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

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.

Doomed for carcinomatosis? An unusual presentation of abdominal tuberculosis.

A 35-year old male from Brazil presented with intermittent abdominal pain. Abdominal computed tomography revealed a nodule adjacent to splenic hilum and multiple abdominal nodules, suspicious of carcinomatosis. The patient underwent gastroscopy and endoscopic ultrasound (EUS), that revealed an ill-defined hypoechogenic lesion adjacent to the spleen and two hypoechogenic subepithelial lesions located in the 4th layer of the stomach and duodenal bulb. Biopsies revealed non-necrotizing granulomatous inflammation with multinucleated giant cells. Soon after, a 18cm palpable mass within the rectus abdominis muscle was identified, and the biopsy was positive for Mycobacterium tuberculosis DNA, confirming the diagnosis of disseminated abdominal tuberculosis.

Endoscopic treatment of disconnected pancreatic duct syndrome secondary to acute necrotizing pancreatitis in a patient with pancreas divisum.

A 50-year old male with acute necrotizing pancreatitis had an unfavorable evolution in the third week of disease, with development of large volume ascites and walled-off necrosis affecting the head and body of pancreas, suspected to be infected, with viable parenchyma in the tail of pancreas. Endoscopic ultrasound guided drainage of the collection was performed through placement of a lumen apposing metal stent. Selective cannulation of main pancreatic duct was only possible though the minor papilla and after contrast media injection a type 1 pancreas divisum and partial disruption of dorsal pancreatic duct were diagnosed.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

Participation in clinical trials increases the detection of pre-malignant lesions during colonoscopy.

BACKGROUND: colorectal adenoma detection has been associated with the effectiveness of cancer prevention. Clinical trials have been designed to determine the role of several interventions to increase the detection of pre-malignant lesions. We hypothesized that colonoscopy in the setting of clinical trials has a higher pre-malignant lesion detection rate. METHODS: a cross-sectional study was performed that compared the detection of pre-malignant lesions in 147 randomly sampled non-research colonoscopies and 294 from the control group of two prospective trials. Outpatients aged 40-79 years, with no personal history of colorectal cancer (CRC) were included. RESULTS: baseline characteristics were similar between the two groups. The pre-malignant lesion detection rate in the trial vs control group was 65.6 % vs 44.2 % (OR 2.411; 95 % CI: 1.608-3.614; p < 0.001), the polyp detection rate was 73.8 % vs 59.9 % (OR 1.889; 95 % CI: 1.242-2.876; p = 0.003), the adenoma detection rate was 62.6 % vs 44.2 % (OR 2.110; 95 % CI: 1.411-3.155; p < 0.001) and the sessile serrated lesion detection rate was 17 % vs 4.1 % (OR 4.816; 95 % CI: 2.014-11.515; p < 0.001). The mean number of pre-malignant and sessile serrated lesions was 1.70 vs 1.06 (p = 0.002) and 0.32 vs 0.06 (p = 0.001) lesions per colonoscopy, respectively. There was no significant change in any of the study outcomes according to the multivariate analysis with each single potential confounder. CONCLUSIONS: patients involved in colonoscopy trials may benefit from higher quality examinations, as shown by the higher detection rates. Institutions should consider supporting clinical research in colonoscopy as a simple means to improve colonoscopy quality and colorectal cancer prevention.

Mode of Delivery Does Not Affect the Risk of Inflammatory Bowel Disease.

BACKGROUND: Recent evidence suggests that exposures in early life that are known to influence microbiome development may affect the risk of developing inflammatory bowel disease (IBD). Cesarean section has been associated with altered colonization of commensal gut flora and is thought to predispose to immune-mediated diseases later in life. AIMS: To evaluate the risk of IBD, Crohn's Disease (CD), and Ulcerative Colitis (UC) according to mode of delivery (C-section vs vaginal delivery). METHODS: A systematic search was performed in PubMed and Embase. The primary outcome was the risk of IBD in individuals delivered vaginally compared to those born by C-section. Secondary outcomes were UC and CD risk according to mode of delivery and IBD risk in individuals born by emergent compared to elective C-section. Publication bias was evaluated by funnel plots and Egger's test. Study's quality was characterized using the Newcastle-Ottawa Scale. RESULTS: Ten studies fulfilled the inclusion criteria, of which seven were population-based. No publication bias was detected. Overall, 14.164 IBD patients and 4.206.763 controls were included. Being born by C-section was not associated with increased risk of IBD [OR 1.01, 95% CI (0.81-1.27), p = 0.92], CD [OR 1.15, 95% CI (0.94-1.42), p = 0.18] or UC [OR 0.94, 95% CI (0.61-1.45), p = 0.79]. No differences were found between emergent and elective C-section in IBD [OR 1.05, 95% CI (0.59-1,87), p = 0.87]. Substantial heterogeneity was found in statistical analysis, and further studies are needed. CONCLUSION: Overall, the risk of developing IBD was not affected by mode of delivery.

