação e Avaliação Contínua.
50
RELATÓRIO UPSI 2005
(a 15 de Fevereiro de 2006)
1. Exames realizados
1.1
Nº de exames requeridos
Pedidos cancelados
Em curso (ainda por efectuar ou por falta de 1 ou mais colheitas)
1.2
Tipos de exame:
1.3
Caracterizações/Identificações genéticas
4
Paternidades
Com 1 pretenso pai, sem análise da mãe
Com 2 filhos
Com 1 pretenso pai e com análise da mãe
Com 2 pretensos pais e com análise da mãe
20
1
118
3
Outros parentescos
Pareceres
8
1
Locais de requisição:
Local
Porto
Vila Nova de Gaia
Lisboa
Santo Tirso
Penafiel
Bragança
Aveiro
Vila do Conde
Ponta Delgada
Ponte da Barca
Arouca
Póvoa de Varzim
Braga
Angra do Heroísmo
Felgueiras
Guimarães
Leiria
Madrid
Mirandela
Oliveira de Azeméis
Paredes
Vale de Cambra
Tribunais
59
22
0
2
4
3
0
7
0
0
3
2
0
0
0
0
0
0
0
0
0
1
Particulares
12
0
25
0
0
0
3
0
1
1
0
0
1
1
1
1
1
1
2
1
1
0
Total
71
22
25
2
4
3
3
7
1
1
3
2
1
1
1
1
1
1
2
1
1
1
Total
103 (66%)
52 (34%)
155
1.4
155
1
17
Tipos de requerentes
Tribunais
Averiguações Oficiosas de Paternidade/Maternidade
Outras
Particulares
92
11
52
51
1.5
Nº exames por requerente
Clientes
Tribunais:
Trib. Família e Menores do Porto
Trib. Família e Menores de Vila Nova de Gaia
Trib. Judicial de Vila Nova de Gaia
Trib. Santo Tirso
Trib. Penafiel
Trib. Bragança
Trib. Vila do Conde
Trib. Arouca
Trib. Póvoa de Varzim
Trib. Vale de Cambra
Total
59
19
3
2
4
3
7
3
2
1
Particulares
Clínica Dr. Joaquim Chaves - Lisboa
Internos (IPATIMUP)
Outros
22
2
28
Total
155
2. Publicações com material da UPSi
2.1
Revistas Internacionais
Alves C, Gusmão L, López-Parra AM, Mesa MS, Amorim A, Arroyo-Pardo E (2005): STR allelic frequencies
for an African population sample (Equatorial Guinea) using AmpFlSTR Identifiler and Powerplex 16 kits. Forensic Sci
Int, 148(2-3): 239-242.
Salas A, Prieto L, Montesino M, Albarrán C, Arroyo E, Paredes-Herrera MR, Di Lonardo AM, Doutremepuich C,
Fernández-Fernández I, González de la Veja A, Alves C, López CM, López-Soto M, Lorente JA, Picornell A, Espinheira
RM, Hernández A, Palácio AM, Espinoza M, Yunis JJ, Pérez-Lezaun A, Pestano JJ, Carril JC, Corach D, Vide MC,
Álvarez-Iglesias V, Pinheiro MF, Whittle MR, Brehm A, Gómez J (2005): Mitochondrial DNA error prophylaxis:
assessing the causes of errors in the GEP’02–03 proficiency testing trial. Forensic Sci Int, 148(2-3): 191-198.
Alonso A, Alves C, Suarez-Mier PM, Albarrán C, Pereira L, Simón LF, Martín P, Garcia O, Gusmão L, Sancho
M, Amorim A (2005): Mitochondrial DNA haplotyping revealed the presence of mixed-up benign and neoplastic
tissue sections from two individuals on the same prostatic biopsy slide. J Clin Pathol, 58: 83-86.
Souto L, Alves C, Gusmão L, Ferreira E, Amorim A, Côrte-Real F, Vieira DN (2005): Population data on 15
autosomal STRs in a sample from East Timor. Forensic Sci Int 155:77-80.
Gusmão L, Gusmao L, Sanchez-Diz P, Calafell F, Martin P, Alonso CA, Alvarez-Fernandez F, Alves C, BorjasFajardo L, Bozzo WR, Bravo ML, Builes JJ, Capilla J, Carvalho M, Castillo C, Catanesi CI, Corach D, Di Lonardo AM,
Espinheira R, Fagundes de Carvalho E, Farfan MJ, Figueiredo HP, Gomes I, Lojo MM, Marino M, Pinheiro MF, Pontes
ML, Prieto V, Ramos-Luis E, Riancho JA, Souza Goes AC, Santapa OA, Sumita DR, Vallejo G, Vidal Rioja L, Vide MC,
Vieira da Silva CI, Whittle MR, Zabala W, Zarrabeitia MT, Alonso A, Carracedo A, Amorim A (2005). Mutation rates at
Y chromosome specific microsatellites. Hum Mutat. 26:520-528.
2.2
Revistas Nacionais
Alves C. O DNA e os testes de paternidade. Saúde, Artes e Letras (SAL), nº 1 (Setembro 2005): 18-22.
3. Actividades/Outros
3.1
Participação no Exercício de Controle de Qualidade de 2005 do Grupo Espanhol e Português da
International Society for Forensic Genetics, GEP-ISFG (Certificado em anexo).
3.2
Participação no Exercício Colaborativo de Interpretação de Sequências de mtDNA do GEP-ISFG
(2005/2006). Resultado a apurar no decurso de 2006.
52
3.3
Organização do encontro anual do GEP-ISFG e da reunião científica do ISFG em Ponta Delgada, Açores, de
13 a 17 de Setembro de 2005.
3.4
Formação de Cíntia Alves no Departamento de Biologia Forense do Instituto Nacional de Medicina Legal,
Delegação de Coimbra, de 29 a 30 de Novembro de 2005, incidindo sobretudo na área das análises preliminares em
diversos tipos de vestígios biológicos.
3.5
Montagem de um novo espaço laboratorial (ainda em curso), independente e apropriado à aplicação das
metodologias na área da investigação genética forense. Concretamente, permitirá à unidade a realização de análises
de perfis genéticos sobre diversos tipos de vestígios biológicos (sangue, sémen, cabelos, saliva, tecidos, etc.) por
aplicação de técnicas de biologia molecular específicas da área forense e sob condições adequadas para impedir e
prevenir a não-contaminação dos locais de trabalho com DNA humano.
53
Unidade de Prestação de Serviços de Susceptibilidade Genética (UPSS)
Relatório de Actividades 2005
Resumo
O objectivo fundamental da UPSS para o ano 2005 foi a consolidação da sua actividade como prestador
de serviços. Este objectivo foi conseguido através:
1. da entrada em funcionamento do laboratório próprio da unidade;
2. da incorporação dos testes de diagnóstico genético previamente existentes no IPATIMUP;
3. da implementação de uma série de novos testes, nomeadamente nas áreas da oncologia e das
doenças cardiovasculares.
