126 research outputs found
Molecular genetics of paediatric versus adult brain tumours
Tese de doutoramento em Ciências da Saúde (especialidade de Ciências da Saúde)Brain
tumours
are
the
leading
cause
of
cancer-‐related
death
in
paediatric
patients
and
are
responsible
for
the
greater
part
of
the
cancer-‐related
years
of
life
lost
across
all
age
groups.
The
more
malignant
histologies
are
the
major
contributors
to
the
high-‐rates
of
mortality
and
morbidity
of
brain
tumours.
In
the
present
thesis
we
focused
our
studies
on
high-‐grade
gliomas
and
medulloblastomas,
the
most
common
malignant
brain
tumours
of
adults
and
childhood
patients,
respectively.
Childhood
and
adult
patients
share
most
of
the
different
brain
tumours’
histological
types,
despite
a
large
variation
in
frequency
across
specific
age
groups.
Nevertheless,
there
is
increasing
evidence
that,
despite
being
histologically
similar,
childhood
and
adult
tumours
have
key
clinical
and
molecular
differences.
The
work
summarized
in
this
thesis
aims
to
disclose
molecular
mechanisms
particular
to
paediatric
and
adult
high-‐grade
gliomas
and
medulloblastomas,
attempting
to
better
understand
the
biology
of
age-‐specific,
histologically
identical,
tumours.
Brain
tumours
are
characterized
by
multiple
genetic
alterations
affecting
receptor
tyrosine
kinase
(RTK)
pathways.
As
the
EGFR
RTK
pathway
is
one
of
the
most
important
signalling
networks
in
high-‐grade
gliomas,
we
aimed
to
study
EGFR
molecular
aberrations
involved
in
protein
activation,
potentially
relevant
for
tumour’s
response
to
EGFR-‐targeted
therapy.
The
role
of
EGFR
in
adult
high-‐grade
gliomas
is
well-‐characterized,
and
in
the
present
thesis
we
studied
a
unique
series
of
Portuguese
patients,
aiming
to
understand
whether
the
EGFR
molecular
alterations
of
these
patients
were
in
line
with
other
populations
in
terms
of
potential
biomarkers
of
EGFR-‐targeted
therapy.
On
the
other
hand,
EGFR
is
thought
to
be
less
significant
in
childhood
tumours,
although
there
is
limited
published
data.
Accordingly,
we
aimed
to
study
the
frequency
and
role
of
EGFR
molecular
alterations
in
paediatric
high-‐grade
gliomas,
aiming
to
evaluate
the
presence
of
molecular
signatures
of
response
to
existing
drugs
in
these
patients.
We
confirmed
that
EGFR
represents
one
of
the
most
frequently
altered
molecules
in
high-‐grade
glioma,
particularly
in
adult
glioblastoma,
and
that
it
is
also
true
for
Portuguese
patients.
In
addition
some
paediatric
tumours,
particularly
anaplastic
oligodendrogliomas,
frequently
presented
EGFR
aberrations
and
therefore
are
also
potential
candidates
for
EGFR-‐targeted
therapy. Microsatellite
instability
(MSI)
frequency
in
brain
tumours
remains
a
controversial
research
topic,
and
there
is
a
lack
of
clarity
in
the
published
literature.
In
this
context,
we
aimed
to
study
MSI
in
high-‐grade
gliomas
and
medulloblastoma
from
adult
and
paediatric
patients
and
identify
MSI
target
genes
potentially
involved
in
MSI-‐related
tumorigenesis.
Our
findings
show
the
presence
of
MSI
in
a
fraction
of
medulloblastoma
and
high-‐grade
gliomas.
Age-‐specific
differences
in
MSI
frequency
were
not
present
in
medulloblastoma,
however
MSI
was
significantly
more
frequent
in
paediatric
high-‐grade
gliomas
than
in
adults
tumours.
Moreover
MSI-‐positivity
was
associated
with
a
stable
genomic
profile.
Overall,
of
the
18
MSI
target-‐genes
studied,
only
three
were
mutated,
all
in
paediatric
in
MSI
tumours,
MBD4
in
one
medulloblastoma,
and
MSH6
and
DNAPKcs
in
high-‐grade
glioma
As
we
failed
to
find
the
MMR
alteration
responsible
for
the
MSI
phenotype,
further
research
is
critical
to
clarify
this
topic.
