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Mutations in the KRAS and NRAS (RAS) genes are negative predictors of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC). The detection of mutations in circulating tumor DNA (ctDNA) has emerged as a less invasive strategy to assess the molecular profile of mCRC patients. We aimed to perform RAS mutational analysis in ctDNA from mCRC patients using BEAMing Digital PCR (OncoBEAM) and Idylla ctDNA qPCR and evaluate the concordance rate with RAS mutational status in tumor tissue and between these two methodologies with different limits of detection.Blood samples were collected from 47 mCRC patients previously tested for RAS mutations in tumor tissue. DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit, and RAS mutation analysis was conducted using OncoBEAM RAS CRC and Idylla ctRAS assays.The overall agreement between tumor tissue and ctDNA analyses was 83% and 78.7% using the OncoBEAM and Idylla assays, respectively, with the concordance being 96.2% and 88.5% in naive treatment patients. The overall agreement between OncoBEAM and Idylla ctDNA analyses was 91.7%.Analysis of ctDNA is a viable strategy for clinical management of mCRC patients. Although the OncoBEAM assay sensitivity is somewhat higher, the fully automated Idylla platform also has good performance, while being cheaper and much less labor-intensive, for the detection of RAS mutations in plasma, either at diagnosis or after progression when considering anti-EGFR treatment rechallenge.

The genomic consequence and clinical interpretation of large duplications are difficult to infer without determining the location and orientation of the duplicated sequence. We aimed to characterize two intragenic duplications detected in two hereditary breast and ovarian cancer syndrome (HBOC) families, namely BRCA1 exon 4 to 6 and BRCA2 exon 17 to 18, previously detected by multiplex ligation probe amplification and initially classified as variants of unknown significance. Using long range PCR, with duplication-specific primers, we were able to ascertain the genomic breakpoints and observed that the two rearrangements occurred in tandem and in direct orientation. The BRCA1 c.134+440_441+870dup and BRCA2 c.7806-2083_8332-1512dup duplications here identified are predicted to cause frameshifts that create a premature stop codon and were reclassified as pathogenic. Furthermore, both families present phenotypic traits typical of HBOC syndrome. We also observed that the genomic breakpoints of these two duplications occurred within highly homologous Alu elements. Concluding, we characterized two in tandem BRCA1 and BRCA2 duplications that likely occurred by Alu-mediated homologous recombination, allowing identification of the underlying cause of the HBOC syndrome in these families.

Gastric cancer (GC) disproportionally affects people of Latino ancestry and represent a leading cause of incidence and mortality disparities in this minority populations. When compared to Non-Latino Whites, Latinos are ~2-fold more likely to be diagnosed with and to die of GC. The goal of our research program is to contribute to GC research in patients of Latino ancestry and advance cancer health disparity research. One of the research projects aims at identifying germline variants associated with GC risk. To do so, we are identifying novel gastric cancer genes and detecting mutations in previously reported GC genes, using whole-exome sequencing, in 500 patients who have been diagnosed with early-onset GC and/or who had GC and family history of cancer. Whole exome sequence data collection was recently completed and is being analyzed with the primary goals of estimating the prevalence and penetrance in known cancer genes as well as the identification of new GC genes. Using in-house exome data and publicly available genetic data, we will identify variants with predicted impact in the phenotype to design a panel that will be used for their detection in gastric preneoplasia biopsies. These variants will be tested using duplex sequencing, a highly sensitive method that can detect very rare mutations with ultra-depth sequencing. We will then select the variants with likely functional consequences for tumor initiation and/or progression to generate isogenic gastric organoids to investigate their effect in proliferation, differentiation, and gene expression. We aim to generate a body of work that can be used for risk assessment, prevention, early diagnostic, and that will lead to better outcomes for GC in this important U.S. minority. Dr. Ana Estrada-Florez is the recipient of the AACR Cancer Health Disparities Fellowship. Citation Format: Ana Patricia Estrada-Florez, Paul Lott, Ted Toal, Nicole Halmai, Elizabeth Quino, Alma Poceros-Coba, Alix Guevara, Fabian Castro, Jhon Suarez, John Mcpherson, Rasika Venkatesh, Hongyong Zhang, Apri Vang, Guadalupe Polanco, Joana Guerra, Graciela Molina, Carol Parra, Adriana Della Valle, Esteban Castelao, Manuel Teixeira, Mabel Bohorquez, Javier Torres, Alejandro Carvajal Corvalan, Luis G. Carvajal-Carmona. Identification of predisposition and progression gastric cancer biomarkers in Latinos [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2274.