Malignancy and mass-forming phenotypes of IgG4-related disease: a challenging diagnosis.

Mass-forming phenotypes of IgG4-related disease (IgG4-RD) mimic malignancy and histological confirmation can be challenging. A woman in her 70s with HIV infection presented with painless obstructive jaundice and weight loss. Magnetic resonance imaging was suggestive of unresectable cholangiocarcinoma. Tumour markers and serum IgG4 were normal. Percutaneous liver biopsy was consistent with IgG4-RD inflammatory pseudotumour, with complete response to glucocorticoid therapy. Two years later, a new episode of obstructive jaundice occurred, with CT showing a solid lesion in the head of the pancreas with double duct sign and encasement of the portal vein. Re-induction therapy was tried without response. Fine-needle biopsy was consistent with pancreatic cancer. Supportive care was offered and the patient died 8 months later, with no signs of disease progression on subsequent imaging. We discuss the challenges of IgG4-RD diagnosis and treatment and the differential diagnosis between mass-forming phenotypes and malignancy, highlighting the difficulties in managing such patients.

Body composition and Crohn's disease behavior: Is adiposity the main game changer?

OBJECTIVE: We investigated the association between body composition upon diagnosis and complicated phenotypes and time until surgery in patients with Crohn's disease (CD). METHODS: We conducted a retrospective cohort study including patients with CD who had a computed tomography enterography or a magnetic resonance enterography performed ≤6 mo of diagnosis. Skeletal muscle and visceral and subcutaneous adipose tissue cross-sectional areas were determined with computed tomography or magnetic resonance images at the third lumbar vertebral level, processed with the sliceOmatic (TomoVison, Magog, QC, Canada) and ABACS plugin. RESULTS: We included 63 patients: 33 (52%) men, median age 35 y. Disease location (L) and behavior (B) according to the Montreal classification were L1 (ileal disease) = 28 (44%), L2 (colonic disease) = 13 (21%), L3(ileocolonic disease) = 18 (28%), L1 + L4 (ileal and isolated upper disease) = 1 (2%), L3 + L4 (ileocolonic and isolated upper disease) = 3 (5%), B1 (non-stricturing) = 39 (62%), B2 (stricturing) = 11 (17%), and B3 (penetrating)= 13 (21%); 20 (32%) patients had perianal disease. Visceral obesity was present in 12 (19%) patients and was associated with higher age of CD onset (median 60 versus 34 y; P = 0.002) and complicated disease behavior (B2 and B3) (66.7% versus 31.7%; P = 0.021). After adjusting for age and perianal disease, total adipose tissue was associated with a 4% increase in the odds of complicated behavior per 10 cm2 of total adipose tissue (odds ratio [OR] = 1.004; 95% confidence interval [CI], 1.00-1.008; P = 0.043). Median follow-up time was 3.35 y, during which 15 (24%) of patients underwent abdominal surgery. Visceral obesity was associated with 5.10-times higher risk of abdominal surgery (95% CI, 1.52-17.09; P = 0.008); after adjusting for disease behavior, visceral obesity maintained a near-significant association with a 2.90-times higher risk of surgery (95% CI, 0.83-10.08; P = 0.09). CONCLUSION: Total fat was associated with complicated disease phenotype and visceral obesity, with higher risk of abdominal surgery and shorter time until surgery.

Early intervention with biologic therapy in Crohn´s disease: how early is early?

BACKGROUND: Early biologic therapy within the first 18-24 months after diagnosis is associated with improved clinical outcomes in Crohn's disease [CD]. However, the definition of the best time to initiate biologic therapy remains unclear. We aimed to assess if there is an optimal timing for early biologic therapy initiation. METHODS: This was a multicentre retrospective cohort study including newly diagnosed CD patients who started anti-tumour necrosis factor [TNF] therapy within 24 months from diagnosis. The timing of initiation of biologic therapy was categorised as ≤6, 7-12, 13-18, and 19-24 months. The primary outcome was CD-related complications defined as a composite of progression of Montreal disease behaviour, CD-related hospitalisations, or CD-related intestinal surgeries. Secondary outcomes included clinical, laboratory, endoscopic, and transmural remission. RESULTS: We included 141 patients where 54%, 26%, 11%, and 9% started biologic therapy at ≤6, 7-12, 13-18, and 19-24 months after diagnosis, respectively. A total of 34 patients [24%] reached the primary outcome: 8% had progression of disease behaviour, 15% were hospitalised, and 9% required surgery. There was no difference in the time to a CD-related complication according to the time of initiation of biologic therapy within the first 24 months. Clinical, endoscopic, and transmural remission was achieved in 85%, 50%, and 29%, respectively, but no differences were found according to the time of initiation of biologic therapy. CONCLUSION: Starting anti-TNF therapy within the first 24 months after diagnosis was associated with a low rate of CD-related complications and high rates of clinical and endoscopic remission, although we found no differences with earlier initiation within this window of opportunity.

