Publications by authors named "Ana Pestana"

19 Publications

  • Page 1 of 1

Genetic Determinants for Prediction of Outcome of Patients with Papillary Thyroid Carcinoma.

Cancers (Basel) 2021 Apr 23;13(9). Epub 2021 Apr 23.

IPATIMUP-Instituto de Patologia e Imunologia Molecular da Universidade do Porto, 4200-135 Porto, Portugal.

Papillary thyroid carcinoma (PTC) usually presents an excellent prognosis, but some patients present with aggressive metastatic disease. BRAF, RAS, and TERT promoter (TERTp) genes are altered in PTC, and their impact on patient outcomes remains controversial. We aimed to determine the role of genetic alterations in PTC patient outcomes (recurrent/persistent disease, structural disease, and disease-specific mortality (DSM)). The series included 241 PTC patients submitted to surgery, between 2002-2015, in a single hospital. DNA was extracted from tissue samples of 287 lesions (primary tumors and metastases). Molecular alterations were detected by Sanger sequencing. Primary tumors presented 143 BRAF, 16 TERTp, and 13 RAS mutations. Isolated TERTp showed increased risk of structural disease (HR = 7.0, < 0.001) and DSM (HR = 10.1, = 0.001). Combined genotypes, BRAF/TERTp (HR = 6.8, = 0.003), BRAF/TERTp (HR = 3.2, = 0.056) and BRAF/TERTp (HR = 2.2, = 0.023) showed increased risk of recurrent/persistent disease. Patients with tumors BRAF/TERTp (HR = 24.2, < 0.001) and BRAF/TERTp (HR = 11.5, = 0.002) showed increased risk of structural disease. DSM was significantly increased in patients with TERTp regardless of BRAF status (BRAF/TERTp, log-rank < 0.001; BRAF/TERTp, log-rank < 0.001). Our results indicate that molecular markers may have a role in predicting PTC patients' outcome. BRAF/TERTp tumors were prone to associate with local aggressiveness (recurrent/persistent disease), whereas TERTp tumors were predisposed to recurrent structural disease and DSM.
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http://dx.doi.org/10.3390/cancers13092048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122921PMC
April 2021

Comprehensive Assessment of mRNA Expression across a Large Cohort of Benign and Malignant Thyroid Tumours.

Cancers (Basel) 2020 Jul 9;12(7). Epub 2020 Jul 9.

Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup), 4200-135 Porto, Portugal.

The presence of promoter (p) mutations in thyroid cancer have been associated with worse prognosis features, whereas the extent and meaning of the expression and activation of in thyroid tumours is still largely unknown. We analysed frozen samples from a series of benign and malignant thyroid tumours, displaying non-aggressive features and low mutational burden in order to evaluate the presence of p mutations and mRNA expression in these settings. In this series, p mutations were found in 2%, only in malignant cases, in larger cancers, and from older patients. mRNA expression was detected in both benign and malignant tumours, with increased frequencies in the malignant tumours with aggressive histotypes, larger tumours, and from older patients. In benign tumours, mRNA expression was found in 17% of the follicular thyroid adenoma (FTA) with increased levels of expression in smaller tumours and associated with the presence of thyroiditis. p mutations and mRNA expression are correlated with worse prognosis features in malignant thyroid tumours, whereas mRNA expression in the benign tumours is associated with the presence of thyroiditis.
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http://dx.doi.org/10.3390/cancers12071846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408963PMC
July 2020

Prognostic Significance of RAS Mutations and P53 Expression in Cutaneous Squamous Cell Carcinomas.