Simple Summary Germline and somatic variant testing of the BRCA1 and BRCA2 genes are important to predict treatment response to PARP inhibitors in ovarian cancer patients. However, germline variants in other genes besides BRCA1 and BRCA2 are associated with ovarian cancer predisposition, which would be missed by a genetic testing aimed only at treatment decision. We aimed to evaluate the yield of clinically actionable germline variants using next-generation sequencing of a customized panel of 10 genes for the analysis of pathology samples of ovarian carcinomas. We identified clinically actionable germline variants in a significantly higher proportion of ovarian cancer patients when compared with genetic testing focused only on BRCA1 and BRCA2. This strategy increases the chance to make available genetic counseling, presymptomatic genetic testing, and gynecological cancer prophylaxis to female relatives who turn out to be healthy carriers of deleterious germline variants. Abstract Since the approval of PARP inhibitors for the treatment of high-grade serous ovarian cancer, in addition to cancer risk assessment, BRCA1 and BRCA2 genetic testing also has therapeutic implications (germline and somatic variants) and should be offered to these patients at diagnosis, irrespective of family history. However, variants in other genes besides BRCA1 and BRCA2 are associated with ovarian cancer predisposition, which would be missed by a genetic testing aimed only at indication for PARP inhibitor treatment. In this study, we aimed to evaluate the yield of clinically actionable germline variants using next-generation sequencing of a customized panel of 10 genes for the analysis of formalin-fixed paraffin-embedded samples from 96 ovarian carcinomas, a strategy that allows the detection of both somatic and germline variants in a single test. In addition to 13.7% of deleterious germline BRCA1/BRCA2 carriers, we identified 7.4% additional patients with pathogenic germline variants in other genes predisposing for ovarian cancer, namely RAD51C, RAD51D, and MSH6, representing 35% of all pathogenic germline variants. We conclude that the strategy of reflex gene-panel tumor testing enables the identification of clinically actionable germline variants in a significantly higher proportion of ovarian cancer patients, which may be valuable information in patients with advanced disease that have run out of approved therapeutic options. Furthermore, this approach increases the chance to make available genetic counseling, presymptomatic genetic testing, and gynecological cancer prophylaxis to female relatives who turn out to be healthy carriers of deleterious germline variants.

Breast cancer is the most frequent event in Li-Fraumeni syndrome associated with germline TP53 variants. Some studies have shown that breast cancers in women with Li-Fraumeni syndrome are commonly HER2-positive, suggesting that HER2 amplification or over-expression in a young woman may be a useful criterion to test for germline variants in the TP53 gene. We assessed the prevalence of germline TP53 variants by Sanger sequencing or next-generation sequencing in 149 women with HER2-positive breast cancer diagnosed until age 40. The pattern of HER2 amplification was evaluated with dual-probe FISH in a subset of breast carcinomas from patients with germline TP53 variants as compared with those of noncarriers. Among 149 women tested, three presented a deleterious TP53 germline variant (2%), with one patient diagnosed at age 31 and the other two with bilateral breast cancer at ages 29/33 and 28/32, respectively. Three of the 36 patients (8.3%) with the first breast cancer diagnosed at age 31 or younger presented a pathogenic TP53 variant. Additionally, all TP53 deleterious variant carriers had a first degree relative diagnosed with different early-onset cancers (frequently not belonging to the Li-Fraumeni syndrome tumor spectrum) diagnosed at age 45 or younger. Higher levels of HER2 amplification were found in breast carcinomas of TP53 pathogenic variant carriers than in those of noncarriers. Deleterious germline TP53 variants account for a small proportion of early-onset HER2-positive breast cancers, but these seem to have higher HER2 amplification ratios. All TP53 pathogenic variant carriers found in this study had the first breast carcinoma diagnosed at age 31 or younger and a first-degree relative with early-onset cancer. Further studies are needed to clarify if HER2 status in early-onset breast cancer patients, in combination with other personal and/or familial cancer history, is useful to update the TP53 testing criteria.

