Caracterização molecular do gene EGFR em pacientes com NSCLC

Trabalho para obtenção do Título de Especialista em Anatomia Patológica, Citológica e TanatológicaN/

A Picture from a National Reference Center for Molecular Pathology

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 large-sized public hospitals in the north of the country were mostly responsible for the reduction in cases (p = 0.0153). Conclusions: Case reduction was observed at hospitals that have mostly been involved in the treatment of COVID-19 and in the north of the country, the region worst-hit by the pandemic. Similar to other studies, our TAT decreased, even with a similar number of routine cases. Thus, we conclude that it is possible to successfully adapt the workflow of a molecular pathology laboratory to new safety standards without losing efficiency.publishersversionpublishe

E-Cadherin (CDH1) and p53 rather than SMAD4 and Caspase-10 germline mutations contribute to genetic predisposition in Portuguese gastric cancer patients

Approximately 30% of all hereditary diffuse gastric cancer (HDGC) families carry CDH1 germline mutations. The other two thirds remain genetically unexplained and are probably caused by alterations in other genes. Using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP)/sequencing, we screened 32 Portuguese families with a history of gastric cancer and 23 patients with early onset gastric cancer for CDH1 germline mutations. In probands negative for CDH1 mutations, we screened genes involved in hereditary cancer syndromes in which gastric cancer may be one of the component tumours, namely p53 (Li-Fraumeni Syndrome) and hMLH1 and hMSH2 (HNPCC). We also screened in these patients for mutations in Caspase-10, a gene inactivated in sporadic gastric cancer, and SMAD4, a gene whose inactivation in mice is associated with signet-ring cell carcinoma of the stomach. One of the families fulfilling the HDGC criteria harboured a CDH1 germline mutation, and one of the families with incomplete criteria harboured a p53 germline mutation. No mutations were identified in hMLH1 and hMSH2, and only sequence variants were found in SMAD4 and Caspase-10. The present work reports for the first time CDH1 germline mutations in Portuguese gastric cancer families, and highlights the need for p53 mutation screening in families lacking CDH1 germline mutations, in a country with one of the highest incidences of gastric cancer in the world. No evidence was found for a role of germline mutations in SMAD4 and Caspase-10 in families lacking CDH1 mutations.http://www.sciencedirect.com/science/article/B6T68-4CP0YVH-5/1/0b2a789fc5dbe341d589aa4cee90b9e

Resistance profile of osimertinib in pre-treated patients with EGFR T790M-mutated non-small cell lung cancer

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 alterations were identified, including C797S mutation (n = 1), PIK3CA mutation (n = 2), MET amplification (n = 1), CTNNB1 mutation (n = 1), and DCTN1-ALK fusion (n = 1). Histological transformation into small cell carcinoma occurred in one patient. Conclusions: This real-world life study highlights the relevance of re-biopsy at the time of disease progression, contributing to understand resistance mechanisms and to guide treatment strategies

"Miocardiopatias associadas à morte súbita".

Mestrado em Medicina LegalMaster Degree in Forensic Medicin

Análise de variantes no gene STK11 e sua influência na resposta à imunoterapia

A imunoterapia é uma das ferramentas terapêuticas de sucesso usadas no tratamento do cancro de pulmão, contudo alguns doentes apresentam progressão da doença ou morte. O gene Serine/ threonineKinase 11 (STK11) está descrito como tendo um papel na fisiologia do sistema imune(SI) e que alterações genéticas no mesmo podem influenciar negativamente a resposta dos pacientes à imunoterapia. Estudar alterações genéticas no gene STK11 em pacientes com cancro de pulmão sujeitos a imunoterapia e a sua possível associação com a resposta do doente à terapêutica. Após extraçãodo DNA de amostras de cancro pulmonar foi realizado um PCR multiplex para amplificação de 9 exões do geneSTK11, os quais foram sequenciados para pesquisa de alterações genéticas. Os pacientes foram divididos em dois grupos: “apresentaram boa resposta à terapia” e “apresentaram má resposta à terapia”, para efetuar curvas de sobrevivência que relacionam resposta à terapia com alterações em STK11. Foram detetadas e classificadas como patogénicas ou provavelmente patogénicas 13 alterações no gene STK11, não tendo sido identificada uma zona do gene ou exão com mais propensão a alterações. Pacientes com alterações no gene STK11 apresentaram pior sobrevida global e pior sobrevida livre de progressão de doença. Estes resultados sugerem que o gene STK11 deve ser estudado na sua totalidade aquando da decisão terapêutica e que deve ser incluído em painéis de Sequenciação de Nova Geração (NGS), uma vez que foram detetadas alterações neste gene com valor preditivo negativo na resposta à imunoterapia.info:eu-repo/semantics/publishedVersio

