Biomarkers for classification and risk assessment of pancreatic cystic neoplasms

Pancreatic cystic lesions (PCLs) are increasing incidental findings due to increased ageing of the population and widespread use of imaging. The main problem in clinical practice has to do with distinguishing the high-risk premalignant and malignant cysts that require surgical treatment from the benign or low-grade dysplastic cysts, which should not be over-treated and might not even require surveillance. The goal of the present work is to perform a comprehensive analysis of biomarkers and diagnostic approaches by Endoscopic Ultrasound with Fine-needle Aspiration (EUS-FNA), in a cohort of patients harboring mostly low-risk cysts under surveillance, which are far more frequent in clinical practice. The PCF analysis performed in this cohort includes studies of genomics (DNA mutations), epigenomics (methylation analysis), metabolomics (glucose), and proteomics (CEA, chromogranin A, NSE), with putative biomarkers encompassing the diagnosis of mucinous and malignant cysts, that require surveillance and surgical resection, respectively. We performed a first meta-analysis comparing current diagnostic methods - CEA and cytology - with KRAS mutations for the diagnosis of mucinous cysts. CEA was the best test for clinically significant cysts (AUC=0.69), cytology performed better in malignant cysts (AUC=0.78), surpassing KRAS mutations (AUC=0.53 and AUC=0.56, respectively). In a second meta-analysis we compared the accuracy of molecular analysis versus micro forceps biopsy (MFB) in the diagnosis of PCLs. The two approaches were identical for diagnosing benign cysts, but molecular analysis was superior for diagnosing both low and high-risk mucinous cysts. In addition to these two meta-analyses, we performed a retrospective study evaluating the added value of KRAS and GNAS mutations in PCF of 52 frozen PCF samples. We conclude that, as compared with conventional tests, these had no added value in the differential diagnosis of PCLs. In another publication, we compared glucose level in PCF with CEA in 82 patients. For mucinous cyst diagnosis, a CEA >192 ng/ml showed an AUC of 0.84 while glucose <50 mg/dl revealed an AUC of 0.86. Besides its higher accuracy, PCF glucose evaluated “on site” with a glucometer is easy, immediate, and requires a minimal amount of PCF. In the next study we sought to determine whether a second EUS-FNA changed the diagnosis or management of pancreatic cysts. We compared the outcome of 105 patients with a single EUS-FNA with that of 23 patients who had a second EUS-FNA. EUS-FNA may be recommended, as it changed management toward surgery in approximately 20% of the patients, particularly with diagnosis of cystic NETs. Following these results, we explored the role of EUS-FNA in small PCLs (<3 cm) in 115 patients with PCLs <3 cm who underwent EUS-FNA. 19/115 were submitted to surgery with 15 malignant or premalignant lesions and the remaining 4 were benign lesions. We conclude that EUS-FNA in lesions <3 cm may improve outcome and cost-effectiveness of surveillance programs, as it confirmed malignancy in 2 out of 5 resected lesions, while it also diagnosed benign cysts who could be released from these programs. In a pilot study with 16 patients, including 4 cystic NETs we aimed at assessing the value of Chromogranin A (CroA) and neuron-specific enolase (NSE) levels in PCF. CroA and NSE levels were higher in cystic NETs with an AUC of 0.94 for CroA and 1 for NSE. These are promising biomarkers to identify pancreatic cystic NETs. Finally, we studied epigenetic changes in the diagnosis of malignant cysts. Methylation changes of GNAS locus were evaluated to understand whether they may contribute to malignant progression of PCLs. Fifty-two samples of PCF were studied. We observed that GNAS locus methylation changes were significantly associated with malignancy. This is the first study to identify methylation changes in the GNAS locus improving diagnosis of malignant PCLs. We end this work proposing a revised diagnostic organogram of PCLs established by current guidelines, that incorporates the results obtained in this dissertation’s research.As lesões quísticas pancreáticas (PCL) têm incidência crescente devido ao envelhecimento da população e ao aumento da utilização dos métodos de imagem. Na prática clínica pretende-se distinguir os quistos mucinosos, de alto risco e malignos, que requerem tratamento cirúrgico, dos quistos benignos ou pré-malignos de baixo risco, que no máximo requerem vigilância. O objetivo do presente trabalho é analisar de forma abrangente, biomarcadores em líquido de quisto pancreático (PCF) obtido por Ecoendoscopia com punção (EUS-FNA), numa coorte de quistos predominantemente de baixo risco sob vigilância imagiológica, que são os mais comuns na prática clínica. A análise de PCF nesta coorte inclui estudos de genómica (mutações no DNA), epigenómica (análise de metilação), metabolómica (glicose) e proteómica (CEA, cromogranina A, NSE), com avaliação de biomarcadores para diagnóstico de quistos mucinosos e quistos malignos, que beneficiam de vigilância e ressecção cirúrgica, respetivamente. Numa primeira meta-análise comparámos a metodologia diagnóstica atual - CEA e citologia - com as mutações do KRAS para diagnóstico dos quistos mucinosos. O CEA foi o melhor teste em quistos clinicamente significativos (AUC=0.69), e a citologia em quistos malignos (AUC=0.78), superando as mutações do KRAS (AUC=0.53 e AUC=0.56, respetivamente). Numa segunda meta-análise comparámos a precisão diagnóstica da análise molecular versus biópsia com micropinça (MFB) no diagnóstico de PCL. As duas abordagens foram idênticas em quistos benignos, mas a análise molecular foi superior em quistos mucinosos tanto de baixo como de alto risco. Além das duas meta-análises, realizámos um estudo retrospetivo para avaliar o valor das mutações do KRAS e do GNAS em 52 amostras de PCF congeladas. Concluímos que não têm valor adicional no diagnóstico diferencial das PCL, relativamente aos testes convencionais. Noutra publicação comparámos o nível de glicose em PCF com o CEA para diagnóstico de quistos mucinosos em 82 doentes. O CEA >192 ng/ml apresentou uma AUC de 0.84 e a glicose <50 mg/dl de 0.86. Além da maior precisão diagnóstica, a glicose avaliada in loco com um glicosímetro, é fácil, imediata e requer um volume mínimo de PCF. No estudo seguinte, avaliámos se uma segunda EUS-FNA alterou o diagnóstico ou a decisão de quistos pancreáticos. Comparámos 105 doentes com uma única EUS-FNA com 23 doentes com uma segunda EUS-FNA. Esta pode ser recomendada, pois cerca de 20% dos doentes foram referenciados para cirurgia após repetição da EUS-FNA, incluindo dois com tumores neuroendócrinos (NET) quísticos. Seguidamente, explorámos o papel da EUS-FNA em pequenas PCL (<3 cm), num estudo com 115 PCL <3 cm. 19/115 foram operadas, correspondendo a 15 lesões malignas ou pré-malignas e 4 benignas. Concluímos que a EUS-FNA em quistos com <3 cm pode melhorar o diagnóstico e o custo-efetividade, pois confirmou malignidade em lesões ressecadas, e diagnosticou quistos benignos que podem ser libertados de vigilância. Num estudo piloto com 16 doentes, incluindo 4 com NET quísticos, avaliámos o valor diagnóstico da cromogranina A (CroA) e da enolase específica neuronal (NSE) em PCF. Os níveis de CroA e NSE foram mais elevados nos NET quísticos, com uma AUC de 0.94 para a CroA e 1 para a NSE. Estes revelaram-se biomarcadores promissores Por fim, estudámos alterações epigenéticas no diagnóstico de quistos malignos. Analisámos a metilação do locus GNAS em PCF para perceber se se associa à progressão maligna de PCL. Estudámos 52 amostras e observámos que a alteração da metilação se associou significativamente a malignidade. Trata-se do primeiro trabalho a avaliar alterações de metilação no locus GNAS no diagnóstico de PCL. Terminamos este trabalho com uma proposta de revisão do organograma de diagnóstico das PCL baseado nas guidelines atuais, que incorpora os resultados desta tese

