European evidence-based guidelines on pancreatic cystic neoplasms

Evidence-based guidelines on the management of pancreatic cystic neoplasms (PCN) are lacking. This guideline is a joint initiative of the European Study Group on Cystic Tumours of the Pancreas, United European Gastroenterology, European Pancreatic Club, European-African Hepato-Pancreato-Biliary Association, European Digestive Surgery, and the European Society of Gastrointestinal Endoscopy. It replaces the 2013 European consensus statement guidelines on PCN. European and non-European experts performed systematic reviews and used GRADE methodology to answer relevant clinical questions on nine topics (biomarkers, radiology, endoscopy, intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN), serous cystic neoplasm, rare cysts, (neo)adjuvant treatment, and pathology). Recommendations include conservative management, relative and absolute indications for surgery. A conservative approach is recommended for asymptomatic MCN and IPMN measuring = 40 mm. Absolute indications for surgery in IPMN, due to the high-risk of malignant transformation, include jaundice, an enhancing mural nodule > 5 mm, and MPD diameter > 10 mm. Lifelong follow-up of IPMN is recommended in patients who are fit for surgery. The European evidence-based guidelines on PCN aim to improve the diagnosis and management of PCN

Stratifying Intraductal Papillary Mucinous Neoplasms by Cyst Fluid Analysis: Present and Future

A significant proportion of patients with intraductal papillary mucinous neoplasms (IPMNs) undergo surgical resection in order to prevent or treat pancreatic cancer at the risk of significant perioperative morbidity. Efforts have been made to stratify the potential risk of malignancy based on the clinical and radiographic features of IPMN to delineate which cysts warrant resection versus observation. An analysis of the cyst fluid obtained by preoperative endoscopic examination appears to be correlative of cyst type and risk, whereas serum markers and radiographic findings have not yet reached a level of sensitivity or specificity that proves they are clinically meaningful. In this review, we investigate the current cyst fluid analysis studies and present those that have shown promise in effectively stratifying high-risk versus low-risk lesions. While new cyst fluid markers continue to be identified, additional efforts in testing panels and marker composites in conjunction with clinical algorithms have also shown promise in distinguishing dysplasia and the risk of malignancy. These should be tested prospectively in order to determine their role in guiding the surveillance of low-risk lesions and to evaluate the new markers detected by proteomics and genetic sequencing