Genes (Basel) 2020 07 6;11(7). Epub 2020 Jul 6.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.

is considered the most commonly-altered gene in cutaneous squamous cell carcinoma (cSCC). Conversely, mutations have been reported in a low percentage of cSCC. The objective of our study was to evaluate the frequency of p53 expression and mutations in cSCC and correlate them with clinicopathological features and patient outcome. We performed immunohistochemistry for p53 and genetic profiling for mutations in a retrospective series of cSCC. The predictive value of p53 expression, mutations, and clinicopathological parameters was assessed using logistic regression models. The overall frequency of mutations was 9.3% (15/162), and 82.1% of the cases (133/162) had p53 overexpression. mutations rate was 3.2% (1/31) of in situ cSCCs and 10.7% (14/131) of invasive cSCCs. mutations were more frequently associated with an infiltrative than an expansive pattern of invasion ( = 0.046). p53 overexpression was a predictor of recurrence in the univariate analysis. Our results indicate that mutations associate with features of local aggressiveness. Larger studies with more recurrent and metastatic cSCCs are necessary to further address the prognostic significance of p53 overexpression in patients' risk stratification.
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http://dx.doi.org/10.3390/genes11070751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397334PMC
July 2020

Evaluation of the role of mitochondria in the non-targeted effects of ionizing radiation using cybrid cellular models.

Sci Rep 2020 04 9;10(1):6131. Epub 2020 Apr 9.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal.

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. The mitochondrial role in orchestrating this biological response needs to be further explored. Cybrids (cytoplasmic hybrids) are useful cell models for studying the involvement of mitochondria in cellular processes. In the present study we used cybrid cell lines to investigate the role of mitochondria in the response to radiation exposure. Cybrid cell lines, derived from the osteosarcoma human cell line 143B, harboring, either wild-type mitochondrial DNA (Cy143Bwt), cells with mitochondria with mutated DNA that causes mitochondrial dysfunction (Cy143Bmut), as well as cells without mitochondrial DNA (mtDNA) (143B-Rho0), were irradiated with 0.2 Gy and 2.0 Gy. Evaluation of the non-targeted (or bystander) effects in non-irradiated cells were assessed by using conditioned media from the irradiated cells. DNA double stranded breaks were assessed with the γH2AX assay. Both directly irradiated cells and cells treated with the conditioned media, showed increased DNA damage. The effect of the irradiated cells media was different according to the cell line it derived from: from Cy143Bwt cells irradiated with 0.2 Gy (low dose) and from Cy143Bmut irradiated with 2.0 Gy (high dose) induced highest DNA damage. Notably, media obtained from cells without mtDNA, the143B-Rho0 cell line, produced no effect in DNA damage. These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. Furthermore, it indicates that cybrid models are valuable tools for radiobiological studies.
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http://dx.doi.org/10.1038/s41598-020-63011-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145863PMC
April 2020

Interaction of Genetic Variations in and Modulates the Risk of Hashimoto's Thyroiditis.

Thyroid 2019 09;29(9):1302-1315

Service of Endocrinology, Diabetology and Metabolism, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

Several single-nucleotide polymorphisms (SNPs) are known to increase the risk of Hashimoto's thyroiditis (HT); such SNPs reside in thyroid-specific genes or in genes related to autoimmunity, inflammation, and/or cellular defense to stress. The transcription factor Nrf2, encoded by , is a master regulator of the cellular antioxidant response. This study aimed to evaluate the impact of genetic variation in on the risk of developing HT. In a case-control candidate gene association study, functional SNPs in the promoter (rs35652124, rs6706649, and rs6721961) were examined either as independent risk factors or in combination with a previously characterized HT risk allele (rs28665122) in the gene , encoding selenoprotein S (SelS). A total of 997 individuals from the north of Portugal (Porto) were enrolled, comprising 481 HT patients and 516 unrelated healthy controls. and SNPs were genotyped using TaqMan assays and Sanger sequencing, respectively. Odds ratios (ORs) were calculated using logistic regression, with adjustment for sex and age. Expression of SelS was analyzed by immunohistochemistry in thyroid tissue from HT patients and control subjects. Molecular interactions between the Nrf2 and SelS pathways were investigated in thyroid tissues from mice and in rat PCCL3 thyroid follicular cells. When all three SNPs were considered together, the presence of one or more minor alleles was associated with a near-significant increased risk (OR = 1.43,  = 0.072). Among subjects harboring only major alleles, there was no increased HT risk associated with heterozygosity or homozygosity for the minor allele. Conversely, in subjects heterozygous or homozygous for the risk allele, the presence of an minor allele significantly increased HT risk by 2.8-fold ( = 0.003). Immunohistochemistry showed reduced expression of SelS in thyroid follicular cells of HT patients. In Nrf2 knockout mice, there was reduced expression of SelS in thyroid follicular cells; conversely, in PCCL3 cells, reducing SelS expression caused reduced activity of Nrf2 signaling. The promoter genotype interacts with the promoter genotype to modulate the risk of HT in a Portuguese population. This interaction may be due to a bidirectional positive feedback between the Nrf2 and SelS pathways.
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http://dx.doi.org/10.1089/thy.2018.0480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760180PMC
September 2019