Nevertheless,
our
studies
provided
evidence
for
a
potential
novel
molecular
pathway
in
a
proportion
of
medulloblastoma
and
paediatric
high-‐grade
gliomas,
associated
with
the
presence
of
MSI.
Overall,
the
work
summarized
in
this
thesis
contributed
to
the
knowledge
of
the
molecular
mechanisms
involved
in
the
development
of
childhood
and
adult
brain
tumours,
and
confirms
that,
despite
being
histologically
indistinguishable,
these
tumours
can
be
molecularly
distinctive.Os
tumores
cerebrais
são
a
principal
causa
de
morte
por
cancro
em
crianças,
sendo
também
os
principais
responsáveis
na
diminuição
de
anos
de
vida
em
doentes
oncológicos
de
todas
as
faixas
etárias.
Os
tumores
de
maior
malignidade
sãos
que
mais
contribuem
para
as
altas
taxas
de
mortalidade
e
morbilidade
características
dos
tumores
cerebrais.
Nesta
tese,
centramos
os
nossos
estudos
em
gliomas
de
alto
grau
e
meduloblastomas,
os
tumores
cerebrais
malignos
mais
frequentes
em
doentes
adultos
e
pediátricos,
respectivamente.
Apesar
dos
diversos
tipos
histológicos
de
tumores
cerebrais
serem
comuns
a
doentes
de
diferentes
idades,
existe
uma
significativa
diferença
na
frequência
com
que
ocorrem
em
adultos
e
crianças.
Além
disso,
há
cada
vez
mais
indícios
de
que,
tumores
histologicamente
semelhantes,
apresentam
diferenças
fundamentais
a
nível
clínico
e
molecular,
dependendo
da
idade
dos
doentes.
Os
tumores
cerebrais
são
caracterizados
por
diversas
alterações
genéticas
que
afectam
os
receptores
de
tirosina
cinase
(RTK).
Sendo
a
via
de
sinalização
do
RTK
EGFR
uma
das
mais
importantes
em
gliomas
de
alto
grau,
estudámos
alterações
moleculares
envolvidas
na
sua
activação
e
potencialmente
importantes
na
resposta
tumoral
à
terapia
dirigida.
O
papel
desta
molécula
em
gliomas
de
alto
grau
de
doentes
adultos
tem
sido
amplamente
descrito,
pelo
que
avaliámos
uma
série
de
tumores
de
doentes
Portugueses
adultos.
Neste
trabalho
pretendemos
perceber
se
as
alterações
de
EGFR
nos
tumores
Portugueses
se
assemelham
às
descritas
noutras
populações,
de
forma
a
avaliar
o
seu
potencial
papel
como
biomarcador
de
terapia
dirigida
ao
EGFR.
Por
outro
lado,
,
apesar
da
escassez
de
dados
publicados,
pensa-‐se
que
a
importância
do
EGFR
em
tumores
pediátricos
seja
limitada.
Para
melhor
esclarecer
este
assunto,
estudámos
a
presença
de
alterações
moleculares
do
EGFR
em
gliomas
pediátricos
de
alto
grau,
com
o
objectivo
de
avaliar
quais
os
potenciais
biomarcadores
na
resposta
a
fármacos
anti-‐EGFR.
Confirmámos
que
o
EGFR
é
uma
das
moléculas
mais
frequentemente
alteradas
em
gliomas
de
alto
grau,
particularmente
em
tumores
adultos,
sendo
isto
também
verdade
para
os
doentes
Portugueses.
Igualmente
os
tumores
pediátricos,
em
particular,
os
oligodendrogliomas
anaplásicos,
apresentam
alterações
nesta
molécula
e
consequentemente,
são
também
potenciais
candidatos
ao
uso
de
fármacos
anti-‐EGFR. A
ocorrência
de
instabilidade
de
microssatélites
(MSI)
em
tumores
cerebrais
é
um
tópico
de
investigação
controverso,
sendo
a
literatura
existente
insuficiente
para
a
esclarecer
este
assunto.