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

Improvement of the diagnosis and management of cystic tumors of the pancreas

Pancreatic cancer is on its course on becoming the second cause of cancer related mortality. Although improvements have been made in the treatment arsenal, still only a minority of patients are able to receive treatment with curative intention. This can be attributed to the fact that most of the pancreatic cancers are diagnosed at a later stage when curative therapy is no longer possible. Thus, efforts are being made to find pancreatic cancer and its precursor lesions at an earlier stage. Intraductal papillary mucinous neoplasms (IPMN) is the most common type of cystic lesion which has the potential for malignant transformation. Hence, an accurate and early diagnosis of this entity could contribute to reverse the dismal trend of pancreatic cancer. Study I & II Aim: To identify and correlate risk factors for advanced histological findings in IPMN patients. Methods: These were retrospective studies in which patients undergoing resection for IPMN during 2008-2015 (study I) and 2004-2017 (study II) were included. Patients characteristics, radiological and histological data were prospectively collected. Results: One-hundred-fifty-two patients were included in study I and 796 patients in study II. In study I, main pancreatic duct (MPD) dilatation of 6-9.9 mm and >10 mm were associated with an increased risk of advanced IPMN histology, such as high grade dysplasia (HGD) and cancer, at odds of 2.92 (CI 1.38–6.20, p=0.005) and 2.65 (CI 1.12–6.25, p=0.02), respectively. In addition, jaundice and elevated levels of CA 19-9 were also associated with higher risk for HGD/cancer at odds of 15.36 (CI 1.94–121.22, p=0.009) and 4.15 (CI 1.90–9.05, p=0.0003), respectively. These associations remained significant at uni- and multivariable regression analysis. In study II, the results showed MPD-dilatation to be the lone significant variable associated with increased probability of HGD or IPMN-cancer at both uni- and multivariable analysis. MPD dilatation of 5-9.9 mm was associated with odds of 2.74 (CI 1.80–4.16) and 4.42 (CI 2.55–7.66) for HGD and IPMN-cancer respectively. MPD-dilatation over 10 mm was associated with greater odds of 6.57 (CI 3.94–10.98) and 15.07 (CI 8.21–27.65) for HGD and IPMN-cancer, respectively. A 5-7 mm diameter of the MPD was determined as the cut-off value to best discriminate between the lesions with low risk of malignancy to those with a high risk. Conclusions: Even a smaller dilatation of the MPD is associated with increased risk of HGD and IPMN-cancer. Dilatation of the MPD and elevated levels of CA 19-9 are important diagnostic markers of advanced histology, thereby facilitating proper selection of patients most suitable for surgery. Study III & IV Aim: To define and validate the metabolic profile of patients with IPMN and serous cystic neoplasm (SCN) and to correlate the metabolite levels to histology and grade of dysplasia. Methods: Plasma and cyst fluid were prospectively collected from patients undergoing resection for IPMN and SCN. Targeted and untargeted analysis of metabolites and lipids species were performed and correlated to histology and clinical parameters. Results: From a cohort of 35 patients in study III and 57 patients in study IV several metabolites and lipids were identified in both cyst fluid and plasma. In study III, the metabolic profile showed significant alterations in the lipid pathways. An integrated metabolomic and lipidomic analysis model was able to discriminate IPMN from SCN up to 100% accuracy. The results in study IV not only validated the results from study III but also found novel metabolites able to discriminate non-cancerous lesions (low-grade IPMN and SCN) from malignant (HGD and cancer). Furthermore, specific metabolites correlated to presence of bacteria sequences in the cyst fluid. Conclusion: Analysis of the metabolic profile in cyst fluid and plasma from IPMN patients has been able to discriminate IPMN from SCN with high accuracy and also to predict the degree of dysplasia within IPMN. This method has shown potential of clinical application which in turn could improve the diagnosis of pancreatic cystic lesions. Study V Aim: To investigate the rate of new recurrence and progression of known IPMNs in the remnant pancreas after pancreatic surgery and to investigate if the grade of dysplasia at first histology affects the risk of recurrence. Methods: This was a retrospective study in which patients undergoing an elective partial pancreatic resection between 2008 and 2017 were included. Patients who underwent total pancreatectomy and/or had less than 2 years of follow-up were excluded. Patient characteristics and data of radiology, histology and recurrence was collected prospectively. Clinical significant recurrence was defined as findings resulting in a change in the management of the lesion. Results: Overall 224 patients were included in the study. The overall recurrence rate was 44.6% (100/224), whereas the clinical significant recurrence rate was 30.8% (69/224). Patients older than 65 years presented 4.4 odds (CI 1.5-13.1) of recurrence and patients with “known IPMN left in remnant” had 2.6 odds (CI 1.12-5.9) of recurrence. Patients with LGD and HGD at first histology without concomitant PDAC had a clinical significant recurrence rate of 11.1% (15/135). No differences regarding risk of recurrence could be found when comparing patients with LGD to HGD (HR 1.1 [CI 0.5-2.2]). Conclusion: Patients with LGD and HGD at first histology harbor a not negligible risk of future malignant transformation and should not be overlooked. The risk is further increased if the patient is older and/or have a previously known IPMN in the remnant. The overall conclusion of the thesis is that the included studies have increased the knowledge on several aspects in the diagnosis and management of cystic tumors of the pancreas. A known radiological diagnostic marker, the dilatation of the main pancreatic duct, has been studied where the results show that even smaller dilatations over 5 mm may indicate malignancy. An upcoming field in metabolomics has been applied on pancreatic cystic neoplasms and has shown great potential as a future diagnostic method. Lastly, the post-operative management of IPMN patients has been studied where the results highlight the need for continued clinical surveillance due the risk of recurrence even after surgery

Development of cancer biomarker assays from dna in various bodily fluids

Abstract DNA from cancer cells can enter surrounding fluid after apoptosis or necrosis. This DNA can be identified by sampling and sequencing the fluid looking for the mutations that gave rise to the cancer, referred to as a liquid biopsy. We have developed several liquid biopsies for the purpose of diagnosis and treatment selection through the use of targeted PCR and high throughput sequencing. We used the SafeSeqS methodology, a molecular barcoding technology previously developed in this lab, to further reduce sequencing errors to aid us in finding these rare mutations. We developed assays that identified genetic mutations from saliva, blood plasma, and pancreatic cysts. We investigating head and neck squamous cell carcinoma (HNSCC) from the oral cavity, oropharynx, hypopharynx, and larynx in 93 patients using a combination of saliva and plasma. We first identified either the E7 gene of HPV 16 or a primary mutation in the tumor and then tried to detect their presence in saliva and plasma. We collected saliva from every patient, while we only had plasma samples from 43 patients. We found that saliva performed best in oral cavity cancers detecting 100% of those patients, while plasma performed similarly across all sites detecting 87% of HNSCC patients. We detected mutations in 96% of patients when both saliva and plasma were available. Pancreatic cyst fluid was used to aid clinicians in the classification of pancreatic cysts. We created an 8 gene panel to look for mutations as well as additional tests for loss of heterozygosity and aneuploidy. We used DNA from fluid captured through endoscopic aspiration as well as from surgically resected cysts. When combined with the typical clinical features we were able to accurately predict which cysts needed surgery with a sensitivity and specificity of 89% and 69% respectively. Each of these projects began as an attempt to aid clinicians in dealing with these various diseases. Our results illustrate that liquid biopsies can be developed into an effective tool for the fight against cancer

Patterns of injury and violence in Yaoundé Cameroon: an analysis of hospital data.