Despite all the knowledge already gathered, the picture of somatic genetic changes in colorectal tumorigenesis is far from complete. Recently, germline and somatic mutations in the exonuclease domain of polymerase epsilon, catalytic subunit (POLE) gene have been reported in a small subset of microsatellite‐stable and hypermutated colorectal carcinomas (CRCs), affecting the proofreading activity of the enzyme and leading to misincorporation of bases during DNA replication. To evaluate the role of POLE mutations in colorectal carcinogenesis, namely in advanced CRC, we searched for somatic mutations by Sanger sequencing in tumor DNA samples from 307 cases. Microsatellite instability and mutation analyses of a panel of oncogenes were performed in the tumors harboring POLE mutations. Three heterozygous mutations were found in two tumors, the c.857C>G, p.Pro286Arg, the c.901G>A, p.Asp301Asn, and the c.1376C>T, p.Ser459Phe. Of the POLE‐mutated CRCs, one tumor was microsatellite‐stable and the other had low microsatellite instability, whereas KRAS and PIK3CA mutations were found in one tumor each. We conclude that POLE somatic mutations exist but are rare in advanced CRC, with further larger studies being necessary to evaluate its biological and clinical implications.

Since the approval of PARP inhibitors for the treatment of high-grade serous ovarian cancer, in addition to cancer risk assessment, BRCA1 and BRCA2 genetic testing also has therapeutic implications (germline and somatic variants) and should be offered to these patients at diagnosis, irrespective of family history. However, variants in other genes besides BRCA1 and BRCA2 are associated with ovarian cancer predisposition, which would be missed by a genetic testing aimed only at indication for PARP inhibitor treatment. In this study, we aimed to evaluate the yield of clinically actionable germline variants using next-generation sequencing of a customized panel of 10 genes for the analysis of formalin-fixed paraffin-embedded samples from 96 ovarian carcinomas, a strategy that allows the detection of both somatic and germline variants in a single test. In addition to 13.7% of deleterious germline BRCA1/BRCA2 carriers, we identified 7.4% additional patients with pathogenic germline variants in other genes predisposing for ovarian cancer, namely RAD51C, RAD51D, and MSH6, representing 35% of all pathogenic germline variants. We conclude that the strategy of reflex gene-panel tumor testing enables the identification of clinically actionable germline variants in a significantly higher proportion of ovarian cancer patients, which may be valuable information in patients with advanced disease that have run out of approved therapeutic options. Furthermore, this approach increases the chance to make available genetic counseling, presymptomatic genetic testing, and gynecological cancer prophylaxis to female relatives who turn out to be healthy carriers of deleterious germline variants.

Massive Open Online Courses (MOOCs) are unlimited web-based courses accessed through computers, smartphones, or other digital devices. Although they have multiple advantages, a common challenge is the low course completion rates. Young family doctors of the World Organization of Family Doctors (WONCA) from more than 20 countries recently completed an initiative that combined social media platforms with a MOOC offered by Harvard University. This resulted in a completion rate five times greater than the baseline rate. We propose perfecting and expanding this method to augment continued medical education, collaboration, and best practice exchange among WONCA's members and participating organizations worldwide.

Interleukin-6 (IL-6) is now known to be not only a major cytokine controlling the immune system but also basic physiological variables such as body weight and metabolism. We recently reported that muscle-specific interleukin-6 deletion influences body weight and body fat in a sex-dependent manner in mice. When compared with littermate floxed controls, males gained less weight whereas females gained more weight after a 12-week high-fat diet treatment (HFD). We herewith report gender-differences of HFD treatment on fast and slow skeletal muscle in muscle-specific IL-6 deficient mice. While gross muscle architecture was normal, in males, HFD resulted in an increased proportion of medium-large size myofibers which was prevented by muscle IL-6 deletion. No modifications of fiber size were observed in females. HFD induced a fiber-type switching in tibialis muscle, increasing the proportion of fast-oxidative fibers and decreasing the fast-glycolytic fibers in female mice which were dependent on muscle IL-6. No changes of fiber types were detected in males. Finally, HFD was associated with increased collagen deposition in both sexes and muscle types. However, this effect was only associated to the presence of muscular IL-6 only on the slow soleus muscle in males. The results demonstrate sex-dependent effects of both HFD and muscle IL-6 deficiency in skeletal muscle.

Constitutional epimutation of the two major mismatch repair genes, MLH1 and MSH2, has been identified as an alternative mechanism that predisposes to the development of Lynch syndrome. In the present work, we aimed to investigate the prevalence of MLH1 constitutional methylation in colorectal cancer (CRC) patients with abnormal expression of the MLH1 protein in their tumors. In a series of 38 patients who met clinical criteria for Lynch syndrome genetic testing, with loss of MLH1 expression in the tumor and with no germline mutations in the MLH1 gene (35/38) or with tumors presenting the BRAF p.Val600Glu mutation (3/38), we screened for constitutional methylation of the MLH1 gene promoter using methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) in various biological samples. We found four (4/38; 10.5%) patients with constitutional methylation in the MLH1 gene promoter. RNA studies demonstrated decreased MLH1 expression in the cases with constitutional methylation when compared with controls. We could infer the mosaic nature of MLH1 constitutional hypermethylation in tissues originated from different embryonic germ layers, and in one family we could show that it occurred de novo. We conclude that constitutional MLH1 methylation occurs in a significant proportion of patients who have loss of MLH1 protein expression in their tumors and no MLH1 pathogenic germline mutation. Furthermore, we provide evidence that MLH1 constitutional hypermethylation is the molecular mechanism behind about 3% of Lynch syndrome families diagnosed in our institution, especially in patients with early onset or multiple primary tumors without significant family history.

