121 research outputs found
Dalla discovery del “Cancer Methylome” alla validazione di nuovi biomarkers epigenetici nelle neoplasie gastrointestinali
No full textABSTRACT (Italiano)
Secondo l’attuale modello di cancerogenesi “epigenetico-staminale” i tumori originano da
un insieme non solo di mutazioni genetiche ma di modificazioni epigenetiche a livello di
geni che regolano le più disparate funzioni cellulari come proliferazione, differenziamento,
apoptosi, stabilità genomica, angiogenesi, invasione e metastasi.
Diversi studi hanno messo in evidenza che le modificazioni epigenetiche (metilazione del
DNA; acetilazione/fosforilazione/metilazione/ubiquitinizzazione degli istoni; non-coding
RNA, modificazioni conformazionali della cromatina) sono presenti sin dalle fasi più precoci
della cancerogenesi, permettendo di individuare nuovi biomarkers di diagnosi, prognosi,
follow-up e predizione di risposta alla terapia. Su questa linea di evidenze abbiamo
condotto uno studio del “cancer methylome” con l’obiettivo di individuare nuovi target
epigenetici.
La fase iniziale di discovery è stato condotta mediante analisi dell’espressione di circa
49.000 geni con tecnologia microarray in 20 linee cellulari derivate da 5 diversi istotipi. Per
mezzo di una strategia, messa a punto con il gruppo della JHU, di smascheramento
farmacologico con l’ agente demetilante 5-aza-2’deossicitidina, è stato possibile
selezionare 200 geni, 25 dei quali erano già noti per la loro metilazione tumore-specifica.
175 rappresentavano nuovi geni potenzialmente metilati. Di questi 175 geni, 82 risultavano
metilati solo nelle linee cellulari, 53 anche nei
tessuti primari di 14 istotipi diversi. Di questi 53
geni 28/53 presentavano una metilazione
tumore-specifica, mentre 25 una metilazione
tessuto-specifica.
Dopo l’analisi high-throughput iniziale di
discovery, abbiamo condotto uno studio di
validazione di specifici target nelle neoplasie
colon-rettali e del pancreas. Tra i geni metilati
in maniera tumore-specifica abbiamo
selezionato i seguenti geni: b4GalT1 per i
tumori del colon (130 casi di adenocarcinoma
del colon, 13 adenomi, and mucosa colica
normale); KIF1A per l’ adenocarcinoma
pancreatico (60 tumori e 10 campioni di
parenchima normale).
Il gene b4GalT1 presenta una sensibilità del
54% (95% CI: 35.1%–72.1%) e una specificità
del 91.7% (95% CI: 74.1%–97.7%). I livelli di metilazione correlano in maniera inversa con i
livelli di espressione dell’ mRNA, non sono state trovate correlazioni tra la metilazione e i
dati di outcome clinico. Il gene KIF1A risulta invece metilato nell’81% degli adenocarcinomi
175 potentially novel
methylated genes
25 known
methylated genes
Methylated in
primary tissue 53/82
Cancer-specific
methylation 28/53
Tissue-specific
Methylation, 25/53 del pancreas ma non nel parenchima normale derivato da soggetti senza malattia
neoplastica evidente. KIF1A presenta metilazione anche nelle lesioni preneoplastiche
IPMN (Intraductal papillary mucinous neoplasm) suggerendo che la metilazione di questo
gene può comparire già nella fasi più precoci del processo neoplastico.
Diversi marcatori molecolari vengono continuamente proposti per una miglior definizione
della diagnosi e della prognosi nel carcinoma del colon retto e del pancreas, ma la loro
reale utilità rimane controversa spesso per la loro scarsa sensibilità e specificità. Il primo
step nella pipeline di discovery di nuovi biomarcatori di screening richiede infatti uno studio
del nuovo target nei tessuti tumorali e in quelli non tumorali per la definizione dei suddetti
parametri. Pertanto alla luce dei risultati ottenuti i marcatori epigenetici di b4GalT1 e KIF1A
rispettivamente ipermetilati in modo significativo nei tumori del colon e del pancreas, in
funzione della loro elevata sensibilità e specificità possono essere considerati dei
“candidate biomarkers” ottimali per lo studio di queste neoplasie e in particolare per ulteriori
studi che ne permettano di definire il ruolo nella diagnosi precoce (screening) e nel followup
(marcatori dei ripresa di malattia).