Endoscopic ultrasound with fine needle aspiration is useful in pancreatic cysts smaller than 3 cm

The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat ivecommons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the dataBackground: In current guidelines, endoscopic ultrasound with fne-needle aspiration (EUS-FNA) is recommended in pancreatic cystic lesions (PCLs) with worrisome features (size≥3 cm, mural nodule, or Wirsung dilation). Objective: To evaluate the diagnostic ability and assess the accuracy of EUS-FNA in PCLs smaller than 3 cm. Methods: Retrospective study of PCLs<3 cm (2007–2016) undergoing EUS-FNA. Clinical, EUS and pancreatic cystic fuid (PCF) data were prospectively registered. Performance of EUS-FNA with PCF analysis for the detection of malignancy and accuracy in surgical cohort were analyzed. Results: We evaluated 115 patients with PCLs<3 cm who underwent EUS-FNA. 19 patients underwent surgery, 7 had malignant, 8 pre-malignant, and the remaining 4 benign lesions. Mass/mural nodule was present in 27% of the cysts, CEA level was higher than 192 ng/mL in 39.4% of patients, and only 35% of cytologic samples were informative. Nevertheless, additional FNA for PCF analysis improved the diagnostic performance of EUS imaging—AUC=0.80 versus AUC=60. Conclusion: EUS-FNA has good accuracy in PCLs<3 cm. It confrmed malignancy even in lesions without worrisome features (nodule/mass), with two in every fve resections showing high-risk/malignant lesions. EUS-FNA was also useful to diagnose benign cysts, possibly allowing surveillance to be stopped in one in every fve patients.This study was supported by a Research Grant from Sociedade Portuguesa de Endoscopia Digestiva in 2018 that had no role in any stage of the study, from design to submission for publication.info:eu-repo/semantics/publishedVersio

Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. FCT-MCTES is also acknowledged for 2020.07660.BD for BBO. Open access funding provided by FCT|FCCN (b-on).info:eu-repo/semantics/publishedVersio

Desenvolvimento de casos clínicos para práticas de simulação em contexto pedagógico