Relatório final do estágio profissionalizante do 6.º ano

Fibrosis is the aberrant deposition of extracellular matrix (ECM) components during tissue healing leading to loss of its architecture and function. Fibrotic diseases are often associated with chronic pathologies and occur in a large variety of vital organs and tissues, including skeletal muscle. In human muscle, fibrosis is most readily associated with the severe muscle wasting disorder Duchenne muscular dystrophy (DMD), caused by loss of dystrophin gene function. In DMD, skeletal muscle degenerates and is infiltrated by inflammatory cells and the functions of the muscle stem cells (satellite cells) become impeded and fibrogenic cells hyperproliferate and are overactivated, leading to the substitution of skeletal muscle with nonfunctional fibrotic tissue. Here, we review new developments in our understanding of the mechanisms leading to fibrosis in DMD and several recent advances towards reverting it, as potential treatments to attenuate disease progression. The authors thank C. Mann and the members of the Cell Biology Group for their helpful discussions and acknowledge funding from MINECO-Spain (SAF2012-38547, FIS-PS09/01267, FIS-PI13/025, and PLE2009-0124), AFM, E-Rare, Fundació MaratóTV3, Duchenne PP-NL, MDA, and EU-FP7 (Myoage, Optistem, and Endostem). Yacine Kharraz and Patrizia Pessina were supported by postdoctoral fellowships from AFM

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a childhood motoneuron disease caused by mutations in the gene encoding for IGHMBP2, an ATPase/Helicase. Paralysis of the diaphragm is an early and prominent clinical sign resulting both from denervation and myopathy. In skeletal muscles, muscle atrophy mainly results from loss of motoneuron cell bodies and axonal degeneration. Although it is well known that loss of motoneurons at the lumbar spinal cord is an early event in the pathogenesis of the disease, it is not clear whether the corresponding proximal axons and NMJs are also early affected. In order to address this question, we have investigated the time course of the disease progression at the level of the motoneuron cell body, prox imal axon (ventral root), distal axon (sciatic nerve), NMJ, and muscle fiber in Nmd2J mice, a mouse model for SMARD1. Our results show an early and apparently parallel loss of motoneurons, proximal axons, and NMJs. In affected muscles, however, denervated fibers coexist with NMJs with normal morphology and unaltered neurotransmission. Furthermore, unaffected axons are able to sprout and reinnervate muscle fibers, suggesting selective vulnerability of neurons to Ighmbp2 deficiency. The preservation of the NMJ morphology and neuro transmission in the Nmd2J mouse until motor axon loss takes place, differs from that observed in SMA mouse models in which NMJ impairment is an early and more general phenomenon in affected muscles.