BackgroundInjuries are quickly becoming a leading cause of death globally, disproportionately affecting sub-Saharan Africa, where reports on the epidemiology of injuries are extremely limited. Reports on the patterns and frequency of injuries are available from Cameroon are also scarce. This study explores the patterns of trauma seen at the emergency ward of the busiest trauma center in Cameroon's capital city.Materials and methodsAdministrative records from January 1, 2007, through December 31, 2007, were retrospectively reviewed; information on age, gender, mechanism of injury, and outcome was abstracted for all trauma patients presenting to the emergency ward. Univariate analysis was performed to assess patterns of injuries in terms of mechanism, date, age, and gender. Bivariate analysis was used to explore potential relationships between demographic variables and mechanism of injury.ResultsA total of 6,234 injured people were seen at the Central Hospital of Yaoundé's emergency ward during the year 2007. Males comprised 71% of those injured, and the mean age of injured patients was 29 years (SD = 14.9). Nearly 60% of the injuries were due to road traffic accidents, 46% of which involved a pedestrian. Intentional injuries were the second most common mechanism of injury (22.5%), 55% of which involved unarmed assault. Patients injured in falls were more likely to be admitted to the hospital (p &lt; 0.001), whereas patients suffering intentional injuries and bites were less likely to be hospitalized (p &lt; 0.001). Males were significantly more likely to be admitted than females (p &lt; 0.001)DiscussionPatterns in terms of age, gender, and mechanism of injury are similar to reports from other countries from the same geographic region, but the magnitude of cases reported is high for a single institution in an African city the size of Yaoundé. As the burden of disease is predicted to increase dramatically in sub-Saharan Africa, immediate efforts in prevention and treatment in Cameroon are strongly warranted

Surgical Treatment of Pancreatic Ductal Adenocarcinoma

This book contains the art and science in current standards of surgical treatment of pancreatic ductal adenocarcinoma. It explains the clinical role of surgical resection during multimodal treatment in patients with pancreatic ductal adenocarcinoma, novel surgical techniques including extended pancreatectomy and minimally invasive surgery, risk of cancer in IPMN, and the clinical importance of liquid biopsy

GENETIC EVOLUTION AND PROGNOSTIC DETERMINANTS OF PANCREATIC CANCER ON LONGITUDINAL LIQUID BIOPSIES

Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates amongst solid tumors. As early detection of PDAC is unusual and typically incidental, most patients present with locally advanced and metastatic disease where effective therapeutic strategies remain a significant unmet need. Specifically, surrogate biomarkers for tumor monitoring of PDAC may lead to improved elucidation of clinical actionability and prognostic potential. On the other hand, tumor tissue is rarely sampled in patients presenting with de novo or recurrent metastatic PDAC, apart from a fine needle aspiration or a core needle biopsy performed for diagnosis. This precludes the opportunity for elucidating molecular underpinnings of cancer recurrence, chemoresistance, and therapeutic decision-making in advanced disease patients over the course of their therapy. For this reason, we aim to use so called “liquid biopsies” in the form of circulating nucleic acids and exosomes as a strategy that is amenable to longitudinal, relatively non-invasive sampling. Circulating tumor DNA and circulating exosomes contain genetic cargo representative of the neoplastic cells from which they are released and can serve as a reliable surrogate of the patient’s tumor DNA, enabling a new way of interrogating cancers. We demonstrate that serial quantitative measurements of these tumor nucleic acid sources in circulation can provide clinically relevant predictive and prognostic information in pancreatic cancer patients, including anticipation of impending disease progression and putative mechanisms of resistance to ongoing therapy. We also describe our ability to specifically capture tumor material in circulation following a comprehensive characterization of the pancreatic cancer exosomal “surfaceome”. By leveraging an immune-capture approach paired with ultrasensitive molecular barcoding techniques, we are able to increase our sensitivity of detection of rare molecules in circulation that are derived from the tumor. Ultimately, this has implications for stratification of patients into therapeutic “buckets” through a personalized approach that may lead to greater survival benefits in PDAC