Nos últimos anos, tem se verificado um grande investimento no apoio a práticas pedagógicas de simulação na educação. Em Portugal, este recurso permanece ainda pouco implementado. A simulação pode ser definida como “”uma técnica que cria uma situação ou ambiente que permite às pessoas experimentarem uma representação de um evento real para a finalidade da prática, aprendizagem, avaliação, teste ou para desenvolver uma compreensão de sistemas ou ações humanas””. A criação do ESSim pretende, entre outros objetivos, desenvolver recursos e competências entre os docentes de diferentes áreas de formação, para a utilização de simulações na educação clínica dos futuros profissionais de saúde. Constituiu-se um grupo de trabalho para a construção de situações clínicas considerando: competências a desenvolver no estudante; casuística que permita interdisciplinaridade; necessidade de um conjunto de perfis para diferentes níveis de formação; e conjunto de características que possa abranger diferentes áreas de formação.info:eu-repo/semantics/publishedVersio

Genetic testing vs microforceps biopsy in pancreatic cysts: Systematic review and meta-analysis

Background: Carcinoembryonic antigen (CEA) and cytology in pancreatic cystic fluid are suboptimal for evaluation of pancreatic cystic neoplasms. Genetic testing and microforceps biopsy are promising tools for pre-operative diagnostic improvement but comparative performance of both methods is unknown. Aim: To compare the accuracy of genetic testing and microforceps biopsy in pancreatic cysts referred for surgery. Methods: We performed a literature search in Medline, Scopus, and Web of Science for studies evaluating genetic testing of cystic fluid and microforceps biopsy of pancreatic cysts, with endoscopic ultrasound with fine-needle aspiration (EUS-FNA) prior to surgery and surgical pathology as reference standard for diagnosis. We evaluated the diagnostic accuracy for: 1- benign cysts; 2- mucinous low-risk cysts; 3- high-risk cysts, and the diagnostic yield and rate of correctly identified cysts with microforceps biopsy and molecular analysis. We also assessed publication bias, heterogeneity, and study quality. Results: Eight studies, including 1206 patients, of which 203 (17%) referred for surgery who met the inclusion criteria were analyzed in the systematic review, and seven studies were included in the meta-analysis. Genetic testing and microforceps biopsies were identical for diagnosis of benign cysts. Molecular analysis was superior for diagnosis of both low and high-risk mucinous cysts, with sensitivities of 0.89 (95%CI: 0.79-0.95) and 0.57 (95%CI: 0.42-0.71), specificities of 0.88 (95%CI: 0.75-0.95) and 0.88 (95%CI: 0.80-0.93) and AUC of 0.9555 and 0.92, respectively. The diagnostic yield was higher in microforceps biopsies than in genetic analysis (0.73 vs 0.54, respectively) but the rates of correctly identified cysts were identical (0.73 with 95%CI: 0.62-0.82 vs 0.71 with 95%CI: 0.49-0.86, respectively). Conclusion: Genetic testing and microforceps biopsies are useful second tests, with identical results in benign pancreatic cysts. Genetic analysis performs better for low- and high-risk cysts but has lower diagnostic yield.info:eu-repo/semantics/publishedVersio

A second endoscopic ultrasound with fine‐needle aspiration for cytology identifies high‐risk pancreatic cysts overlooked by current guidelines

© 2020 Wiley Periodicals LLCBackground: Endoscopic ultrasound with fine-needle aspiration (EUS-FNA) is recommended for diagnosis of pancreatic cystic lesions (PCLs). Its role in surveillance is unclear. Our goal was to determine if a second EUS-FNA changes diagnosis or management of PCLs. Methods: A retrospective analysis of an EUS database, searching for EUS-FNAs in PCLs from 2007 to 2017 was performed. Demographics, cyst characteristics, and FNA results were compared in patients under surveillance, performing a single or two consecutive EUS-FNAs. Results: Of 203 PCLs referred for EUS-FNA, surveillance was decided in 128 (63%). Data of 105 (82%) patients with a single EUS-FNA were compared with 23 (18%) with two EUS-FNAs during surveillance. Patients were younger in this latter group (P = .055), whereas CEA levels were marginally higher (P = .078) and a mass/nodule were more frequent (P = .006). The mean time between EUSFNAs was 38 months (4.7-118.8) for 18 patients maintaining surveillance vs 18 months (2.9-56.9) in the four referred for surgery (P = NS) after two EUS-FNAs (two NETs, one IPMN-HGD, and one MCN-LG). A high correlation in CEA level between consecutive EUS-FNAs (r 2 = 0.945, P < .01) was present, with a change of category observed (cut-off level = 192 ng/mL) in two patients only. Of four patients with a second EUS-FNA with conclusive cytology, two had NETs confirmed on resection. Conclusions: Repeating EUS-FNA in surveillance of PCLs with clinical suspicion of malignancy increased neoplasm diagnoses, changing decision toward surgery in almost 20% of patients while excluding IPMNs with mucin nodules from unnecessary resections. A second EUS-FNA for cytology appears justified in some PCLs, particularly for diagnosing NETs.info:eu-repo/semantics/publishedVersio