Em paralelo a UPSS procedeu à organização de um “reception front desk” que funciona como estrutura
de recepção e registo de casos e como estrutura de interface com profissionais de saúde e doentes.
Para 2006 a UPSS estabeleceu como principais objectivos:
1. Aumentar o número de testes de diagnóstic genético realizados em áreas que consideramos
chave para a unidade, como a gastrenterologia, oncologia e cardiovascular;
2. Dar continuidade ao programa de estabelecimento e implementação de novos testes;
3. Implementar um programa de acreditação da unidade.
Publicações com material da UPSS
Oliveira C, Velho S, Domingo E, Preto A, Hofstra RM, Hamelin R, Yamamoto H, Seruca R, Schwartz S
Jr. Concomitant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI
sporadic colorectal cancer. Oncogene. 2005; 24: 7630-4.
Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S Jr, Duval A, Carneiro F, Machado
JC, Hamelin R, Seruca R. The prevalence of PIK3CA mutations in gastric and colon cancer. Eur J
Cancer. 2005; 41: 1649-54
Domingo E, Niessen RC, Oliveira C, Alhopuro P, Moutinho C, Espin E, Armengol M, Sijmons RH,
Kleibeuker JH, Seruca R, Aaltonen LA, Imai K, Yamamoto H, Schwartz S Jr, Hofstra RM. BRAF-V600E
is not involved in the colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2
genes. Oncogene. 2005; 24: 3995-8.
Exames realizados na UPSS
Exame
Cancro do cólon - Pesquisa de instabilidade microssatélites
Cancro difuso do estômago hereditário - Mutações caderina-E
Cancro do estômago - Genotipagem Helicobacter pylori
Cancro do estômago - Genotipagem de polimorfismos pró-inflamatórios
Doença de Crohn - Polimorfismos do gene CARD15/NOD2
Síndrome Li-Fraumeni e Cancro esporádico - Mutações p53
Delecções cromossómicas
Cancro do Cólo de Útero - Detecção de HPV por PCR
Sarcomas, PNET e Ewing - Translocações cromossómicas
Neuroblastoma - Amplificação do gene NMYC
Cancro da mama - Pesquisa de mutações dos genes BRCA 1 e 2
MEN - Pesquisa de mutações do gene RET
Pesquisa de mutações do gene OTC
Actividade enzimática de TPMT
Nº de testes
20
5
2
1
3
1
1
6
8
2
15
4
5
0
54
Diagnóstico de deficiência proteica α1-antitripsina
Diagnóstico molecular de X-Frágil
Diagnóstico molecular de Síndromes de Prader-Willi e Angelman
Pesquisa de UBE3A no Síndrome de Angelman
Total de exames
95
3
64
10
245
Novos exames implementados na UPSS
Área Cardio-vascular
•
Hipercolesterolemia familiar - Detecção de mutações nos genes LDLR, APOB e PCSK9;
•
Enfarte miocárdio e trombose venosa - Detecção dos polimorfismos Factor V Leiden, Factor II
(Protrombina) e MTHFR;
•
Miocardiopatia hipertrófica e miocardiopatia dilatada - Detecção de mutações nos genes MYH7,
MYBPC3 e TNNT2;
•
Arritmias cardíacas (Síndrome QT-longo, síndrome QT-curto e síndrome de Brugada);
•
Detecção de mutações nos genes SCN5A, KCNQ1 e KCNH2;
•
Síndrome de Marfan - Detecção de mutações nos genes FBN1, TGFBR1 e TGFBR2.
Área Oncologia
•
Cancro do Pulmão - Mutações EGFR;
•
GIST - Pesquisa de mutações do gene KIT
55
5. RECÉM-DOUTORADOS
Ana Carvalho
Estudou a Survivina, uma proteína que tem gerado grandes
expectativas na área clínica, por constituir um possível novo alvo
para a terapia do cancro. Analisou células que não têm survivina
o que permitiu concluir que esta é essential para o sucesso da
mitose. Na ausência de Survivina os cromossomas apresentam
problemas na formação da placa metafásica, e depois da
segregação cromossómica, o processo de citocinese falha,
originando células multinucleadas. Devido ao facto desta
proteína desempenhar um papel essencial na citocinese,
investigou-se o potencial envolvimento no fenótipo de
multinucleação observado nas células de ReedSternberg/Hodgkin, características de um tipo particular de
linfomas, a Doença de Hodgkin. Este estudo demonstrou
contudo, que esta proteína não está directamente envolvida no
processo de multinucleação característico destas células.
Bioquímica, doutorou-se em 2 de Fevereiro de 2005 com a tese "Cellular and molecular analysis of survivin and the
involvement of chromosomal passengers in the multinucleation phenotype of Hodgkin's Disease". Actualmente está
a fazer um pós-doutoramento no Ludwig Institute for Cancer Research em San Diego.
56
Carla Carrilho
Identificou pela primeira vez os tipos de HPV associados a
neoplasias do colo do útero em Moçambique e concluiu-se que são
muito frequentes as infecções múltiplas por diversos tipos de vírus
(HPVs 16,18,31,33,35,45 e 58). Estes resultados apontam para a
necessidade de serem produzidas vacinas profilácticas
multivalentes para prevenir esta neoplasia em África. Foi ainda
possível identificar alguns marcadores (P53, Ki67, alguns carbohidratos e queratinas) com utilidade para o diagnóstico de lesões
mais agressivas em fases iniciais do processo de cancerização.
Médica,
doutorou-se em Fevereiro de 2005 com a tese “Cervix cancer in Mozambique: role of human Papilloma
vírus (HPV) in the ethiopathogenesis of cervix cancer and evaluation of the usefulness of some markers in the
diagnosis”. Actualmente é Professora de Patologia na Universidade Eduardo Mondlane e Directora do Departamento
de Anatomia Patológica do Hospital Central de Maputo.
57
Carla Costa
Demonstrou que a expressão do VEGFR-1 pelas células de
carcinoma mamário é um parâmetro patológico que poderá ter
relevância terapêutica. Estes estudos foram efectuados em
ratinhos nos quais foram implantadas células tumorais mamárias
humanas e estes tratados com um agente bloqueador do VEGFR-1
humano. Esta terapia anti-VEGFR-1 resultou na regressão do
crescimento tumoral.
Bióloga,
doutorou-se em 7 de Março de 2005 com a tese “Influência de factores angiogénicos e células
endoteliais/hematopoiéticas percursoras derivadas da medula óssea no crescimento de tumores sólidos”.
Actualmente é Professora Auxiliar de Biologia Celular na Faculdade de Medicina do Porto.