OPNa Overexpression Is Associated with Matrix Calcification in Thyroid Cancer Cell Lines.

Int J Mol Sci 2018 Sep 30;19(10). Epub 2018 Sep 30.

i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.

Osteopontin (OPN) spliced variants (OPN-SV: OPNa, OPNb, and OPNc) are aberrantly expressed in tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer (TC). PTC often presents with desmoplasia and dystrophic calcification, including psammoma bodies (PB). This work aimed to investigate total OPN (tOPN) and OPN-SV expression and their association with the presence of PB in the PTC classical variants (cPTC), as well as the involvement of OPN-SV in matrix calcification of TC cell lines. We found that cPTC samples presenting PB showed higher OPN expression levels. In TC cell lines, OPNa overexpression promotes higher matrix calcification and collagen synthesis when compared to that of clones overexpressing OPNb or OPNc. In response to OPN knockdown, calcification was inhibited, paralleled with the downregulation of calcification markers. In conclusion, our data evidenced that OPN expression is associated with the presence of PB in cPTC samples. Among the OPN-SV, OPNa is the main contributor to matrix calcification in tested TC cells, providing clues to a better understanding on the biology and ethiopathogenesis of the calcification process in TC cells.
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http://dx.doi.org/10.3390/ijms19102990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213506PMC
September 2018

TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma.

J Am Acad Dermatol 2019 Mar 28;80(3):660-669.e6. Epub 2018 Aug 28.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal; Medical Faculty, University of Porto, Porto, Portugal. Electronic address:

Background: Telomerase reverse transcriptase gene (TERT) promoter (TERTp) mutations have been reported as potential predictors of poor prognosis in several cancers, but the prognostic value of TERTp mutations for cutaneous squamous cell carcinoma (cSCC) has not been determined.

Objective: To evaluate the frequency of TERTp mutations and correlate it with clinicopathologic features and patient outcome.

Methods: We performed genetic profiling of TERTp mutations in a retrospective series of cSCCs. The predictive value of TERTp mutations and clinicopathologic parameters were assessed by using logistic regression models.

Results: A total of 152 cSCCs from 122 patients were analyzed for TERTp mutations; the mutation rate was 31.6% (48 of 152), and it was higher in invasive cSCC (42 of 121 [34.7%]) than in in situ cSCC (6 of 31 [19.4%]). Age older than 75 years (odds ratio [OR], 14.84; P = .013] and TERTp mutation (OR, 8.11; P = .002) were independent predictors of local recurrence. TERTp mutation (OR, 15.89; P = .022) was independently associated with higher risk of lymph node metastasis.

Limitations: The restricted number of metastatic cases.

Conclusion: TERTp mutations may prove to be a molecular biomarker with prognostic significance in invasive cSCC, but larger studies are needed.
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http://dx.doi.org/10.1016/j.jaad.2018.08.032DOI Listing
March 2019

TERTp mutation is associated with a shorter progression free survival in patients with aggressive histology subtypes of follicular-cell derived thyroid carcinoma.

Endocrine 2018 09 15;61(3):489-498. Epub 2018 Jun 15.

Programa de Pós Graduação em Endocrinologia da Faculdade de Medicina da Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Purpose: Evaluate the impact of TERTp mutation on the outcomes after initial treatment of 45 patients with thyroid carcinomas derived from follicular cells (TCDFC) with aggressive histology, in which the role of this mutation is not yet well defined.

Methods: Analysis of the presence of TERTp (-124C > T and -146C > T), BRAF (V600E), and NRAS (Q 61R) mutations by Sanger sequencing and analysis of their correlation with the patient's outcomes.