Trabalho final de mestrado integrado em Medicina área científica de Neurologia, apresentado à Faculdade de Medicina da Universidade de Coimbra Introdução: A Doença de Huntington é uma doença neurodegenerativa autossómica dominante resultante de uma expansão de repetições do trinucleotídeo Citosina-Adenina- Guanina no gene responsável pela codificação da proteína huntingtina. Embora as alterações motoras decorrentes determinem o início da doença, vários estudos sugerem que estas são precedidas por distúrbios cognitivos e psiquiátricos, em cerca de 15 anos. A avaliação da função cognitiva geral dos doentes necessita de baterias complexas, sendo o Mini Mental State Exam (MMSE) o teste de rastreio mais frequentemente usado. Por outro lado, o Montreal Cognitive Assessment (MoCA) é um teste de rastreio cognitivo mais recente que apresenta uma sensibilidade superior na deteção de defeito cognitivo ligeiro, pelo que tem sido considerada a sua utilização para a deteção das alterações cognitivas precoces nos doentes com Doença de Huntington. Objetivo: Comparar o MMSE com o MoCA como testes de rastreio para a disfunção cognitiva na Doença de Huntington, em doentes em diferentes estadios de severidade de doença. Métodos: O MMSE e o MoCA foram administrados a 37 doentes com diagnóstico genético de Doença de Huntington, consecutivamente avaliados na Consulta de Neurogenética do Serviço de Neurologia dos Hospitais da Universidade de Coimbra, e a 40 controlos emparelhados no que respeita a idade, sexo e escolaridade, sem qualquer patologia neurodegenerativa Resultados: Os grupos de controlo e de doentes não apresentaram diferenças na distribuição das características demográficas. As pontuações totais do MMSE e do MoCA apresentaram valores significativamente inferiores no grupo de doentes (p = 0,002 e p = 0,003, respetivamente). Estratificando as amostras por nível de escolaridade apenas se encontram diferenças entre os dois grupos nas pontuações do MMSE para escolaridade inferior ou igual a 11 anos. Utilizando os pontos de corte determinados para a população portuguesa, o MoCA apresentou uma sensibilidade significativamente superior ao MMSE (64,9% vs 21,6%; p < 0,001) para detetar declínio cognitivo nos doentes. Na análise de curvas Reciever Operating Characteristics obtiveram-se resultados estatisticamente significativos registados apenas no MoCA nas áreas Visuoespacial, Linguagem e Função Executiva/Atenção (p = 0,01, p = 0,039 e p = 0,018, respetivamente). Conclusões: Com base nos pontos de corte definidos para a população portuguesa, o MoCA é um teste breve de rastreio com maior sensibilidade na deteção de defeitos cognitivos na Doença de Huntington comparativamente ao MMSE, requisitando tarefas que implicam o funcionamento de áreas cerebrais caracteristicamente afetadas nesta doença. Devido ao facto de avaliar domínios como a Função Executiva/Atenção, revela-se mais adequado à deteção de défice cognitivo ligeiro nos doentes com Doença de Huntington, devendo ser preferencialmente utilizado em relação ao MMSE Introduction: Huntington’s Disease is an autosomal dominant neurodegenerative disorder caused by a trinucleotide Cytosine-Adenine-Guanine repeat expansion in the gene encoding for huntingtin protein. Although motor abnormalities determine the beginning of the disease, several studies suggest cognitive and psychiatric impairments 15 years earlier. In order to obtain an evaluation of the patients’ global cognitive function, complex batteries of exams are needed. For this purpose, the Mini Mental State Exam (MMSE) is the screening test most frequently used. On the other hand, the Montreal Cognitive Assessment (MoCA) is a recent screening test with higher sensitivity for mild cognitive impairment and it has been studied its application for the detection of the earliest cognitive impairments in patients with Huntington’s Disease. Objective: Compare the MMSE with the MoCA as screening tests for the cognitive impairment in the Huntington’s Disease, in patients in different stages of disease’s severity. Methods: The MMSE and the MoCA were performed in 37 patients with genetic diagnosis of the Huntington’s Disease, regularly evaluated in the Neurogenetic Consultation of the Neurology Service in Coimbra’s University Hospital, as well as in 40 control subjects with no neurodegenerative pathology, using age, gender and education as comparison factors in the selection criteria. Results: Both groups (subjects with Huntington’s Disease and control subjects) showed no differences in the distribution of the demographic characteristics. The MMSE and MoCA’s total scores showed lower values in the Huntington’s Disease group (p=0,002 and p=0,003 respectively). Samples’ description according to educational level showed differences between the two groups in the MMSE scores only, for an education lower or equal to 11 years. With the cut-offs determined to the Portuguese population, the MoCA presented a superior sensitivity to detect cognitive decline among Huntington’s Disease patients in comparison to the MMSE (64,9% vs 21,6 %; p

Mestrado em Engenharia Biomédica Submitted by Maria da Luz Antunes (mluz.antunes@estesl.ipl.pt) on 2021-05-20T11:17:16Z No. of bitstreams: 1 Análise do desempenho cognitivo e motor e sua relação com a atrofia do cerebelo em pacientes com esclerose múltipla surto-remissão.pdf: 3119007 bytes, checksum: 37e75428f4b59ad83848af3a135d6cb8 (MD5) Made available in DSpace on 2021-05-20T11:17:16Z (GMT). No. of bitstreams: 1 Análise do desempenho cognitivo e motor e sua relação com a atrofia do cerebelo em pacientes com esclerose múltipla surto-remissão.pdf: 3119007 bytes, checksum: 37e75428f4b59ad83848af3a135d6cb8 (MD5) Previous issue date: 2020-06 N/A