58
Cristina Faleiro
O carcinoma epitelial do ovário é a principal causa de morte por
tumores do trato genital feminino. Neste estudo de tumores
epiteliais do ovário, o comportamento clínico destes tumores foi
variável de acordo com a imunoexpressão das proteínas caderinaE e cateninas. A expressão reduzida das β-cateninas associa-se a
tumores com características clínico patológicas de mau
prognóstico, nomeadamente tumores indiferenciados, carcinomas
serosos e de células claras, metastização peritoneal, estádios
avançados e volume da doença residual após cirurgia de
citorredução. Na análise da sobrevivência global, a perda da
imunoexpressão da caderina–E e da β-catenina associam-se a pior
prognóstico. A identificação precoce de variáveis moleculares de
mau prognóstico, permitem aos clínicos identificar grupos de risco
que beneficiam de terapêuticas agressivas.
Bióloga,
doutorou-se em 9 de Novembro de 2005 com a tese “O Complexo E-Caderina e Cateninas em tumores
epiteliais do ovário”. Actualmente está a fazer um pós-doutoramento no IPO do Porto.
59
Jacinta Serpa
A interface entre os humanos e muitos microorganismos é feita
através de dois sistemas, chamados Lewis e Secretor. Os dois
sistemas enzimáticos de que estamos a falar determinam a adição
(ou não) de fucoses às nossas mucinas e condicionam maior ou
menor facilidade de adquirirmos infecções. O estudo explorou os
polimorfismos dos genes responsáveis pelas variações interindividuais dos fenótipos tendo-se identificado polimorfismos já
conhecidos e, no caso da enzima FUT2 que determina o estado
secretor, identificaram-se e caracterizaram-se em termos
funcionais duas novas variantes do gene. Demonstrou-se ainda
que a metilação do promotor do gene FUT3, que condiciona a
expressão dos antigénios Lewis, é modulada por metilação. Para
além de se ter avançado muito nas relações entre genótipos e
fenótipos nestes sistemas os resultados permitirão esclarecer
melhor a determinação da susceptibilidade às infecções
bacterianas (por exemplo pelo Helicobacter pylori) e víricas.
Bióloga,
doutorou-se em 15 de Dezembro de 2005 com a tese “Fenótipo/genótipo secretor e Lewis e a sua
relevância para a infecção por Helicobacter pylori”. Actualmente está a fazer um pós-doutoramento no IPO de
Lisboa.
60
Manuela Lacerda
Estudou em dois componentes do carcinoma mamário, o
componente intra-ductal e o componente invasivo e também nas
metástases, a expressão de marcadores que têm sido
considerados relevantes na progressão do cancro da mama, tais
como: receptores hormonais (estrogénios e progesterona),
receptores de factores de crescimento, marcadores de proliferação
celular, a expressão da proteína p53 e proteínas reguladoras da
apoptose. Concluiu-se que o grau do componente intra-ductal é
preditivo do grau do componente invasivo e há essencialmente
dois tipos de carcinomas invasivos da mama.
Médica, doutorou-se em Julho de 2005 com a tese “Estudo da progressão no carcinoma mamário utilizando como
modelo o carcinoma mamário invasivo com predomínio do componente intra-ductal”. Era e continua a ser Directora
do Departamento de Patologia do IPO de Coimbra.
61
Nuno Lunet
Neste estudo foram abordados aspectos da epidemiologia do
cancro gástrico, desde a sua relevância como problema de Saúde
Pública a um nível local, até ao contributo de exposições
ambientais, incluindo alimentação, outros estilos de vida e a
infecção por Helicobacter pylori, para a ocorrência de cancro do
estômago com diferentes localizações anatómicas e tipos
histológicos. Demonstrou-se que a associação entre a infecção
por Helicobacter pylori e o cancro do estômago depende da
exposição ao tabaco e ao álcool. Demonstrou-se ainda que o
elevado consumo de frutas e vegetais se associa a um menor
risco de cancro de estômago.
Licenciado
em Farmácia, doutorou-se em 4 de Julho de 2005 com a tese “Diet, lifestyles and gastric cancer”.
Actualmente é Professor Auxiliar Convidado de Epidemiologia na Faculdade de Medicina da Universidade do Porto.
62
Patrícia Castro
Estudou as alterações no número de cromossomas (aneuploidia)
bem como as alterações cromossómicas estruturais (rearranjos)
numa extensa série de tumores da tireóide. Identificou pela
primeira vez uma associação entre a aneuploidia e um subtipo de
tumores da tireóide (adenomas fetais).
Mostrou que um subtipo de carcinoma papilar da tireóide
apresenta alterações moleculares que o aproximam do carcinoma
folicular, estas características estão associadas a factores de
agressividade.
Bióloga,
doutorou-se em 29 de Novembro de 2005 com a tese “Caracterização do fenótipo de instabilidade
cromossómica em tumores foliculares da tireóide”. Actualmente está a fazer um pós-doutoramento no IPATIMUP.
63
Patrícia Mesquita
A mucina MUC2 é habitualmente expressa no intestino e, no
estômago, a sua expressão é aberrante em situações de
metaplasia intestinal e em cerca de 30% das neoplasias.
Identificou-se o factor de transcrição (CDX2) essencial para
desencadear esta expressão aberrante de MUC2 em células
gástricas e mapearam-se os locais do promotor do gene MUC2
utilizados pelo CDX2. Demonstrou-se ainda que a hipometilação
da região promotora do gene MUC2, em locais específicos, é
fundamental para que o gene possa ser expresso. Avançou-se
assim substancialmente no conhecimento dos mecanismos
reguladores das lesões de metaplasia intestinal que, em muitos
casos, precedem o aparecimento de carcinoma gástrico.
Bioquímica,
doutorou-se em 9 de Novembro de 2005 com a tese “Regulação do gene da mucina MUC2 em
carcinoma gástrico e linhas celulares de carcinoma gástrico – papel da metilação e de factores de transcrição”.
Actualmente está a fazer um pós-doutoramento na Estação Zootécnica Nacional, em Santarém.
64
Sandra Beleza
Caracterizou as linhagens masculinas (baseadas em marcadores
genéticos do cromossoma Y) e femininas (pelo estudo do DNA
mitocondrial) em Cabinda, Angola, com o objectivo de avaliar o
impacto genético dos portugueses, decorrente dos
Descobrimentos. Verificou que, como já tinha sido descrito para
outras ex-colónias, este impacto foi nulo no componente feminino
e significativo no masculino (foi mesmo o dobro relativamente ao
detectado em Moçambique, na costa leste africana). Identificou
ainda as linhagens feminina (L1c) e masculina (E3a) com maior
impacto na migração Bantu para o sul de África, migração que reestruturou extensamente a diversidade genética na África subSaariana. Numa componente aplicada à Genética Forense, avaliou
o modo mais eficiente de incluir novos marcadores microssatélites
para uma melhor definição de haplótipos do cromossoma Y,
concluíndo que está dependente do grupo populacional em
questão, e, que no caso português, dada a ausência de
estruturação populacional, não se justifica a divisão em regiões
(norte, centro e sul) devendo ser usada uma base de dados
forense única.