Results: Forty-five patients with aggressive histopathologic variants were included in the study. Of these, 68.9% had aggressive variants of papillary thyroid cancer (PTC), 22.2% had poorly differentiated thyroid carcinoma (PDTC)/insular carcinoma, and 8.9% had invasive follicular thyroid cancer (FTC) with Hurthle cell features (Hurthle cell carcinoma). Lymph node metastases were present in 46.7% and distant metastases in 54.6%. The response to the initial therapy was excellent in 45.5% and structurally incomplete in 50%. During the follow-up period (median of 56 months; 5-360 months), 47.7% presented with disease progression and 17.8% experienced disease-related death. In 53.3% of the cases at least one molecular alteration (TERTp in 33.4%, BRAF in 24.5%, RAS in 8.9%) was detected. In the multivariate analysis, TERTp mutation was the factor associated with the highest risk (6 times) of having structural disease after initial therapy (p = 0.01), followed by vascular invasion (p = 0.02), gross extrathyroidal extension (ETE) (p = 0.02) and distant metastasis (p = 0.04). Regarding mutational status, only TERTp mutation was associated with disease progression, and diminished disease progression-free survival (PFS). The presence of distant metastasis, vascular invasion and gross ETE were significantly associated with the risk of disease progression.

Conclusions: TERTp mutation appears be an indicator of both persistence and progression of structural disease after initial therapy in aggressive variants of TCDFC, and associates with a shorter progression free survival regardless of the therapy employed.
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http://dx.doi.org/10.1007/s12020-018-1642-0DOI Listing
September 2018

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and mRNA Expression.

Int J Mol Sci 2018 May 13;19(5). Epub 2018 May 13.

Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto 4099-002, Portugal.

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase mRNA levels, whereas Torin2 caused a ~6 fold increase in mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of mRNA expression.
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http://dx.doi.org/10.3390/ijms19051448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983778PMC
May 2018

CRABP1, C1QL1 and LCN2 are biomarkers of differentiated thyroid carcinoma, and predict extrathyroidal extension.

BMC Cancer 2018 01 10;18(1):68. Epub 2018 Jan 10.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal.

Background: The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.

Methods: We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.

Results: We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.

Conclusions: We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.
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http://dx.doi.org/10.1186/s12885-017-3948-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763897PMC
January 2018

NIS expression in thyroid tumors, relation with prognosis clinicopathological and molecular features.

Endocr Connect 2018 Jan;7(1):78-90

Instituto de Investigação e Inovação em Saúde (i3S)Porto, Portugal

Thyroid cancer therapy is based on surgery followed by radioiodine treatment. The incorporation of radioiodine by cancer cells is mediated by sodium iodide symporter (NIS) (codified by the gene), that is functional only when targeted to the cell membrane. We aimed to evaluate if NIS expression in thyroid primary tumors would be helpful in predicting tumor behavior, response to therapy and prognosis. NIS expression was addressed by qPCR and immunohistochemistry. In order to validate our data, we also studied expression on 378 primary papillary thyroid carcinomas from The Cancer Genome Atlas (TCGA) database. In our series, expression was lower in carcinomas with vascular invasion and with extrathyroidal extension and in those harboring V600E mutation. Analysis of expression from TCGA database confirmed our results. Furthermore, it showed that larger tumors, with locoregional recurrences and/or distant metastases or harboring , and/or promoter (p) mutations presented significantly less expression. Regarding immunohistochemistry, 12/211 of the cases demonstrated NIS in the membrane of tumor cells, those cases showed variable outcomes concerning therapy success, prognosis and all but one were wild type for , and p mutations. mRNA lower expression is associated with features of aggressiveness and with key genetic alterations involving , and p. Mutations in these genes seem to decrease protein expression and its targeting to the cell membrane. mRNA expression is more informative than NIS immunohistochemical expression regarding tumor aggressiveness and prognostic features.
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http://dx.doi.org/10.1530/EC-17-0302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754505PMC
January 2018

TERT biology and function in cancer: beyond immortalisation.

J Mol Endocrinol 2017 02 5;58(2):R129-R146. Epub 2017 Jan 5.