Neste
contexto,
estudámos
a
presença
de
MSI
em
gliomas
de
alto
grau
e
meduloblastomas
de
doentes
adultos
e
pediátricos,
assim
como
os
genes-‐alvo
potencialmente
envolvidos
na
tumorigénese
relacionada
com
MSI.
Os
nossos
resultados
demonstram
a
presença
de
MSI
numa
fracção
de
meduloblastomas
e
gliomas
de
alto
grau.
Em
meduloblastomas
não
se
observaram
variações
na
presença
de
MSI
em
doentes
de
diferentes
idades,
no
entanto
em
gliomas
de
alto
grau,
a
frequência
de
MSI
é
estatisticamente
mais
elevada
nos
tumores
pediátricos
.
Além
disso,
em
gliomas,
a
presença
de
MSI
foi
associada
a
um
perfil
genómico
estável.
Dos
mais
de
18
genes-‐alvo
analisados,
foram
encontradas
mutações
apenas
em
três
e
somente
em
tumores
pediátricos:
MBD4
num
meduloblastoma
e
MSH6
e
DNAPKcs
em
gliomas
de
alto
grau.
.
O
estudo
das
moléculas
de
“mismatch
repair”
não
clarificou
qual
o
seu
papel
no
fenótipo
de
MSI,
observado
nestes
doentes.
No
entanto,
os
nossos
estudos
evidenciam
a
presença
de
uma
potencial
nova
via
molecular
em
alguns
meduloblastomas
e
gliomas
de
alto
grau
associada
à
presença
de
MSI.
Concluindo,
o
trabalho
resumido
nesta
tese,
contribuiu
para
o
conhecimento
das
mecanismos
molecular
envolvidos
no
desenvolvimento
de
tumores
cerebrais
pediátricos
e
adultos,
e
confirma
que
apesar
de
histologicamente
semelhantes,
podem
ser
molecularmente
distintos
SPINT2 deregulation in prostate carcinoma
SPINT2 is a tumor suppressor gene that inhibits proteases implicated in cancer progression, like HGFA, hepsin and matriptase. Loss of SPINT2 expression in tumors has been associated with gene promoter hypermethylation; however, little is known about the mechanisms of SPINT2 deregulation in prostate cancer (PCa). We aimed to analyze SPINT2 expression levels and understand the possible regulation by SPINT2 promoter hypermethylation in PCa. In a cohort of 57 cases including non-neoplastic and PCa tissues, SPINT2 expression and promoter methylation was analyzed by immunohistochemistry and methylation-specific PCR, respectively. Methylation status of the SPINT2 promoter was also evaluated by bisulfite sequencing and 5-aza-2’-deoxycytidine treatment. Oncomine and TCGA databases were used to perform in silico PCa analysis of SPINT2 mRNA and methylation levels. A reduction in SPINT2 expression levels from nonneoplastic to PCa tissues was observed; however, none of the cases exhibited SPINT2 promoter methylation. Both bisulfite sequencing and 5-aza demonstrated that SPINT2 promoter is not methylated in PCa cells. Bioinformatics approaches did not show downregulation of SPINT2 at the mRNA level and, in corroboration with our results, SPINT2 promoter region is reported to be unmethylated. Our study suggests an involvement of SPINT2 in PCa tumorigenesis, probably in association with a post-translational regulation of SPINT2.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the ICVS internal research funds of participating authors and by FCT project, ref. PTDC/SAUONC/115513/2009. F.P. received fellowship from the FCT, ref. SFRH/BD/81369/2011 and M.VP from the ON.2 SR&TD Integrated Program (N-01-01-01-24-01-07), ref. UMINHO/ BPD/36/2013
Analysis of EGFR Overexpression, EGFR gene amplification and the EGFRvIII Mutation in portuguese high-grade gliomas
Background: Patients with malignant gliomas do not respond to any current therapy. Epidermal growth factor receptor (EGFR) controls several oncogenic processes, being
frequently up-regulated in gliomas due to overexpression, gene amplification and gene mutation. EGFR inhibitors are being tried in gliomas, yet the molecular determinants of therapeutic response are unclear. Materials and Methods: EGFR overexpression, EGFRvIII mutation and EGFR amplification were determined by immunohistochemistry and chromogenic in situ hybridization (CISH) in 27 primary glioblastomas (GBM), 24 anaplastic oligodendrogliomas (AO) and four
anaplastic oligoastrocytomas (AOA). Results: EGFR overexpression was associated with EGFR amplification, being found in 48% and 53% GBM, 33% and 40% AO and 75%
and 67% AOA, respectively. EGFRvIII was found in 22% GBM, 8% AO and was absent in AOA. No association was observed between EGFR alterations and patient survival.