Secondary Sclerosing Cholangitis in a Critically Ill Patient with Severe SARS-CoV-2 Infection: A Possibly Emergent Entity during the Current Global Pandemic.

A 46-year-old woman without previous history of hepatobiliary disease was admitted to the intensive care unit due to SARS-CoV-2 infection. Admission blood tests revealed impending hyperinflammation in the context of systemic inflammatory response syndrome. She required 12 days of mechanical ventilation and vasopressor support. After admission, liver function tests became deranged in a cholestatic pattern and continued to worsen despite overall clinical improvement. Magnetic resonance cholangiopancreatography revealed liver abscesses, intrahepatic bile duct dilation with multiple strictures and some linear repletion defects at the bifurcation of the common hepatic duct. During endoscopic retrograde cholangiopancreatography, biliary casts were retrieved confirming the diagnosis of secondary sclerosing cholangitis in the critically ill patient triggered by a severe SARS-CoV-2 infection. Other causes of cholestasis and secondary sclerosing cholangitis were properly excluded. We present an illustrative case and discuss the current literature, focusing on SARS-CoV-2 infection contribution to the development of this potentially underdiagnosed and severe condition.

Uma mulher de 46 anos sem antecedentes de patologia hepatobiliar foi admitida na unidade de cuidados intensivos no contexto de infeção por SARS-CoV-2. Apresentava alterações analíticas interpretadas no contexto de síndrome de resposta inflamatória sistémica. Houve necessidade de suporte vasopressor e ventilação mecânica invasiva durante 12 dias. Após a admissão, verificou-se uma alteração das provas hepáticas com padrão colestático, com agravamento contínuo apesar da melhoria do quadro infecioso. A colangiografia por ressonância magnética revelou a presença de abcessos hepáticos, dilatação das vias biliares intrahepáticas com múltiplas estenoses e com alguns defeitos de repleção lineares na bifurcação do ducto hepático comum. Na colangiopancreatografia endoscópica retrógrada foram removidos cilindros bilares da via biliar, confirmando o diagnóstico de colangite esclerosante secundária associada aos cuidados intensivos, no contexto de uma infeção grave por SARS-CoV-2. Foram excluídas outras causas de colestase e colangite esclerosante secundária de forma exaustiva. Apresentamos um caso clínico ilustrativo com respetiva iconografia e revisão da literatura, com especial enfoque na contribuição da infeção por SARS-CoV-2 no desenvolvimento desta entidade clínica, potencialmente grave e subdiagnosticada.

Histological Inflammation in the Endoscopically Uninflamed Mucosa is Associated With Worse Outcomes in Limited Ulcerative Colitis.

BACKGROUND: The Montreal classification categorizes patients with ulcerative colitis (UC) based on their macroscopic disease extent. Independent of endoscopic extent, biopsies through all colonic segments should be retrieved during index colonoscopy. However, the prognostic value of histological inflammation at diagnosis in the inflamed and uninflamed regions of the colon has never been assessed. METHODS: This was a multicenter retrospective cohort study of newly diagnosed patients with treatment-naïve proctitis and left-sided UC. Biopsies from at least 2 colonic segments (endoscopically inflamed and uninflamed mucosa) were retrieved and reviewed by 2 pathologists. Histological features in the endoscopically inflamed and uninflamed mucosa were scored using the Nancy score. The primary outcomes were disease complications (proximal disease extension, need for hospitalization or colectomy) and higher therapeutic requirements (need for steroids or for therapy escalation). RESULTS: Overall, 93 treatment-naïve patients were included, with a median follow-up of 44 months (range, 2-329). The prevalence of any histological inflammation above the endoscopic margin was 71%. Proximal disease extension was more frequent in patients with histological inflammation in the endoscopically uninflamed mucosa at diagnosis (21.5% vs 3.4%, P = 0.04). Histological involvement above the endoscopic margin was the only predictor associated with an earlier need for therapy escalation (adjusted hazard ratio, 3.69; 95% confidence interval, 1.05-13.0); P = 0.04) and disease complications (adjusted hazard ratio, 4.79; 95% confidence interval, 1.10-20.9; P = 0.04). CONCLUSIONS: The presence of histological inflammation in the endoscopically uninflamed mucosa at the time of diagnosis was associated with worse outcomes in limited UC.