Bióloga,
doutorou-se em Maio de 2005 com a tese “Phylogenetic and demographic history of two human
populations revealed by the analysis of two non-recombining segments of the genome: Y-chromosome and
mitochondrial DNA”. Actualmente está a fazer um pós-doutoramento no IPATIMUP e no Departamento de
Antropologia da Universidade de Toronto.
65
6. ESTAGIÁRIOS
ESTRANGEIROS
NO
IPATIMUP
2005
Beatriz
Martinez
Colombia
Silvia Jiménez
Colombia
Mercedes Aler
Espanha
Paula Diz
Espanha
Ana Parra
Espanha
Ekaterina Rojkova
Russia
Suna Erkilic
Turquia
Wen Xiaogang
China
Ulises Toscanini
Argentina
Amanda Arruda
Brasil
Paula Farina
Brasil
Sabeh Frigi
Tunísia
Carlos Botelho
Brasil
Rafael Vilela
Brasil
Dayse da Silva
Maísa Quintal
Brasil
Brasil
Juliano Freitas
Brasil
Origem dos estagiários em 2005
66
7. TRABALHOS PUBLICADOS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Almeida R, Almeida J, Shoshkes M, Mendes N, Mesquita P, Silva E, Van Seuningen I, Reis CA, Santos-Silva F,
David L. OCT-1 is over-expressed in intestinal metaplasia and intestinal gastric carcinomas and binds to, but does not
transactivate, CDX2 in gastric cells. Journal of Pathology 207:396-401, 2005 IF:5.3
Alonso A, Alves C, Suarez-Mier MP, Albarran C, Pereira L, Fernandez DE Simon L, Martin P, Garcia O, Gusmão L,
Sancho M, Amorim A: Mitochondrial DNA haplotyping revealed the presence of mixed up benign and neoplastic
tissue sections from two individuals on the same prostatic biopsy slide. Journal of Clinical Pathology 58:83-86, 2005
IF:2.6
Alves C, Gusmão L, Lopez-Parra AM, Mesa MS, Amorim A, Arroyo-Pardo E: STR allelic frequencies for an African
population sample (Equatorial Guinea) using AmpFlSTR Identifiler and Powerplex 16 kits. Forensic Science
International 148:239-242, 2005 IF:1.4
Amorim A, Pereira L: Pros and cons in the use of SNPs in forensic kinship investigation: a comparative analysis with
STRs. Forensic Science International 150:17-21, 2005 IF:1.4
Arroyo-Pardo E, Gusmão L, López-Parra AM, Baeza C, Mesa MS, Amorim A: Genetic variability of 16 Y-chromosome
STRs in a sample from Equatorial Guinea (Central Africa). Forensic Science International 149:109-113, 2005 IF:1.4
Basto D, Trovisco V, Lopes JM, Martins A, Pardal F, Soares P, Reis RM: Mutation analysis of B-RAF gene in human
gliomas. Acta Neuropathologica 109:207-210, 2005 IF:2.5
Beleza S, Gusmão L, Amorim A, Carracedo A, Salas A: The genetic legacy of western Bantu migrations. Human
Genetics 117:366-375, 2005 IF:4.3
Cameselle-Teijeiro J, Abdulkader I, Soares P, Alfonsín-Barreiro N, Moldes-Boullosa J, Sobrinho-Simões M: Cystic
tumor of the atrioventricular node of the heart appears to be the heart equivalent of the solid cell nests
(ultimobranchial rests) of the thyroid. American Journal of Clinical Pathology 123:369-375, 2005 IF:2.7
Cameselle-Teijeiro J, Preto A, Soares P, Sobrinho-Simoes M. A stem cell role for thyroid solid cell nests. Human
Pathology 36:590-591, 2005 IF:3.4
Carneiro F, Sobrinho-Simões M: Hereditary diffuse gastric cancer: Lessons from histopathology. Advances in
Anatomic Pathology 12:151-152, 2005 IF:1.7
Carrilho C, Cirnes L, Alberto M, Buane L, Mendes N, David L: Distribution of HPV infection and tumor markers in
cervical intraepithelial neoplasia from cone biopsies of Mozambican women. Journal of Clinical Pathology 58: 61-68,
2005 IF:2.6
Carvalho I, Milanezi F, Martins A, Reis RM, Schmitt F. Overexpression of platelet-derived growth factor receptor alpha
in breast cancer is associated with tumour progression. Breast Cancer Research 7: R788-R795, 2005 IF:3.0
Carvalho-Silva DR, Tarazona-Santos E, Rocha J, Pena SDJ, Santos FR. Y chromosome diversity in Brazilians:
switching perspectives from slow to fast evolving markers. Genetica 126:1-10, 2005. IF: 2.0
Castro P, Eknæs M, Teixeira MR, Danielsen H, Soares P, Lothe R, Sobrinho-Simões M: Adenomas and follicular
carcinomas of the thyroid display two major patterns of chromosomal changes. Journal of Pathology 206:305-311,
2005 IF:5.3
Castro P, Roque L, Magalhães J, Sobrinho-Simões M: A subset of the follicular variant of papillary thyroid carcinoma
harbors the PAX8-PPARg. International Journal of Surgical Pathology 5:333-340, 2005 IF:0.8
Cherni L, Pereira L, Goios A, Yacoubi-Loueslati B, Khodjet el Khil H, Gomes I, Gusmão L, Alves C, Slama A, Amorim
A, BenAmmar Elgaaied A: Y-chromosomal STR haplotypes in three ethnic groups and one cosmopolitan population
from Tunisia. Forensic Science International 152:95-99, 2005 IF:1.4
Cherni L, Yaacoubi-Loueslati B, Pereira L, Alves C, Khodjet El Kill H, Ben Ammar EL, Gaaied A, Amorim A: Data for
15 autosomal STR markers (Powerplex 16 System) from two Tunisian populations: Kesra (Berber) and Zriba (Arab).