Conclusion: We characterized, for the first time, EGFR molecular alterations in Portuguese patients with malignant
glioma and identified a subpopulation of patients presenting putative biomarkers for EGFR-based therapies
Analysis of microsatellite instability in medulloblastoma
Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.M.V.-P. is the recipient of a Ph.D. fellowship (SFRH/BD/29145/2006), and I.A. is the recipient
of a research fellowship (SFRH/BI/33160/2007) from
Fundação para a Ciência e Tecnologia, Portugal. This
study was partially supported by a grant from Clinical
de Radioterapia do Porto, Portugal
Study of hTERT and Histone 3 Mutations in Medulloblastoma
CNPq/Universal
(475358/2011-2), Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP; 2012/19590-0) and Fundação para a Ciência
e Tecnologia (FCT; PTDC/SAU-ONC/115513/2009) grants to
R.M.R. The project was cofinanced by Programa Operacional
Regional do Norte (ON.2-O Novo Norte), Quadro de Referência
Estratégico Nacional (QREN) and Fundo Europeu de Desenvol-
vimento Regional (FEDER). M.V.-P. is the recipient of an FCT
Post-Doctorate Research Fellowship (SFRH/BPD/104290/2014)Hotspot activating mutations of the telomerase reverse transcriptase (hTERT) promoter region were recently described in several tumor types. These mutations lead to enhanced expression of telomerase, being responsible for telomere maintenance and allowing continuous cell division. Additionally, there are alternative telomere maintenance mechanisms, associated with histone H3 mutations, responsible for disrupting the histone code and affecting the regulation of transcription. Here, we investigated the clinical relevance of these mechanistically related molecules in nnedulloblastoma. Sixty-nine medulloblastomas, formalin fixed and paraffin embedded, from a cohort of patients aged 1.5-70 years, were used to investigate the hotspot mutations of the hTERT promoter region, i.e. H3F3A and HIST1H3B, using Sanger sequencing. We successfully sequenced hTERT in all 69 medulloblastoma samples and identified a total of 19 mutated cases (27.5%). c.-124:G>A and c.-146:G>A mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p < 0.0001), being found only in 5 patients <20 years of age. In addition, hTERT-mutated tumors were more frequently recurrent (p = 0.026) and hTERT mutations were significantly enriched in tumors located in the right cerebellar hemisphere (p = 0.039). No mutations were found on the H3F3A or HIST1H3B genes. hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in nnedulloblastoma.This study was partially supported by CNPq/Universal (475358/2011-2), Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP; 2012/19590-0) and Fundacao para a Ciencia e Tecnologia (FCT; PTDC/SAU-ONC/115513/2009) grants to R.M.R. The project was cofinanced by Programa Operacional Regional do Norte (ON.2-O Novo Norte), Quadro de Referencia Estrategico Nacional (QREN) and Fundo Europeu de Desenvolvimento Regional (FEDER). M.V.-P. is the recipient of an FCT Post-Doctorate Research Fellowship (SFRH/BPD/104290/2014).info:eu-repo/semantics/publishedVersio
Portuguese propolis antitumoral activity in melanoma involves ROS production and induction of apoptosis
Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40–60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.Foundation for Science and Technology (FCT)—projects UIDB/04050/2020, UDBI/04033/2020, UIDB/50026/2020, UIDP/50026/2020, UIDB/50006/2020, UIDP/50006/2020, and by the project NORTE-01-0145-FEDER-000055, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.P.C., C.B.M. and A.S.F. are recipients of FCT grants (2020.05779.BD, SFRH/BD/145955/2019, and PD/BD/128276/2017, respectively
Genetic variants of vascular endothelial growth factor predict risk and survival of gliomas