Institute of Molecular Pathology and ImmunologyUniversity of Porto (IPATIMUP), Porto, Portugal

Evasion of replicative senescence and proliferation without restriction, sometimes designated as immortalisation, is one of the hallmarks of cancer that may be attained through reactivation of telomerase in somatic cells. In contrast to most normal cells in which there is lack of telomerase activity, upregulation of TERT transcription/activity is detected in 80-90% of malignant tumours. In several types of cancer, there is a relationship between the presence of TERT promoter mutations, TERT mRNA expression and clinicopathological features, but the biological bridge between the occurrence of TERT promoter mutations and the aggressive/invasive features displayed by the tumours remains unidentified. We and others have associated the presence of TERT promoter mutations with metastisation/survival in several types of cancer. In follicular cell-derived thyroid cancer, such mutations are associated with worse prognostic features (age of patients, tumour size and tumour stage) as well as with distant metastases, worse response to treatment and poorer survival. In this review, we analyse the data reported in several studies that imply TERT transcription reactivation/activity with cell proliferation, tumour invasion and metastisation. A particular attention is given to the putative connections between TERT transcriptional reactivation and signalling pathways frequently altered in cancer, such as c-MYC, NF-κB and B-Catenin.
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http://dx.doi.org/10.1530/JME-16-0195DOI Listing
February 2017

TERT promoter mutations: a genetic signature of benign and malignant thyroid tumours occurring in the context of tinea capitis irradiation.

Eur J Endocrinol 2017 Jan 19;176(1):49-55. Epub 2016 Oct 19.

IPATIMUP - Institute of Molecular Pathology and Immunology of the University of PortoPorto, Portugal.

Objective: The aim of this study is to evaluate the frequency and molecular characteristics of TERTp mutations in thyroid adenomas and carcinomas occurring in the low-dose radiation exposure tinea capitis setting.

Design And Methods: Twenty-seven patients with 34 well-differentiated thyroid carcinomas and 28 patients with 29 follicular adenomas diagnosed in a Portuguese tinea capitis cohort were studied. Blood samples were obtained from all the patients. Screening for TERTp mutations was performed by PCR amplification followed by Sanger sequencing. A series of 33 sporadic thyroid adenomas was used as control.

Results: TERTp mutations were detected in six of the 28 patients with adenoma (21.4%) and in four of the 27 patients with carcinoma (14.8%). Three tumours (two carcinomas and one adenoma) had the tandem mutation -124/-125 GG>AA (30.0%), whereas the remaining seven had the -124G > A. The 20.7% frequency of TERTp mutations in adenomas contrasts with the absence of mutations in the adenomas from the control group and from most series on record, whereas the one found in carcinomas (11.8%) is similar to those reported in the literature for sporadic carcinomas.

Conclusion: TERTp mutations, including the tandem mutation -124/-125 GG>AA not described previously in thyroid tumours, appear to represent a genetic signature for thyroid tumours in patients submitted to low-dose X-ray irradiation. The high frequency of TERTp mutations in the adenomas of our cohort contrasts with their absence in sporadically occurring, as well as in adenomas of the Chernobyl series.
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http://dx.doi.org/10.1530/EJE-16-0740DOI Listing
January 2017

pmTOR is a marker of aggressiveness in papillary thyroid carcinomas.

Surgery 2016 12 26;160(6):1582-1590. Epub 2016 Aug 26.

Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), University of Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), University of Porto, Porto, Portugal; Medical Faculty, University of Porto, Porto, Portugal; Department of Pathology and Oncology, Medical Faculty of the University of Porto, Porto, Portugal. Electronic address:

Background: Activation of the mTOR pathway has been observed in thyroid cancer, but the biologic consequences regarding tumor behavior and patient prognosis remain poorly explored.

Methods: We aimed to evaluate the associations of the mTOR pathway with clinicopathologic and molecular features and prognosis through the immunocharacterization of pmTOR and pS6 expression (as readouts of the pathway) in a series of 191 papillary thyroid carcinomas.