Forensic Science International 147:101-106, 2005 IF:1.4
Cherni L, Yacoubi Loueslati B, Pereira L, Ennafaâ H, Amorim A, El Gaaied ABA. Female gene pools of Berber and
Arab neighboring communities in Central Tunisia: microstructure of mtDNA variation in North Africa.Human Biology
77:61-70,2005 IF:1.2
Coelho M, Luiselli D, Bertorelle G, Lopes AI, Seixas S, Destro-Bisol G, Rocha J: Microsatellite variation and evolution
of human lactase persistence. Human Genetics 117:329-339, 2005 IF:4.3
De Souza Goes AC, De Carvalho EF, Gomes I, Da Silva DA, GIL EH, Amorim A, Gusmão L: Population and mutation
analysis of 17 Y-STR loci from Rio de Janeiro (Brazil). International Journal of Legal Medicine 119:70-76, 2005 IF:2.1
Diederiche M, Martín P, Amorim A, Corte-Real F, Gusmão L: A case of double alleles at three Y-STR loci: forensic
implications. International Journal of Legal Medicine 119:223-225, 2005 IF:2.1
Dolle L, Oliveira MJ, Bruyneel E, Hondermarck H, Bracke M. Nerve Growth Factor mediates its pro-invasive effect in
parallel with the release of a soluble E-cadherin fragment from breast cancer MCF-7/AZ cells. Journal of Dairy
Research 72(S1):20-26, 2005 IF:1.2
Domingo E, Niessen RC, Oliveira C, Alhopuro P, Moutinho C, Espín E, Armengol M, Sijmons RH, Kleibeuker JH,
Seruca R, Aaltonen LA, Imai K, Yamamoto H, Schwartz SJr, Hofstra R MW: BRAF-V600E in not involved in the
67
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25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
colorectal tumorigenesis of HNPCC in patients with functional MLH1 and MSH2 genes. Oncogene 24:3995-3998,
2005 IF:6.3
Duarte IS, Santos AM, Sousa H, Catarino R, Pinto D, Matos A, Pereira D, Moutinho J, Canedo P, Machado JC,
Medeiros R. G-308A TNF-alpha polymorphism is associated with an increased risk of Invasive cervical cancer.
Biochemical and Biophysical Research Communications 334: 588-592, 2005 IF:2.9
Dufloth RM, Carvalho S, Heinrich JK, Shinzato JY, Santos C, Zeferino LC, Schmitt F. Analysis of BRCA1 and BRCA2
mutations in Brazilian breast cancer patients with positive family history. São Paulo Medical Journal 123:192-197,
2005
Dufloth RM, Costa S, Schmitt F, Zeferino LC. DNA repair gene polymorphisms and susceptibility to familial breast
cancer in a group of patients from Campinas, Brazil. Genetics and Molecular Research 4:771-782, 2005
Fernando O, Mota P, Lima M, Silva C, Montiel R, Amorim A, Prata MJ. Peopling of the Azores Islands (Portugal): data
from the Y chromosome.Human Biolology 77:189-199, 2005 IF:1.2
Ferreira AC, Almeida S, Tavares M, Canedo P, Pereira F, Regalo G, Figueiredo C, Trindade E, Seruca R, Carneiro F,
Amil J, Machado JC, Tavarela-Veloso F: NOD2/CARD15 and TNFA, but not IL1B and IL1RN, are associated with
Crohn’s disease. Inflammatory Bowel Disease 11:331-339, 2005 IF:3.5
Ferreira JEA, Mello PA, Magalhães AV, Botelho CHA, Naves LA, Nosé V, Schmitt F . Caracterização clínica e
imunoistoquímica dos adenomas clinicamente não-funcionantes de hipófise. Arquivos de Neuro-Psiquiatria 63:10701078, 2005 IF:0.4
Ferreira P, Oliveira MJ, Beraldi E, Mateus AR, Nakajima T, Gleave M, Yokota J, Carneiro F, Huntsman D, Seruca R,
Suriano G.Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro. Experimental
Cell Research 310:99-104, 2005 IF:4.0
Figueiredo C, Machado JC, Yamaoka Y. Pathogenesis of Helicobacter pylori infection. Helicobacter 10 (Suppl 1):1420, 2005 IF:2.3
Garcia-Rostan G, Costa AM, Pereira-Castro I, Salvatore G, Hernandez R, Hermsem MJ, Herrero A, Fusco A,
Cameselle-Teijeiro J, Santoro M. Mutation of the PIK3CA gene in anaplastic thyroid cancer. Cancer Research
65:10199-10207, 2005 IF:7.7
Goios A, Nogueira C, Pereira C, Vilarinho L, Amorim A, Pereira L: MtDNA single macrodeletions associated with
myopathies: absence of haplogroup related increased risk. Journal of Inherited Metabolic Disease 28:769-778, 2005
IF:1.6
Gorringe KL, Chin SF, Pharoah P, Staines JM, Oliveira C, Edwards PA, Caldas C: Evidence that both genetic
instability and selection contribute to the accumulation of chromosome alterations in cancer. Carcinogenesis 26:923930, 2005 IF:5.4
Gouveia AM, Pimenta AP, Lopes JM, Capelinha AF, Ferreira SS, Valbuena C, Oliveira MC: Esophageal GIST:
therapeutic implications of an uncommon presentation of a rare tumor. Diseases of the Esophagus 18:70-73, 2005
IF:0.8
Granja NM, Begnami M, Bertolan J, Longatto-Filho A, Schmitt FC: Desmoplastic small round cell tumour: cytological
and immunocytochemical features. Cytojournal 2: 6, 2005
Granja NM, Ricardo SA, Longatto A, Alves VA, Bedrossian CW, Wiley E, Schmitt FC: Potential use of loss of
heterozygosity in pleural effusions of breast cancer metastases using the microsatellite marker of the 16q22.1 region
of the CDH1 gene. Analytical Quantitative Cytology and Histology 27: 61-66, 2005 IF:0.9
Guida T, Salvatore G, Faviana P, Giannini R, Garcia-Rostan G, Provitera L, Basolo F, Fusco A, Carlomagno F,
Santoro M. Mitogenic effects of the up-regulation of minichromosome maintenance proteins in anaplastic thyroid
carcinoma. Journal of Clinical Endocrinology & Metabolism 90:4703-4709, 2005 IF:5.8
Gusmão L, Butler JM, Carracedo A, Gill P, Kayser M, Mayr WR, Morling N, Prinz M, Roewer L, Tyler-Smith C,
Schneider PM: DNA commission of the International Society of Forensic Genetics (ISFG): an update of the
recommendations on the use of Y-STRs in forensic analysis. International Journal of Legal Medicine 26:1-10, 2005
IF:2.1
Gusmao L, Sanchez-Diz P, Calafell F, Martin P, Alonso CA, Alvarez-Fernandez F, Alves C, Borjas-Fajardo L, Bozzo
WR, Bravo ML, Builes JJ, Capilla J, Carvalho M, Castillo C, Catanesi CI, Corach D, Di Lonardo AM, Espinheira R,
Fagundes de Carvalho E, Farfan MJ, Figueiredo HP, Gomes I, Lojo MM, Marino M, Pinheiro MF, Pontes ML, Prieto V,
Ramos-Luis E, Riancho JA, Souza Goes AC, Santapa OA, Sumita DR, Vallejo G, Vidal Rioja L, Vide MC, Vieira da
Silva CI, Whittle MR, Zabala W, Zarrabeitia MT, Alonso A, Carracedo A, Amorim A. Mutation rates at Y chromosome
specific microsatellites. Human Mutation 26:520-528, 2005 IF:6.8
Kagan E, Ragupathi G, Yi SS, Reis CA, Gildersleeve J, Kahne D, Clausen H, Danishefsky SJ, Livingston PO:
Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide
epithelial cancer antigen Tn. Cancer Immunology, Immunotherapy 54:424-430, 2005 IF:3.5
Lima J, Trovisco V, Soares P, Máximo V, Magalhães J, Salvatore G, Santoro M, Bogdanova T, Tronko M, Abrosimov
M, Jeremiah S, Thomas G, Williams D, Sobrinho-Simões M. Reply to: Low prevalence of BRAF mutations in
radiation-induced thyroid tumors in contrast to sporadic papillary carcinomas. Cancer Letters 230:149-150, 2005
IF:3.0
68
43. Longatto-Filho A, Albergaria A, Paredes J, Moreira MA, Milanezi F, Schmitt FC: P-cadherin expression in glandular
lesions of the uterine cervix detected by liquid-based cytology. Cytopathology 16: 88-93, 2005 IF:1.0