The vascular endothelial growth factor regulates angiogenesis that is increased in glioma. VEGF polymorphisms are thought to modulate vascular endothelial growth factor plasma levels and therefore may be implicated in glioma risk. We aimed to clarify the role of VEGF and von Willebrand factor polymorphisms in glioma susceptibility and prognosis. A case-control study of 126 glioma patients and 180 cancer-free controls was performed. Using Sequenom MassARRAY platform, 11 VEGF and 1 VWF polymorphisms were genotyped. Unconditional multivariate logistic regression models were used to calculate odds ratios and 95% confidence intervals. The associations between polymorphisms and survival were evaluated using a Cox regression model. Bonferroni's adjustment was used to correct for multiple testing. The VEGF polymorphism rs833061 was strongly associated with increased risk for glioma (odds ratio = 164.85) and glioblastoma (odds ratio = 155.66), confirmed after Bonferroni correction. Also, the VEGF polymorphisms rs3024994, rs2010963, and particularly the homozygous carriers of rs1005230 were associated with a worse prognosis for glioma and glioblastoma. Our data support a role of VEGF and VWF polymorphisms as glioma biomarkers, with additional potential relevance for molecular stratification of patients for anti-angiogenic therapies.FCT -Fundação para a Ciência e a Tecnologia(NORTE-01-0145-FEDER-000013)info:eu-repo/semantics/publishedVersio
Copy number profiling of Brazilian astrocytomas
Copy number alterations (CNA) are one of the driving mechanisms of glioma tumorigenesis, and are currently used as important biomarkers in the routine setting. Therefore, we performed CNA profiling of 65 astrocytomas of distinct malignant grades (WHO grade I-IV) of Brazilian origin, using array-CGH and microsatellite instability analysis (MSI), and investigated their correlation with TERT and IDH1 mutational status and clinico-pathological features. Furthermore, in silico analysis using the Oncomine database was performed to validate our findings and extend the findings to gene expression level. We found that the number of genomic alterations increases in accordance with glioma grade. In glioblastomas (GBM), the most common alterations were gene amplifications (PDGFRA, KIT, KDR, EGFR, and MET) and deletions (CDKN2A and PTEN). Log-rank analysis correlated EGFR amplification and/or chr7 gain with better survival of the patients. MSI was observed in 11% of GBMs. A total of 69% of GBMs presented TERT mutation, whereas IDH1 mutation was most frequent in diffuse (85.7%) and anaplastic (100%) astrocytomas. The combination of 1p19q deletion and TERT and IDH1 mutational status separated tumor groups that showed distinct age of diagnosis and outcome. In silico validation pointed to less explored genes that may be worthy of future investigation, such as CDK2, DMRTA1, and MTAP. Herein, using an extensive integrated analysis, we indicated potentially important genes, not extensively studied in gliomas, that could be further explored to assess their biological and clinical impact in astrocytomas.This study was partially supported by the Universal/National Counsel of Technological and Scientific Development (CNPq) (475358/2011-2 – R.M.R.), São Paulo Research Foundation (FAPESP) (2012/19590-0 and 2016/09105-8 – R.M.R.) and the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-ONC/115513/2009-FCMO-01-0124FEDER-015949). L.T.B. was recipient of FAPESP fellowships (2011/ 08523-7 and 2012/08287-4), N.C.C.was recipient of a FAPESP fellowship (2013/25787-3), M.L.S. was recipient of a CNPq/Programa Institucional de Bolsas de Iniciação Científica (PIBIC) fellowship (100707/ 2014-9), W.M. was recipient of FAPESP (2013/15515-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/ Programa de Suporte à Pós-Graduação de Instituições de Ensino Particulares (Prosup) fellowships, and M.V.P. was a Postdoctoral research fellow under the FCT project PTDC/SAU-ONC/115513/2009. R.M.R. has a CNPq scholarship. C.J. and A.M. acknowledge National Health Service funding to the National Institute for Health Research Biomedical Research Centre at The Royal Marsden and the Institute of Cancer Research.info:eu-repo/semantics/publishedVersio
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