Results: pmTOR expression was associated with distant metastases (P = .05) and persistence of disease (P = .05). Cases with greater expression of pmTOR were submitted to more I treatments (r[102] = 0.2; P = .02) and a greater cumulative dose of radioactive iodine (r[100] = 0.3; P = .01). Positive pmTOR expression showed to be an independent risk factor for distant metastases (odds ratio = 18.2; 95% confidence interval 2.1-157.9; P = .01). In contrast, pS6 expression was associated with absence of extrathyroid extension (P = .001), well-defined tumor margins (P = .05), and wild-type BRAF status (P = .01). There was no correlation between the expression of pmTOR and pS6 expression (r[140] = 0.1; P = .3).

Conclusion: pmTOR expression is an indicator of aggressive, metastatic papillary thyroid carcinoma, being possibly implicated in refractoriness to therapy, while pS6 expression is associated with less aggressive pathologic features. Further studies are needed to understand better the biologic consequences of activation of the mTOR pathway in the behavior of thyroid cancer, namely the contribution of other pmTOR downstream effectors.
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http://dx.doi.org/10.1016/j.surg.2016.06.050DOI Listing
December 2016

TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromes.

Sci Rep 2016 07 14;6:29714. Epub 2016 Jul 14.

Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, 4200-135, Portugal.

One of the hallmarks of cancer is its unlimited replicative potential that needs a compensatory mechanism for the consequential telomere erosion. Telomerase promoter (TERTp) mutations were recently reported as a novel mechanism for telomerase re-activation/expression in order to maintain telomere length. Pancreatic endocrine tumors (PETs) were so far recognized to rely mainly on the alternative lengthening of telomeres (ALT) mechanism. It was our objective to study if TERTp mutations were present in pancreatic endocrine tumors (PET) and could represent an alternative mechanism to ALT. TERTp mutations were detected in 7% of the cases studied and were mainly associated to patients harbouring hereditary syndromes. In vitro, using PET-derived cell lines and by luciferase reporter assay, these mutations confer a 2 to 4-fold increase in telomerase transcription activity. These novel alterations are able to recruit ETS transcription factor members, in particular GABP-α and ETV1, to the newly generated binding sites. We report for the first time TERTp mutations in PETs and PET-derived cell lines. Additionally, our data indicate that these mutations serve as an alternative mechanism and in an exclusive manner to ALT, in particular in patients with hereditary syndromes.
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http://dx.doi.org/10.1038/srep29714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944231PMC
July 2016

Osteopontin-a splice variant is overexpressed in papillary thyroid carcinoma and modulates invasive behavior.

Oncotarget 2016 Aug;7(32):52003-52016

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.

Osteopontin (OPN) is a matricellular protein overexpressed in cancer cells and modulates tumorigenesis and metastasis, including in thyroid cancer (TC). The contribution of each OPN splice variant (OPN-SV), named OPNa, OPNb and OPNc, in TC is currently unknown. This study evaluates the expression of total OPN (tOPN) and OPN-SV in TC tissues and cell lines, their correlation with clinicopathological, molecular features and their functional roles. We showed that tOPN and OPNa are overexpressed in classic papillary thyroid carcinoma (cPTC) in relation to adjacent thyroid, adenoma and follicular variant of papillary thyroid carcinoma (fvPTC) tissues. In cPTC, OPNa overexpression is associated with larger tumor size, vascular invasion, extrathyroid extension and BRAFV600E mutation. We found that TC cell lines overexpressing OPNa exhibited increased proliferation, migration, motility and in vivo invasion. Conditioned medium secreted from cells overexpressing OPNa induce MMP2 and MMP9 metalloproteinases activity. In summary, we described the expression pattern of OPN-SV in cPTC samples and the key role of OPNa expression on activating TC tumor progression features. Our findings highlight OPNa variant as TC biomarker, besides being a putative target for cPTC therapeutic approaches.
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http://dx.doi.org/10.18632/oncotarget.10468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239531PMC
August 2016

Osteopontin expression is correlated with differentiation and good prognosis in medullary thyroid carcinoma.

Eur J Endocrinol 2016 Apr 25;174(4):551-61. Epub 2016 Jan 25.