Inflammatory Bowel Disease Patients' Perspectives during COVID-19 Pandemic: Results from a Portuguese Survey.

INTRODUCTION: Patients with inflammatory bowel disease (IBD) do not seem to be at increased risk of infection by SARS-CoV-2, but there is a concern whether immunosuppressive therapy may be associated with more severe disease. Several clinical practice recommendations have been published to help guide IBD care during the COVID-19 pandemic. Nonetheless, few studies have addressed patients' perspectives and fears. We aimed to evaluate Portuguese IBD patients' perspectives on the clinical management of their disease during the SARS-CoV-2 pandemic as well as the impact on their professional life. METHODS: An anonymous electronic survey was created using REDCap and was distributed by the Portuguese Association of Inflammatory Bowel Disease (APDI) between May and August 2020. Patients' perspectives on immunosuppressive therapy, disease management, interaction with gastroenterology departments, and the impact of the pandemic in their professional life were assessed. Patients' proposals to improve medical care were also evaluated. Descriptive analysis and logistic regression were performed. RESULTS: A total of 137 participants answered the survey (79.6% females, mean age 41.7 ± 12.1 years). Although having IBD and receiving treatment with immunosuppressors (thiopurines, steroids, or biologics) were considered promotors of anxiety, most patients (85.4%) agreed that disease remission was a priority and only a minority of patients interrupted their treatment during the pandemic. In multivariate analysis, active disease, biologic treatment, and use of corticosteroids in the last 3 months were perceived by the patients as high-risk features for increased risk of SARS-Cov-2 infection and more severe disease. Fifty-nine patients (44%) believed that their follow-up was influenced by the pandemic and only 58.8% felt that they had the opportunity to discuss their therapeutic options with their doctor. Sixty-three patients (46.0%) were working from home during the pandemic, although this decision was related to IBD and immunosuppressive therapy in only 36.5 and 39.7% of the cases, respectively. Areas where care could have been improved during the pandemic were identified by patients, namely enhancement of the communication with IBD professionals, conciliation of telemedicine with face-to-face appointments, and facilitation of the interaction between patients and employers. CONCLUSION: Most patients agreed that maintaining IBD remission is crucial, and only a minority of the patients stopped their treatment as per their own initiative. IBD status only had a small influence on patients' professional activity during the COVID-19 outbreak, with most changes being related to the pandemic itself.

Preclinical disease and preventive strategies in IBD.

There is growing evidence that inflammatory bowel disease has a preclinical phase that precedes disease symptoms, during which immune system activation has already occurred and inflammatory pathways have been primed, setting the stage for disease to expand and leading up to clinical diagnosis. Gaining insight to this pre-diagnosis period could improve our knowledge about disease pathogenesis and potentially result in the detection of biomarkers that could predict disease development. The ultimate goal of such research is to identify a population at risk for developing IBD, so preventive strategies could be implemented and disease prevention be achieved.

Participation in clinical trials increases the detection of pre-malignant lesions during colonoscopy

Background: colorectal adenoma detection has been associated with the effectiveness of cancer prevention. Clinical trials have been designed to determine the role of several interventions to increase the detection of pre-malignant lesions. We hypothesized that colonoscopy in the setting of clinical trials has a higher pre-malignant lesion detection rate.Methods: a cross-sectional study was performed that compared the detection of pre-malignant lesions in 147 randomly sampled non-research colonoscopies and 294 from the control group of two prospective trials. Outpatients aged 40-79 years, with no personal history of colorectal cancer (CRC) were included.Results: baseline characteristics were similar between the two groups. The pre-malignant lesion detection rate in the trial vs control group was 65.6 % vs 44.2 % (OR 2.411; 95 % CI: 1.608-3.614; p < 0.001), the polyp detection rate was 73.8 % vs 59.9 % (OR 1.889; 95 % CI: 1.242-2.876; p = 0.003), the adenoma detection rate was 62.6 % vs 44.2 % (OR 2.110; 95 % CI: 1.411-3.155; p < 0.001) and the sessile serrated lesion detection rate was 17 % vs 4.1 % (OR 4.816; 95 % CI: 2.014-11.515; p < 0.001). The mean number of pre-malignant and sessile serrated lesions was 1.70 vs 1.06 (p = 0.002) and 0.32 vs 0.06 (p = 0.001) lesions per colonoscopy, respectively. There was no significant change in any of the study outcomes according to the multivariate analysis with each single potential confounder.Conclusions: patients involved in colonoscopy trials may benefit from higher quality examinations, as shown by the higher detection rates. Institutions should consider supporting clinical research in colonoscopy as a simple means to improve colonoscopy quality and colorectal cancer prevention. (AU)