<it>BRAF</it>, <it>KRAS </it>and <it>PIK3CA </it>mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

<p>Abstract</p> <p>Background</p> <p><it>BRAF</it>, <it>KRAS </it>and <it>PIK3CA </it>mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to <it>KRAS </it>and <it>PIK3CA </it>mutations, <it>BRAF </it>mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), <it>MLH1 </it>methylation and microsatellite instability (MSI). We aimed at determine the frequency of <it>KRAS</it>, <it>BRAF </it>and <it>PIK3CA </it>mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, <it>MLH1 </it>methylation and MSI were also studied.</p> <p>Methods</p> <p>Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and <it>MLH1 </it>methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies.</p> <p>Results</p> <p><it>KRAS</it>, <it>PIK3CA </it>or <it>BRAF </it>occur in 71% of polyps and were mutually exclusive. <it>KRAS </it>mutations occur in 35% of polyps. <it>PIK3CA </it>was found in one of the polyps. V600E <it>BRAF </it>mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for <it>BRAF</it>. <it>MLH1 </it>methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that <it>KRAS </it>and <it>PIK3CA </it>are likely to precede both types of CRC. <it>BRAF </it>mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (<it>P </it>= 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (<it>P </it>= 0.0191).</p> <p>Conclusion</p> <p>Our results show that <it>BRAF</it>, <it>KRAS </it>and <it>PIK3CA </it>mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that <it>BRAF </it>mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of <it>BRAF </it>mutations. These results support that <it>BRAF </it>mutations harbour a mild oncogenic effect in comparison to <it>KRAS </it>and suggest that <it>BRAF </it>mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC.</p

E-Cadherin (CDH1) and p53 rather than SMAD4 and Caspase-10 germline mutations contribute to genetic predisposition in Portuguese gastric cancer patients

Approximately 30% of all hereditary diffuse gastric cancer (HDGC) families carry CDH1 germline mutations. The other two thirds remain genetically unexplained and are probably caused by alterations in other genes. Using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP)/sequencing, we screened 32 Portuguese families with a history of gastric cancer and 23 patients with early onset gastric cancer for CDH1 germline mutations. In probands negative for CDH1 mutations, we screened genes involved in hereditary cancer syndromes in which gastric cancer may be one of the component tumours, namely p53 (Li-Fraumeni Syndrome) and hMLH1 and hMSH2 (HNPCC). We also screened in these patients for mutations in Caspase-10, a gene inactivated in sporadic gastric cancer, and SMAD4, a gene whose inactivation in mice is associated with signet-ring cell carcinoma of the stomach. One of the families fulfilling the HDGC criteria harboured a CDH1 germline mutation, and one of the families with incomplete criteria harboured a p53 germline mutation. No mutations were identified in hMLH1 and hMSH2, and only sequence variants were found in SMAD4 and Caspase-10. The present work reports for the first time CDH1 germline mutations in Portuguese gastric cancer families, and highlights the need for p53 mutation screening in families lacking CDH1 germline mutations, in a country with one of the highest incidences of gastric cancer in the world. No evidence was found for a role of germline mutations in SMAD4 and Caspase-10 in families lacking CDH1 mutations.http://www.sciencedirect.com/science/article/B6T68-4CP0YVH-5/1/0b2a789fc5dbe341d589aa4cee90b9e

Effects of different storage conditions of sequencing products with formamide in the quality of sequences.

DNA sequencing is widely used in molecular diagnosis and good sequence quality is crucial to a correct interpretation. It has been described that formamide quality decrease and sequencing reactions exposure to light, heat and/or oxygen can cause irregular peaks of cytosine and guanine in electropherograms. In a previous study, we concluded that despite the presence of this artifact when formamide is stored under non-ideal conditions, it does not significantly reduce the quality of the sequences.info:eu-repo/semantics/publishedVersio