Instituto de Investigação e Inovacão em SaúdeUniversidade do Porto, 4200-135 Porto, PortugalInstitute of Molecular Pathology and Immunology of the University of Porto (Ipatimup) - Cancer BiologyRua Dr Roberto Frias, s/n, 4200-465 Porto, PortugalMedical FacultyUniversity of Porto, Al. Professor Hernâni Monteiro, P-4200 Porto, PortugalUnidade de Investigação em Patobiologia Molecular (UIPM)Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG), Rua Professor Lima Basto, 1099-023 Lisboa, PortugalMolecular Pathology Service of the Portuguese Institute of Oncology of Coimbra FGEPE, Avenue. Bissaya Barreto, 98, 3000-075 Coimbra, PortugalDepartment of PathologyHospital de S. João, Al. Professor Hernâni Monteiro, P-4200 Porto, PortugalResearch CoordinationNational Institute of Cancer, Rio de Janeiro 22743-051, BrazilNatural Sciences DepartmentHealth and Humanities Institute, Fluminense Federal University, Rio das Ostras, Rio de Janeiro 28895-532, Brazil Instituto de Investigação e Inovacão em SaúdeUniversidade do Porto, 4200-135 Porto, PortugalInstitute of Molecular Pathology and Immunology of the University of Porto (Ipatimup) - Cancer BiologyRua Dr Roberto Frias, s/n, 4200-465 Porto, PortugalMedical FacultyUniversity of Porto, Al. Professor Hernâni Monteiro, P-4200 Porto, PortugalUnidade de Investigação em Patobiologia Molecular (UIPM)Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG), Rua Professor Lima Basto, 1099-023 Lisboa, PortugalMolecular Pathology Service of the Portuguese Institute of Oncology of Coimbra FGEPE, Avenue. Bissaya Barreto, 98, 3000-075 Coimbra, PortugalDepartment of PathologyHospital de S. João, Al. Professor Hernâni Monteiro, P-4200 Porto, PortugalResearch CoordinationNational Institute of Cancer, Rio de Janeiro 22743-051, BrazilNatural Sciences DepartmentHealth and Humanities Institute, Fluminense Federal University, Rio das Ostras, Rio de Janeiro 28895-532, Brazil Instituto de Investigação e In

Background: Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established.

Objective: The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC).

Methods: OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours.

Results: OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT).

Conclusion: The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.
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http://dx.doi.org/10.1530/EJE-15-0577DOI Listing
April 2016

Low frequency of TERT promoter mutations in gastrointestinal stromal tumors (GISTs).

Eur J Hum Genet 2015 Jun 24;23(6):877-9. Epub 2014 Sep 24.

1] Molecular Oncology Research Center, Barretos Cancer Hospital, Sao Paulo, Brazil [2] Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho, Braga, Portugal [3] ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Portugal.

Somatic mutations in the promoter region of telomerase reverse transcriptase (TERT) gene, mainly at positions c.-124 and c.-146 bp, are frequent in several human cancers; yet its presence in gastrointestinal stromal tumor (GIST) has not been reported to date. Herein, we searched for the presence and clinicopathological association of TERT promoter mutations in genomic DNA from 130 bona fide GISTs. We found TERT promoter mutations in 3.8% (5/130) of GISTs. The c.-124C>T mutation was the most common event, present in 2.3% (3/130), and the c.-146C>T mutation in 1.5% (2/130) of GISTs. No significant association was observed between TERT promoter mutation and patient's clinicopathological features. The present study establishes the low frequency (4%) of TERT promoter mutations in GISTs. Further studies are required to confirm our findings and to elucidate the hypothetical biological and clinical impact of TERT promoter mutation in GIST pathogenesis.
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http://dx.doi.org/10.1038/ejhg.2014.195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795049PMC
June 2015

Successful pregnancy occurring with interferon-alpha therapy in chronic myeloid leukemia.

Acta Obstet Gynecol Scand 2005 Mar;84(3):300-1

Department of Obstetrics and Maternal-Fetal Medicine/Clinical Hematology, Hospital Geral de Santo António, Porto, Portugal.

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http://dx.doi.org/10.1111/j.0001-6349.2005.0358b.xDOI Listing
March 2005
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