44. Longatto-Filho A, Martins A, Costa SMA, Schmitt FC: VEGFR-3 expression in breast cancer tissue is not restricted to
lymphatic vessels. Pathology Research and Practice 201:93-99, 2005 IF:0.7
45. Lopes R, Castro I, Pontes P, Candeias J, Lemoine N, Sambade C: Expression profile of survivin in acute leukemias:
the importance of differential splicing. Leukemia 19:1284-1286, 2005 IF:5.8
46. Loueslati BY, Cherni L, Khodjet-Elkhil H, Ennafaa H, Pereira L, Amorim A, Ben Ayed F, Ben Ammar Elgaaied A.
Islands Inside an Island: Reproductive Isolates on Jerba Island. American Journal of Human Biology 18:149-153,
2005 IF:1.2
47. Lynch HT, Grady W, Suriano G, Huntsman D: Gastric cancer: New genetic developments. Journal of Surgical
Oncology 90:114-133,2005 IF:1.8
48. Magro F, Pereira P, Carneiro F, Lopes JM, Teixeira A, Sarmento C, Lima JP, Saraiva AC, Teixeira A, TavarelaVeloso F: Colon stenosis in a patient with ulcerative colitis as a manifestation of mixed Mullerian tumor of the
peritoneum. Scandinavian Journal of Gastroenterology 40:1251-1254, 2005 IF:1.8
49. Magro F, Pereira P, Carneiro F, Tavarela-Veloso F: Reactive hepatitis in a patient with Crohn’s disease successfully
treated with infliximab: does tumor necrosis factor alpha play a role in reactive hepatitis? Inflammatory Bowel Disease
11: 88-90, 2005 IF:3.5
50. Magro F, Pereira P, Carneiro F, Tavarela-Veloso F: Unusual presentation of tuberculosis after infliximab therapy.
Inflammatory Bowel Disease 11: 82-84, 2005 IF:3.5
51. Maia L, Dinis J, Cravo M, Claro I, Baltazar C, Fonseca I, Veloso T, Capelinha AF, Carneiro F, Nobre-Leitao C.Who
takes the lead in the development of ulcerative colitis-associated colorectal cancers: mutator, suppressor, or
methylator pathway? Cancer Genetics and Cytogenetics 162:68-73, 2005 IF:1.6
52. Marques T, David L, Reis C, Nogueira A. Topographic expression of MUC5AC and MUC6 in the gastric mucosa
infected by Helicobacter pylori and in associated diseases. Pathology Research and Practice 201:665-672, 2005
IF:0.7
53. Martinez B, Caraballo L, Gusmao L, Amorim A, Carracedo A. Autosomic STR population data in two Caribbean
samples from Colombia. Forensic Science International 152:79-81, 2005. IF:1.4
54. Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F. P63, Cytokeratin 5 and P-Cadherin: three molecular
markers to distinguish basal phenotype in breast carcinomas. Virchows Archiv 447: 688-694, 2005. IF:2.2
55. Máximo V, Botelho T, Capela J, Soares P, Lima J, Taveira A, Amaro T, Barbosa AP, Preto A, Harach HR, Williams D,
Sobrinho-Simões M. Somatic and germline mutation in GRIM19, a dual function gene involved in mitochondrial
metabolism and cell death is linked to mitochondrion-rich (Hürthle cell) tumours of the thyroid. British Journal of
Cancer 92:1892-1998, 2005 IF:3.7
56. Maximo V, Lima J, Soares P, Botelho T, Gomes L, Sobrinho-Simoes M. Mitochondrial D-Loop instability in thyroid
tumours is not a marker of malignancy. Mitochondrion 5:333-340, 2005 IF:1.5
57. Mergulhao P, Magro F, Pereira P, Correia R, Lopes JM, Magalhaes J, Dias JM, Carneiro F, Tavarela-Veloso F.
Gingival hyperplasia as a first manifestation of Crohn's disease. Digestive Diseases Sciences 50:1946-1949, 2005
IF:1.4
58. Montiel R, Bettencourt C, Silva C, Santos C, Prata MJ, Lima M. Analysis of Y-chromosome Variability and its
Comparison with mtDNA Variability Reveals Different Demographic Histories Between Islands in the Azores
Archipelago (Portugal). Annals of Human Genetics 69:135-144, 2005 IF:2.7
59. Oliveira C, Moreira H, Seruca R, Cardoso de Oliveira M, Carneiro F: Role of pathology in the identification of
Hereditary Diffuse Gastric Cancer: Report of a Portuguese family. Virchows Archiv 446:181-184, 2005 IF:2.2
60. Oliveira C, Velho S, Domingo E, Preto A, Hofstra RMW, Hamelin R, Yamamoto H, Seruca R, Schwartz Jr S:
Concomintant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI sporadic colorectal
cancer. Oncogene 24:7630-7634, 2005 IF:6.3
61. Oliveira E, Alves S, Quental S, Ferreira F, Norton L, Costa V, Amorim A, Prata MJ. The MTHFR C677T and A1298C
polymorphisms and susceptibility to childhood acute lymphoblastic leukemia in Portugal. Journal of Pediatric
Hematology/Oncology. 27:425-429, 2005 IF:1.2
62. Oliveira MJ, Lauwaet T, De Bruyne G, Mareel M, Leroy A. Listeria monocytogenes produces a pro-invasive factor that
signals via ErbB2/ErbB3 heterodimers. Journal of Cancer Research and Clinical Oncology 131:49-59, 2005 IF:2.4
63. Oliveira MS, Fraga AG, Torrado E, Castro AG, Pereira JP, Longatto-Filho A, Milanezi F, Schmitt FC, Wayne MM,
Portaels F, Silva MT, Pedrosa J. Infection with Mycobacterium ulcerans induces persistent inflammatory responses in
mice. Infection and Immunity 73: 6299-6310, 2005. IF:4.0
64. Paredes J, Albergaria A, Oliveira JT, Jeronimo C, Milanezi F, Schmitt FC. P-cadherin overexpression is an indicator of
clinical outcome in invasive breast carcinomas and is associated with CDH3 promoter hypomethylation. Clinical
Cancer Research 11:5869-5877, 2005 IF:5.6
65. Pereira F, Pereira L, van Asch B, Bradley DG, Amorim A: The mtDNA catalogue of all Portuguese autochthonous goat
(Capra hircus) breeds: high diversity of female lineages at the western fringe of European distribution. Molecular
Ecology 14:2313-2318, 2005 IF:4.4
69
66. Pereira L, Cunha C, Alves C, Amorim A: African female heritage in Iberia: a reassessment of mtDNA lineage
distribution in present times. Human Biology 77:213-229, 2005 IF:1.2
67. Pereira L, Gonçalves J, Goios A, Rocha T, Amorim A: Human mtDNA haplogroups and reduced male fertility: real
association or hidden population sub-structuring. International Journal of Andrology 28:241-247, 2005 IF:1.9
68. Pereira L, Richards M, Goios A, Alonso A, Albarran C, Garcia O, Behar Dm, Golge M, Hatina J, Al-Gazali L, Bradley
Dg, Macaulay V, Amorim A: High-resolution mtDNA evidence for the late-glacial resettlement of Europe from an
Iberian refugium. Genome Research 15: 19-24, 2005 IF:10.4
69. Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG, Espinel-Ingroff A. Comparison of two probes for testing
susceptibilities of pathogenic yeasts to voriconazole, itraconazole, and caspofungin by flow cytometry. Journal of
Clinical Microbiology 43:4674-4679, 2005 IF:3.4
70. Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG: Safe susceptibility testing of Mycobacterium tuberculosis by flow
cytometry with the fluorescent nucleic acid stain SYTO 16. Journal of Medical Microbiology 54:77-81, 2005 IF:2.5
71. Pina-Vaz C, Rodrigues AG, Costa-de-Oliveira S, Ricardo E, Mardh PA. Potent synergic effect between ibuprofen and
azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry.