ABSTRACT (Inglese)
Solid human tumors arise and progress through aberrant function of various genes that
positively and negatively regulate many aspects of cell function, including proliferation,
apoptosis, genome stability, angiogenesis, invasion and metastasis. Studies in animals and
in humans have demonstrated that these epigenetic changes are an early event in
carcinogenesis and are present in the precursor lesions of a variety of cancers including
colon and pancreatic lesions. In an effort to identify important tumor suppressor genes
silenced by promoter methylation, in the first phase of the present study I have conducted a
genome-wide promoter screening study, in collaboration with Dr. David Sidransky’s group
(Johns Hopkins University), in cancer cell lines and 13 different tumor histotypes . The
approach coupled probabilistic search algorithms in the entire human genome with an
established pharmacologic unmasking strategy in cancer cell lines for unbiased and precise
global localization of tumor-specific methylated genes. The computational approach
followed by microarray analysis allowed to identify 200 genes predicted to be methylated.
25 were previously reported as harbouring cancer specific promoter methylation after a
literature search. The remaining 175 genes were tested for promoter methylation by
bisulphite sequencing (BS) or methylation-specific PCR (MSP) in one or more cell line that
exhibited re-expression after demethylating treatment. Promoter methylation of 82 genes
(82/175) (47%) was documented in cell lines based on identification of =50% methylated
CpG sites in the CpG island. Out of 82 genes which showed methylation in cell lines,
promoter methylation was detected in 53 (65%) genes in primary tumor tissues. After
testing corresponding age matched normal tissues, 28 of these genes (28/175; 16%) were
identified to be methylated in a cancer-specific manner. After the initial high-throughput
discovery analysis, a high-resolution validation study on specific target has been conducted
in colorectal and pancreatic cancer. Among cancer-specific methylated genes we selected
b4GalT1 for colorectal cancer (130 colorectal adenocarcinomas, 13 adenomas, and normal
tissue) and KIF1A for pancreatic adenocarcinoma (60 tumor samples and normal
parenchyma).
b4GalT1 resulted to have a 54% sensitivity (95% CI: 35.1%–72.1%) and a specificity of
91.7% (95% CI: 74.1%–97.7%). An inverse correlation was observed between methylation
and B4GALT1 mRNA expression levels. Moreover significant differences in methylation
levels and frequencies of B4GALT1 was demonstrated in invasive colorectal lesions as
compared with normal mucosa (p = 0.0001) and in carcinoma samples as compared with
adenoma (p = 0.009).
KIF1A was also found to be specifically methylated in 81% of a pancreatic adenocarcinoma
cases and not in normal parenchyma derived from cancer-free subject. Additionally KIF1A
resulted to be methylated in IPMN (Intraductal papillary mucinous neoplasm) which are
considered preneoplastic lesions, suggesting that methylation of this gene in pancreatic
cancer may occur since the early phases.
These results suggest that the B4GALT1 and KIF1A represent new and valuable candidate
epigenetic biomarkers respectively of colorectal and pancreatic cancer
TP53 Mutations in Head and Neck Cancer
No full textThe authors reply: In response to Beutner and
colleagues, it is true that the Affymetrix p53 gene
chip does not identify all mutations, but the later
version of the chip, which we used, does identify
single-base deletions and insertions that would
account for many of the possible frameshift mutations. In fact, 12 frameshift mutations were
included in our cohort (in 12 of 224 patients
[5.4%]), 10 of which we scored as “disruptive” in
that they resulted in a “stop” sequence later within the gene. The proportion of frameshift mutations that we found in this cohort closely resembles that in our earlier study of 129 head and neck
cancers analyzed by direct sequencing of TP53,
1
in which 7% of all mutations were frameshifts.
The International Agency for Reseach on Cancer
currently reports that 17.2% of TP53 mutations in
head and neck cancer are deletions and insertions.2
The 10 patients with disruptive frameshift mutations in our current cohort had a median survival of 2.2 years — nearly identical to the median
survival of 2.0 years for the 75 patients with other
disruptive mutations (P=0.95). It is logical to ex1.
