Luís Cirnes - Academia.edu (original) (raw)

Papers by Luís Cirnes

Diagnostic Cytopathology, 1998

Background: Osimertinib efficacy in pre-treated patients with epidermal growth factor receptor (E... more Background: Osimertinib efficacy in pre-treated patients with epidermal growth factor receptor (EGFR) T790M-mutated non-small cell lung cancer (NSCLC) has been demonstrated in clinical trials, but real-world data, particularly regarding resistance profile, remains limited. This study aims to analyze the resistance mechanisms acquired after treatment with Osimertinib. Methods: Clinical outcomes and molecular results from re-biopsies at the time of osimertinib progression of EGFR T790M-mutated NSCLC patient were analyzed. Results: Twenty-one patients with stage IV adenocarcinoma were included [median 69 years; 57.1% female; 85.7% never-smokers; 23.8% ECOG performance status (PS) ≥2]. Median PFS and OS were 13.4 (95% CI: 8.0–18.9) and 26.4 (95% IC: 8.9–43.8) months, respectively. At the time of analysis, 10 patients had tumor progression (47.6%). T790M loss occurred in 50%, being associated with earlier progression (median PFS 8.1 vs. 21.4 months, p = 0.011). Diverse molecular alterati...

Revista do Grupo de Estudos do Cancro do Pulmão

BioMed Research International

Introduction. The Portuguese healthcare system had to adapt at short notice to the COVID-19 pande... more Introduction. The Portuguese healthcare system had to adapt at short notice to the COVID-19 pandemic. We implemented workflow changes to our molecular pathology laboratory, a national reference center, to maximize safety and productivity. We assess the impact this situation had on our caseload and what conclusions can be drawn about the wider impact of the pandemic in oncological therapy in Portugal. Material and Methods. We reviewed our database for all oncological molecular tests requested between March and April of 2019 and 2020. For each case, we recorded age, sex, region of the country, requesting institution, sample type, testing method, and turnaround time (TAT). A comparison between years was made. Results. The total number of tests decreased from 421 in 2019 to 319 in 2020 (p=0.0027). The greatest reduction was in clinical trial-related cases. Routine cases were similar between years (267 vs. 256). TAT was higher in 2019 (mean 15 days vs. 12.3 days; p=0.0003). Medium- to la...

Cancers

Identification of targetable molecular changes is essential for selecting appropriate treatment i... more Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting ...

Archivos de bronconeumologia, Jan 24, 2017

Patients with activating somatic mutations in the Epidermal Growth Factor Receptor (EGFR) have be... more Patients with activating somatic mutations in the Epidermal Growth Factor Receptor (EGFR) have better clinical outcomes when treated with Tyrosine Kinase Inhibitors (TKI) over chemotherapy. However, the impact of the use of TKIs on overall survival outside clinical trials is not well established. To characterize and analyze the overall survival of a Caucasian population with NSCLC and EGFR mutations. A retrospective cohort analysis of patients with NSCLC screened for EGFR mutations (exons 18-21) between October 2009 and July 2013 was conducted. Clinical and pathological characteristics, mutational EGFR status, treatment and overall survival were evaluated. From the 285 patients which performed screening for EGFR mutations, 54 (18.9%) had mutations, 25 (46.3%) of which in exon 19 and 20 of which (37.0%) in exon 21. The occurrence of mutations was associated with female sex and non-smoking habits (both, P<.001). The median survival of the global population was 12.0 months, with a b...

The Veterinary Journal, 2009

The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fra... more The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fragment control region of canine mammary mixed tumours could be used as clonal markers that identified the cell population of origin. Ten benign mixed mammary tumours and nine carcinomas arising from benign mixed tumours were microdissected and DNA from epithelial and mesenchymal tumour cells and from normal mammary tissue was examined for sequence variations in a fragment of the hypervariable control region. Identical sequence variants in both the epithelial and mesenchymal components (as well as in the corresponding normal tissue) were found in 80% of the benign mixed tumours and in 89% of the carcinomas arising from benign mixed tumours suggesting a shared clonal origin. The distinctive sequence alterations identified in the epithelial and mesenchymal components of 15.8% of all 19 tumours examined, suggests the possibility that a minority of mammary tumours are polyclonal in origin or that early clonal divergence occurs. Increased mutation within the mtDNA D-loop fragment of mixed tumour components was not observed.