Journal of Antimicrobial Chemotherapy 56:678-685, 2005 IF:3.6
72. Regalo G, Wright NA, Machado JC. Trefoil factors: from ulceration to neoplasia. Cellular and Molecular Life Sciences
62: 2910-2915, 2005 IF:4.8
73. Reis C, Carneiro E, Fonseca J, Pereira P, Vaz R, Pinto R, Capelinha AF, Lopes JM, Salgado A: Epithelioid
hemangioendothelioma and multiple thoraco-lumbar lateral meningoceles: two rare pathological entities in a patient
with NF-1. Neuroradiology 47:165-169, 2005 IF:1.5
74. Reis RM, Martins A, Ribeiro SA, Basto D, Longatto-Filho A, Schmitt FC, Lopes JM. Molecular characterization of
PDGFR-alpha/PDGF-A and c-KIT/SCF in gliosarcomas. Cellular Oncology 27:319-326, 2005.
75. Reis RM, Reis-Filho JS, Longatto Filho A, Tomarev S, Silva P, Lopes JM. Differential Prox-1 and CD 31 expression in
mucousae, cutaneous and soft tissue vascular lesions and tumors. Pathology Research and Practice 201:771-776,
2005 IF:0.7
76. Reis-Filho JS, Milanezi F, Carvalho S, Simpson PT, Steele D, Savage K, Lambros MB, Pereira EM, Nesland JM,
Lakhani SR, Schmitt FC. Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and
overexpression: immunohistochemical and chromogenic in situ hybridization analysis. Breast Cancer Research
7:R1028-1035, 2005 IF 3.0
77. Reis-Filho JS, Schmitt FC. Fluorescence in situ hybridization, comparative genomic hybridization, and other molecular
biology techniques in the analysis of effusions. Diagnostic Cytopathology 33:294-299, 2005 IF:1.0
78. Rodrigues AG, Araujo R, Pina-Vaz C: Human albumin promotes germination, hyphal growth and antifungal resistence
by Aspergillus fumigatus. Medical Mycology 43:711-717, 2005 IF 1.4
79. Salas A, Prieto L, Montesino M, Albarrán C, Arroyo E, Paredes-Herrera MR, Di Lonardo AM, Doutremepuich C,
Fernández-Fernández L, González De La Veja A, Alves C, López CM, López-Soto M, Lorente JA, Picornell A,
Espinheira RM, Hernández A, Palácio AM, Espinoza M, Yunis JJ, Pérez-Lezaun A, Pestano JJ, Carril JC, Corach D,
Vide MC, Álvarez-Iglesias V, Pinheiro MF, Whittle MR, Brehm A, Gómez J: Mitochondrial DNA error prophylaxis:
assessing the causes of errors in the GEP’02–03 proficiency testing trial. Forensic Science International 148:191-198,
2005 IF:1.4
80. Sampaio P, Gusmao L, Correia A, Alves C, Rodrigues AG, Pina-Vaz C, Amorim A, Pais C. New microsatellite
multiplex PCR for Candida albicans strain typing reveals microevolutionary changes. Journal of Clinical Microbiology
43:3869-3876, 2005 IF:3.4
81. Santos-Silva F, Fonseca A, Caffrey T, Carvalho F, Mesquita P, Reis C, Almeida R, David L, Hollingsworth M:
Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR
polymorphism. Glycobiology 15:511-517, 2005 IF:4.1
82. Silva LF, Ribeiro D, David L, Felino A: Oral osteossarcoma: case report. Oral Oncology 41:195-197, 2005 IF:2.0
83. Simpson PT, Reis-Filho JS, Gale T, Lakhani SR. Molecular evolution of breast cancer. Journal of Pathology 205:248254, 2005 IF: 5.3
84. Slovin SF, Ragupathi G, Fernandez C, Jefferson MP, Diani M, Wilton AS, Powell S, Spassova M, Reis C, Clausen H,
Danishefsky S, Livingston P, Scher HI. A bivalent conjugate vaccine in the treatment of biochemically relapsed
prostate cancer: a study of glycosylated MUC-2-KLH and Globo H-KLH conjugate vaccines given with the new semisynthetic saponin immunological adjuvant GPI-0100 OR QS-21. Vaccine 23:3114-122, 2005 IF:2.8
85. Sobrinho-Simões M, Magalhães J, Fonseca E, Amendoeira I: Diagnostic pitfalls of thyroid pathology. Current
Diagnostic Pathology 11:52-59, 2005.