2.
trapolate that the majority of any frameshift mutations we may have missed would also be disruptive, and their identification would therefore
serve to reassign some patients with poor outcomes from the wild-type group to the disruptive-mutation group, further enhancing the distinction in outcome based on mutation status that
we were able to identify.
We agree with Perrone and colleagues that the
evidence indicates a distinctive genetic pathway
for tumorigenesis in the setting of HPV-related
oropharyngeal tumors. The number of cases in
their series and ours is insufficient to comment
with statistical confidence regarding the prognostic effect of a TP53 mutation when it is present
with HPV DNA, as compared with either alteration in isolation. Since as many as 70% of tonsil
or tongue-base cancers have HPV, and HPV inactivates wild-type p53, a TP53 mutation would be
expected to play a relatively minor role in this
subgroup of squamous-cell carcinomas of the head
and neck
Up-regulation of NHEJ activity in colonic adenomas and colorectal cancers: correlation with progression of disease
No full textBilateral consecutive rupture of the quadriceps tendon in a man with BstUI polymorphism of the COL5A1 gene.
No full textA genetic component has been implicated in tendinopathies involving tendon rupture. Type V collagen, a quantitatively minor fibrillar collagen which forms heterotypic fibrils with type I collagen, plays a role in the regulation of the size and configuration of fibrils of the much more abundant component type I collagen. To date, no data on the genetic component of bilateral rupture of the quadriceps tendon have been reported. We describe the presence of BstUI polymorphism of the COL5A1 gene in a man with bilateral rupture of the quadriceps tendon. The COL5A1 (the variant rs12722, BstUI RFLP) can be a candidate gene associated with the development of bilateral quadriceps tendon rupture
Luana, loura de olhos claros
No full textDestacando a importância da análise qualitativa no estudo da arte e da comunicação, a autora analisa como a opção estética por uma atriz loura na construção da personagem Luana, na novela O Rei do Gado, veiculada pela Rede Globo, no segundo semestre de 1996, reafirma a aspiração ao branqueamento e identidade com o europeu, que permanecem no imaginário nacional.While pointing out the importance of qualitative analysis in the study of art and communication, the author analyses in what way the aestheticdecision for a blond actress in the elaboration of the character Luana of the soap-opera O Rei do Gado, broadcasted on the TV network Rede Globo, in the second half of l996, reinforces the aspiration for whitening and for an identification with the European, which is still present in the national imaginary
Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck
No full textPurpose: Squamous cell carcinomas of theheadandneck(HNSCC) oftenharbor p53mutations,
but p53 protein degradation by the viral oncoprotein E6 may supercede p53 mutations in human
papillomavirus 16 (HPV16)^ positive tumors. The prevalence of p53 mutations in HPV-positive
HNSCCs is indeed lower, but in some tumors these alterations coexist.The purpose of this study
was to discernwhetherHNSCCs differ in the type ofp53 mutations as a function ofHPV16 status.
Experimental Design: The study was nested within a prospective multicenter study (ECOGE
4393/RTOGR9614) of patients with HNSCC treated surgically with curative intent.Tumors from
one study center were used to construct a tissue microarray.The tumors were well characterized
with respect to p53 mutational status. The tissue microarray was evaluated by HPV16 in situ
hybridization. HPV16 analysis was also done on a select group of tonsillar carcinomas known to
harbor disruptive p53 mutations defined as stop mutations or nonconservative mutations within
the DNA binding domain.
Results: HPV16 was detected in12 of 89 (13%) HNSCCs. By tumor site, HPV16 was detected in
12 of 21 (57%) tumors fromthe palatine/lingual tonsils, but in none of 68 tumors fromnontonsillar
sites (P < 0.00001). Both HPV16-positive and HPV16-negative HNSCCs harbored p53 mutations
(25% versus 52%), but disruptive mutations were only encountered in HPV16-negative
carcinomas. Of seven tonsillar carcinomas with disruptive p53 mutations, none were HPV16 positive,
in contrast to HPV16-positive tonsillar carcinomas without disruptive p53 mutations (0%
versus 57%; P = 0.008).