A presente comunicação descreve as modificações usadas para extrair e amplificar o DNA mitocondri... more A presente comunicação descreve as modificações usadas para extrair e amplificar o DNA mitocondrial obtido de amostras de tumores mamários caninos fixados em formol tamponado a 10% e incluídos em parafina. Os componentes epiteliais e mesenquimais (condromixóide e condróide), bem como a mama normal adjacente, foram microdissectados manualmente de 19 tumores mamários (10 tumores mistos benignos e nove carcinomas em tumores mistos). O DNA foi extraído utilizando-se o Invisorb ® Spin Tissue Mini Kit com modificações do protocolo proposto pelo fabricante. Um fragmento de 273-pb foi amplificado por reação em cadeia da polimerase (PCR) e seqüenciado em seqüenciador automático. O fragmento foi analisado em 100% das amostras, entretanto modificações como lise adicional, redução do volume das soluções de extração e PCR, aumento da temperatura de anelamento e do número de ciclos de amplificação foram necessárias. Em seis amostras os produtos iniciais de PCR foram diluídos e reamplificados para obtenção de sucesso.

PLOS ONE, 2015

This study consisted in the comparison of the prevalence of Helicobacter pylori (H. pylori) prese... more This study consisted in the comparison of the prevalence of Helicobacter pylori (H. pylori) present in the stomach and in saliva of a sample of Portuguese adolescents and the assessment of the association between H. pylori infection with socio-demographic variables and prevalence of dental caries.

European Journal of Human Genetics, 2014

Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-car... more Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n=15) to optimize the strategy and (2) validation set (n=20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS-Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.European Journal of Human Genetics advance online publication, 4 June 2014; doi:10.1038/ejhg.2014.97.

Thyroid, 2007

Objective: Our aim was to characterize the molecular and genotypic profile of eight thyroid carci... more Objective: Our aim was to characterize the molecular and genotypic profile of eight thyroid carcinoma-derived cell lines-TPC1, FB2, B-CPAP, K1, XTC-1, C643, 8505C, and Hth74-in order to use them as in vitro models of thyroid carcinogenesis. Design: We evaluated the expression of five thyroid-specific genes (Tg, TSHr, TPO, PAX8, and TTF-1) to establish the cell lineage and to assess the differentiation status of each of the cell lines. We screened for mutations in the most relevant oncogenes=tumor suppressor genes affected in thyroid carcinogenesis: RAS, BRAF, CTNNB1, and TP53 along with RET=PTC rearrangements. Considering the putative relevance in general carcinogenesis, we have also studied other molecules such as EGFR, PI3K, RAF-1, and THRB. To determine the genetic identity of the cell lines, we performed genotypic analysis. Main outcome: The panel of cell lines we have studied displayed activation of several oncogenes (BRAF, RAS, RET=PTC) and inactivation of tumor suppressor genes (TP53) known to be important for thyroid carcinogenesis. Two of the cell lines-TPC1 and FB2-shared the same genotypic profile, probably representing clones of an ancestor cell line (TPC1). Conclusion: Due to their different molecular alterations, these cell lines represent a valuable tool to study the molecular mechanisms underlying thyroid carcinogenesis. We suggest that genotypic analyses should be included as a routine procedure to guarantee the uniqueness of each cell line used in research.

Pathology - Research and Practice, 1998

The aim of this study was to evaluate the concordance between the presence of p53 mutations in br... more The aim of this study was to evaluate the concordance between the presence of p53 mutations in breast carcinomas expressing the protein by immunohistochemistry. A series of 60 breast carcinomas was evaluated by immunohistochemistry using monoclonal antibodies against p53 protein (DO 7 and PAb 1801). Twenty cases classified as being positive for p53 according to the current approach (if 5% or more of neoplastic cells contained reaction product in the nucleus) were used for molecular studies. These cases were re-assessed semi-quantitatively using a scoring system based on intensity and percentage of stained cells. DNA was phenol-chloroform extracted from microdissected normal and tumour cells obtained from formalin-fixed, paraffin-embedded tissue sections. Mutations in the p53 gene were analysed by SSCP (single strand conformational polymorphism) with primers covering exons 2-3 to 11. Ten out of the 20 p53-positive cases presented mutations detected by SSCP analysis. Mutations have been found in several exons ranging from exon 4 to exon 10. We observed a positive relationship between the presence of mutations and immunohistochemical evaluation of p53 protein expression using a semiquantitative scoring system. All cases with more than 2/3 stained tumour cells and strong intensity of staining exhibited p53 mutations. At variance, no p53 mutations were found in cases with less than 1/3 stained tumour cells and moderate intensity of staining. Therefore, only the identification of positivity for p53 detected by immunohistochemistry did not always reflect the detection of p53 mutations in breast cancer, however the use of a semi-quantitative approach seems to be useful as an indicator of the presence of mutation.

Oncogene, 2007

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an ... more The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Laboratory Investigation, 2000

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in targ... more Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Metϩ) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Metϩ and MSI-H/Metϩ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC

The Journal of Pathology, 2004

Gastric cancer is thought to result from a combination of environmental factors and accumulation ... more Gastric cancer is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, consequently affecting mainly older patients (>50 years of age). Less than 10% of patients present with the disease before 45 years of age, and these young patients are thought to develop carcinomas with a different molecular genetic profile to that of sporadic carcinomas occurring at a later age.