86. Sobrinho-Simões M, Maximo V, Vieira de Castro I, Fonseca E, Soares P, Garcia-Rostan G, Cardoso de Oliveira M.
Hurthle (oncocytic) cell tumors of thyroid: etiopathogenesis, diagnosis and clinical significance. International Journal
of Surgical Pathology 13:29-35, 2005 IF:0.8
87. Sobrinho-Simoes M, Preto A, Rocha AS, Castro P, Maximo V, Fonseca E, Soares P. Molecular pathology of welldifferentiated thyroid carcinomas. Virchows Archiv 447:787-793, 2005 IF:2.2
70
88. Sobrinho-Simões M: BRAF Mutations in thyroid carcinomas: phenotype-genotype correlations. Advances in Anatomic
Pathology 12:106-107, 2005 IF:1.7
89. Souto L, Alves C, Gusmão L, Ferreira E, Amorim A, Côrte-Real F, Vieira DN: Population analysis of 15 STRs on a
sample from East-Timor. Forensic Science International 155:77-80, 2005 IF:1.4
90. Suriano G, Vrcelj N, Senz J, Ferreira P, Masoudi H, Cox K, Nabais S, Lopes C, Machado JC, Seruca R, Carneiro F,
Huntsman DG: beta-Catenin (CTNNB1) gene amplification: A new mechanism of protein overexpression in cancer.
Genes Chromosomes Cancer 42:238-246, 2005 IF:4.3
91. Suriano G, Yew S, Ferreira P, Senz J, Kaurah P, Ford JM, Longacre TA, Norton JA, Chun N, Young S, Oliveira MJ,
Macgillivray B, Rao A, Sears D, Jackson CE, Boyd J, Yee C, Deters C, Pai GS, Hammond LS, McGivern BJ,
Medgyesy D, Sartz D, Arun B, Oelschlager BK, Upton MP, Neufeld-Kaiser W, Silva OE, Donenberg TR, Kooby DA,
Sharma S, Jonsson BA, Gronberg H, Gallinger S, Seruca R, Lynch H, Huntsman DG. Characterization of a recurrent
germ line mutation of the E-cadherin gene: implications for genetic testing and clinical management. Clinical Cancer
Research 11:5401-5409, 2005 IF:5.6
92. Trovisco V, Soares P, Preto A, Vieira de Castro I, Lima J, Castro P, Máximo V, Botelho T, Moreira S, Meireles AM,
Magalhães J, Abrosimov A, Cameselle-Teijeiro J, Sobrinho-Simões M: Type and prevalence of BRAF mutations are
closely associated to papillary thyroid carcinoma histotype and patients’ age but not with tumour aggressiveness.
Virchows Archiv 446:589-595, 2005 IF:2.2
93. Trovisco V, Soares P, Soares R, Magalhães J, Sá-Couto P, Sobrinho-Simões M: A new BRAF gene mutation
detected in a case of a solid variant of papillary thyroid carcinoma. Human Pathology 36:694-697, 2005 IF:3.4
94. van Asch B, Pereira L, Pereira F, Santa-Rita P, Lima M, Amorim A. MtDNA diversity among four Portuguese
autochthonous dog breeds: a fine-scale characterisation.BMC Genetics 6: 37, 2005 IF:0.9
95. Van Marck V, Stove C, Van Den Bossche K, Stove V, Paredes J, Vander Haeghen Y, Bracke MP-cadherin promotes
cell-cell adhesion and counteracts invasion in human melanoma. Cancer Research 65:8774-8783, 2005 IF:7.7
96. Vasconcelos MH, Maia LF, Sousa C, Beleza SS and Guimaraes JE. Evidence for a specific intracellular localization of
an antisense oligonucleotide in K562 cells. The Japanese Pharmacological Society. 99:105-8, 2005 IF:1.7
97. Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz Jr S, Duval A, Carneiro F, Machado JC, Hamelin
R, Raquel Seruca R: PIK3CA, KRAS AND BRAF mutations in MSI and MSS gastrointestinal carcinomas. European
Journal of Cancer 41:1649-1654, 2005 IF:3.3
98. Vilarinho L, Cardoso ML, Gaspar P, Barbot C, Azevedo L, Diogo L, Santos M, Carrilho I, Fineza I, Kok F, Chorao R,
Alegria P, Martins E, Teixeira J, Cabral Fernandes H, Verhoeven NM, Salomons GS, Santorelli FM, Cabral P, Amorim
A, Jakobs C. Novel L2HGDH mutations in 21 patients with L-2-hydroxyglutaric aciduria of Portuguese origin. Human
Mutation 26:395-396, 2005 IF:6.8
99. Willems S, Carneiro F, Geboes K: Gastric carcinoma with osteoclast-like giant cells and lymphoepithelioma-like
carcinoma of the stomach: two of a kind? Histopathology 47:331-333, 2005 IF:3.0
71
8. REVISTAS
CIENTÍFICAS
INTERNACIONAIS COM
MEMBROS DO
IPATIMUP
NOS SEUS EDITORIAL
BOARDS
Acta Cytologica (SCI PRINTERS & PUBL INC)
Advances in Anatomic Pathology (LIPPINCOTT WILLIAMS & WILKINS)
Archives of Pathology and Laboratory Medicine (COLLEGE OF AMERICAN PATHOLOGISTS)
Breast Cancer Research (BIOMED CENTRAL LTD)
Cancer Genetics and Cytogenetics (ELSEVIER SCI IRELAND LTD)
Critical Review in Oncogenesis (BEGELL HOUSE, USA)
Current Diagnostic Pathology (CHURCHILL LIVINGSTONE)
Cytojournal (BIOMED CENTRAL)
Cytopathology (BLACKWELL PUBLISHING LTD)
Diagnostic Cytopathology (WILEY-LISS)
Endocrine Pathology (BLACKWELL PUBLISHING LTD)
European Journal of Cancer Prevention (LIPPINCOTT WILLIAMS & WILKINS)
Forensic Science International (ELSEVIER SCI IRELAND LTD)
Hereditary Cancer in Clinical Practice (UICC GLOBAL CANCER CONTROL)
Histopathology (BLACKWELL PUBLISHING LTD)
Human Biology (WAYNE STATE UNIVERSITY PRESS)
International Journal of Surgical Pathology (WESTMINSTER PUBL INC)
Journal of Cellular and Molecular Medicine (CAROL DAVILA UNIV PRESS)
Journal of Pathology (JON WILEY & SONS LTD)
Pathology Research & Practice (URBAN & FISCHER VERLAG)
Seminars in Diagnostic Pathology (W B SAUNDERS CO)
Ultrastructural Pathology (TAYLOR & FRANCIS INC)
Virchows Archiv (SPRINGER)
72
9. NÚCLEO DE AMIGOS
DO
IPATIMUP
Allianz Portugal
Amorim Inv. E Participações SGPS
Banco BPI
Bayer Portugal
Banco Espírito Santo
Bial - Portela & C.ª
Forter Térmica Industrial
Fundação Millennium BCP
GlaxoSmithKline Prod. Farmacêuticos
Merck Sharp & Dohme
Mota Engil, SGPS
Novartis Farma - Prod. Farmacêuticos
Olinveste SGPS
Portgás - Soc. de Prod. e Distr. de Gás
Portucel SGPS
RAR - Sociedade de Controle (Holding)
Roche Farmacêutica Química
SAG GEST - Soluções Automóvel Globais
Sonae SGPS
Têxtil Manuel Gonçalves
Unicer
73