Conclusions: Although HPV16 and mutated p53 may coexist in a subset of HNSCCs, HPV16
and disruptive p53 mutations seem to be nonoverlapping events. A less calamitous genetic profile,
including the absence of disruptive p53 mutations, may underlie the emerging clinical profile
of HPV16-positive HNSCC such as improved patient outcome
Detectable BRAF mutation in serum DNA samples from patients with papillary thyroid carcinomas
No full textBackground. An activating point mutation of the
BRAF oncogene results in a V600E amino acid missense mutation
found in a majority of papillary thyroid carcinomas (PTC).
Methods. In this study, 28 matched tumor and serum samples
obtained from patients with both benign and malignant
thyroid disorders were analyzed for BRAF mutation using a
gap-ligase chain reaction technique.
Results. The BRAF mutation was absent in tumor DNA
samples obtained from patients with benign adenomas, follicular
neoplasms or carcinoma, and thyroid lymphoma. In contrast,
5 of 14 PTC tumors were positive for the BRAF mutation.
Moreover, 3 of 14 patients with PTC were positive for BRAF
mutation in serum and tumor. Of these 3 patients, 2 had lymph
node metastasis and 2 had PTC in background of the Hashimoto’s
thyroiditis.
Conclusions. The detection of free circulating mutant BRAF
in patients with PTC is possible and future studies are warranted
to determine its clinical significance
Current understanding and clinical utility of miRNAs regulation of colon cancer stem cells
No full textCancer stem cells (CSCs) in colorectal tumorigenesis are suggested to be responsible for initiation, development and propagation of colorectal cancer (CRC) and have been extensively characterized by the expression of phenotypic determinants, such as surface or intracellular proteins. The generation of CSCs is likely due to a dysregulation of the signaling pathways that principally control self-renewal and pluripotency in normal intestinal stem cells (ISCs) through different (epi)genetic changes that define cell fate, identity, and phenotype of CSCs. These aspects are currently under intense investigation. In the framework of the oncogenic signaling pathways controlled by microRNAs (miRNAs) during CRC development, a plethora of data suggests that miRNAs can play a key role in several regulatory pathways involving CSCs biology, epithelial-mesenchymal transition (EMT), angiogenesis, metastatization, and pharmacoresistance. This review examines the most relevant evidences about the role of miRNAs in the etiology of CRC, through the regulation of colon CSCs and the principal differences between colorectal CSCs and benign stem cells. In this perspective, the utility of the principal CSCs-related miRNAs changes is explored, emphasizing their use as potential biomarkers to aid in diagnosis, prognosis and predicting response to therapy in CRC patients, but also as promising targets for more effective and personalized anti-CRC treatments
Alpha-1 Antitrypsin Deficiency: Current Perspective from Genetics to Diagnosis and Therapeutic Approaches.
No full textMitochondrial and Nuclear DNA Variants in Amyotrophic Lateral Sclerosis: Enrichment in the Mitochondrial Control Region and Sirtuin Pathway Genes in Spinal Cord Tissue
No full textAmyotrophic Lateral Sclerosis (ALS) is a progressive disease with prevalent mitochondrial dysfunctions affecting both upper and lower motor neurons in the motor cortex, brainstem, and spinal cord. Despite mitochondria having their own genome (mtDNA), in humans, most mitochondrial genes are encoded by the nuclear genome (nDNA). Our study aimed to simultaneously screen for nDNA and mtDNA genomes to assess for specific variant enrichment in ALS compared to control tissues. Here, we analysed whole exome (WES) and whole genome (WGS) sequencing data from spinal cord tissues, respectively, of 6 and 12 human donors. A total of 31,257 and 301,241 variants in nuclear-encoded mitochondrial genes were identified from WES and WGS, respectively, while mtDNA reads accounted for 73 and 332 variants. Despite technical differences, both datasets consistently revealed a specific enrichment of variants in the mitochondrial Control Region (CR) and in several of these genes directly associated with mitochondrial dynamics or with Sirtuin pathway genes within ALS tissues. Overall, our data support the hypothesis of a variant burden in specific genes, highlighting potential actionable targets for therapeutic interventions in ALS
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