The Journal of Pathology, 2003

Transforming growth factor beta (TGFβ) is a potent inhibitor of cell growth, whose action is tran... more Transforming growth factor beta (TGFβ) is a potent inhibitor of cell growth, whose action is transduced through interaction between type I (RI) and type II (RII) receptors. Abnormal expression of these receptors has been identified in several human cancers and was found to be associated with resistance to TGFβ. TGFβ RII mutations occur in many types of malignancy. TGFβ RI hypermethylation has been suggested as a cause of abnormal or absent expression of this receptor in cancer. This study has analysed the methylation status of the promoter region of the TGFβ RI gene using a methylation-sensitive enzyme followed by polymerase

International Journal of Cancer, 1999

Microsatellite instability (MSI) has been reported to occur in a wide variety of sporadic tumours... more Microsatellite instability (MSI) has been reported to occur in a wide variety of sporadic tumours, such as colorectal and gastric cancers. MSI positivity has been associated with a particular clinico-pathologic profile, including the presence of abundant lymphoid infiltration, poor differentiation and a relatively good outcome for the patients. Since medullary breast carcinomas (MBCs) share these clinico-pathologic features with the MSI-positive tumours described above, we evaluated MSI in this particular histologic type of breast cancer. DNA of 24 MBC cases was extracted from formalinfixed, paraffin-embedded tissue. The presence of MSI was analysed using BAT-26. We also searched mutations in 2 target genes: TGF-␤ RII and BAX. Five cases of the series were also analysed for 1 (CA) dinucleotide tandem repeat sequence (D1S158), 8 tetranucleotide repeat sequences (D3S1358, D5S818, D7S820, D8S1179, D13S317, D21S11, FGA and VWA) and 1 pentanucleotide repeat (dAAAAT), localized in intron 1 of p53 gene. We found 2 carcinomas (8.3%) with BAT-26 instability. None of the cases had mutations in the ''target genes'', TGF-␤ RII and BAX, including the 2 cases with BAT-26 instability. No MSI was observed using the panel of tetra-and pentanucleotide markers. Loss of heterozygosity was found in some loci. No significant difference in mean MIB-1 index according to RER status was observed. The low frequency of MSI in MBC is similar to that of other histologic types of breast cancer. Although MBCs share some clinico-pathologic features with colorectal and gastric carcinomas, which exhibit a high frequency of MSI, the underlying genetic events leading to this breast tumour are different from those leading to tumours of the digestive tract. Int.

Familial Cancer, 2009

The Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant disease caused by TP53 germline muta... more The Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant disease caused by TP53 germline mutations. This study aimed to characterize the TP53 mutational spectrum in patients suspected to have LFS in Portugal and to evaluate the influence of the MDM2-SNP309 and TP53-72Arg variants and of telomere length on age of tumor onset. Probands were primarily selected using the classical LFS criteria (two cases) or the more sensitive Chompret Li-Fraumeni-like (LFL) criteria (13 cases), but 12 additional patients that did not comply with those LFS or LFL criteria were included in the analysis based on clinical suspicion (LFS suspects). Nine of the 27 probands (33.3%) presented germline TP53 mutations, two of them occurring de novo and two of them being novel. Three of the nine TP53 mutations were found in families that did not comply with any of the commonly used criteria for TP53 testing, leaving room to recommend the use of less stringent criteria. An association was found between the presence of the TP53-72Arg (but not the MDM2-SNP309) variant and earlier age of onset in TP53 carriers. A negative correlation between telomere length and age of cancer onset was found in patients with germline TP53 mutation, whereas no such correlation was found in controls or in patients with wild-type TP53.

European Journal of Surgical Oncology (EJSO), 2007

Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin... more Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin germline mutations. One-third of these mutations are of the missense type, representing a burden in genetic counselling. A new germline missense mutation (P373L) was recently identified in a HDGC Italian family. The present work aimed at addressing the disease-causative nature of the P373L mutant. Assessment of the P373L mutation effect was based on cell aggregation and invasion assays. LOH analysis at the E-cadherin locus, search for somatic E-cadherin mutations and for promoter hypermethylation were performed to identify the mechanism of inactivation of the E-cadherin wild-type allele in the tumour. In vitro the P373L germline mutation impaired the E-cadherin functions. E-cadherin promoter hypermethylation was observed in the tumour of the P373L mutation carrier. We conclude that the combination of clinical, in vitro and molecular genetic data is helpful for establishing an accurate analysis of HDGC-associated CDH1 germline missense mutations and subsequently for appropriate clinical management of asymptomatic mutation carriers.