A registry-based study on universal screening for defective mismatch repair in colorectal cancer in Denmark highlights disparities in screening uptake and counselling referrals.

Universal screening for defective mismatch repair (dMMR) in colorectal cancer utilizes immunohistochemical staining for MLH1, MSH2, MSH6 and PSM2. Additionally, BRAF V600E mutations status and MLH1 hypermethylation should be performed to distinguish germline and somatic dMMR alterations. A decade of Danish population-based registries has been analysed regarding screening uptake, detection rate and referral to genetic counselling. MMR testing was performed in 71·8% (N = 34,664) of newly diagnosed colorectal cancers with an increasing trend to 88·8% coverage in the study's final year. The likelihood of undergoing MMR testing was reduced in males with 2% (95% CI 0·4-2·7, p = 0·008), with 4·1% in patients above age 70 years (95% CI 1·5-6·6, p = 0·003) compared in patients below age 51 years, with 16·3% in rectal cancers (95% CI 15·1-17·6, p < 0·001) and 1·4% left-sided colon cancers (95% CI 0·1-1·7, p = 0·03) compared to right-sided colon cancers. Tumour stage II and III increased the likelihood of being tested, with 3·7% for stage II (95% CI 2·2-5·6, p < 0·001) and 3·3% for stage III tumours (95% CI 1·8-4·8, p < 0·001) compared to stage I tumours, whereas the likelihood for stage IV tumours is reduced by 35·7% (95% CI 34·2-37·2, p < 0·001). Test rates significantly differed between the Danish health care regions. dMMR was identified in 15·1% (95% CI 14·8-15·6, p < 0·001) cases with somatic MMR inactivation in 6·7% of the cases. 8·3% tumours showed hereditary dMMR expression patterns, and 20·0% of those were referred to genetic counselling. Despite the high uptake rates, we found disparities between patient groups and missed opportunities for genetic diagnostics.

Diarrhoea-causing microorganisms are rare in adult patients undergoing diagnostic laparoscopy for suspected appendicitis: a prospective observational cohort study.

We investigated if diarrhoea-causing bacteria, including Yersinia species, could mimic the symptoms of appendicitis and lead to surgery. This prospective observational cohort study (NCT03349814) included adult patients undergoing surgery for suspected appendicitis. Rectal swabs were analysed with polymerase chain reaction (PCR) for Yersinia, Campylobacter, Salmonella, Shigella and Aeromonas spp. Blood samples were analysed routinely and with an in-house ELISA serological test for Yersinia enterocolitica antibodies. We compared patients without appendicitis and patients with appendicitis confirmed by histopathology. The outcomes included PCR-confirmed infection with Yersinia spp., serologic-confirmed infection with Y. enterocolitica, PCR-confirmed infection with other diarrhoea-causing bacteria and Enterobius vermicularis confirmed by histopathology. A total of 224 patients were included, 51 without and 173 with appendicitis, and followed for 10 days. PCR-confirmed infection with Yersinia spp. was found in one patient (2%) without appendicitis and no patients (0%) with appendicitis (p = 0.23). Serology was positive for Y. enterocolitica for the same patient without appendicitis and two patients with appendicitis (p = 0.54). Campylobacter spp. were detected in 4% vs 1% (p = 0.13) of patients without and with appendicitis, respectively. Infection with Yersinia spp. and other diarrhoea-causing microorganisms in adult patients undergoing surgery for suspected appendicitis was rare.

Severe ulcerative oesophagitis caused by primary Epstein-Barr virus infection in an immunocompetent individual.

Epstein-Barr virus (EBV) infects the vast majority of the human population. The primary infection in immunocompetent individuals is typically asymptomatic or presenting as infectious mononucleosis. Here, an 18-year-old man without medical history was admitted with mild non-specific symptoms of infection presenting primarily with severe dysphagia and epigastric pain. Gastroscopy revealed severe, extensive, ulcerative oesophagitis with suspicion of Crohn's disease. However, a diagnosis of primary EBV infection presenting as severe ulcerative oesophagitis and without systemic symptoms of infectious mononucleosis was made based on dynamic changes in EBV serology (shift from IgM to IgG positivity), EBV-specific immunohistochemical staining, and PCR analysis of biopsy specimens. This rare manifestation of primary EBV in an immunocompetent patient was treated symptomatically and resolved within a few weeks, and should be considered a differential diagnosis at otherwise unexplained ulcerative oesophagitis in younger individuals.

Frequency and coexistence of KRAS, NRAS, BRAF and PIK3CA mutations and occurrence of MMR deficiency in Danish colorectal cancer patients.

The MAPK signalling genes KRAS, NRAS and BRAF and the PIK3CA gene are routinely investigated for mutations in the diagnostic routine of colorectal cancer. Few studies have reported co-existing mutations in these genes with clinical relevance, while some have been previously regarded as mutually exclusive. We set to investigate the frequency and co-occurrent mutations in these targets, and the occurrence of mismatch repair deficiency (dMMR) in a large cohort of Danish colorectal cancers. 1000 colorectal tumours were sequenced as part of our diagnostic workflow for KRAS, NRAS, BRAF and PIK3CA mutations using next-generation sequencing (NGS) and analysed by immunohistochemistry (IHC) for loss of the MMR proteins, MLH1, PMS2, MSH2 and MSH6. Co-existing mutations in 12 patients (1.2%) occurred as multiple mutations in the same gene or spread across several genes (KRAS, NRAS and/or BRAF). The frequency of single mutations in the genes occurred with a frequency similar to previously reported, except for a higher frequency of BRAF mutations (18.0%). We found dMMR in 14.6% of the cases with a majority lacking expression of both MLH1 and PMS2. BRAF mutations were only present in dMMR cases involving MLH1 and/or PMS2. Our findings suggest that co-existing mutations occur, except for the hotspot BRAF V600E, which is mutually exclusive with KRAS/NRAS mutations. Therefore, instead of single gene alterations from the MAPK signalling, assessing co-occurrence of mutations within one or more of those genes should also be accounted. This may impact future oncological treatments and should be considered in the diagnostic workflow.

Phospho-ERK levels as predictors for chemotherapy of rectal carcinoma.

Treatment of rectal cancer has been vastly improved by advances in surgery and radiochemotherapy but remains an important cause of morbidity and mortality worldwide. A particular problem is the lack of predictive markers that can help to individualize treatment. The growth- and apoptosis-regulating signaling molecules ERK 1 and 2 are important to cancer growth and progression. They are activated through phosphorylation, which is initiated by a cascade involving the EGF receptor and RAS as upstream regulators. Moreover, in vitro studies indicate that phospho-ERKs interfere with 5-fluorouracil-based chemotherapy. Recently, we showed that high levels of phospho-ERKs in rectal cancer cells predict poor responses to neoadjuvant (preoperative) radiochemotherapy. We now report that preoperative phospho-ERK levels also can subdivide high-risk rectal cancer patients into a favorable and a poor prognostic group with respect to recurrence-free survival. Importantly, phospho-ERK levels were of predictive significance only in high-risk patients, who received adjuvant (postoperative) chemotherapy, but not in high-risk patients not receiving such therapy. Our results suggest that high cancer cell levels of phospho-ERK predict poor responsiveness to both preoperative and postoperative chemotherapy of rectal cancer.

Digital image analysis of pan-cytokeratin stained tumor slides for evaluation of tumor budding in pT1/pT2 colorectal cancer: Results of a feasibility study.

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users. Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides. Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p < 0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p = 0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p = 0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3-4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.

Early metastatic colorectal cancers show increased tissue expression of miR-17/92 cluster members in the invasive tumor front.

Accurate prediction of regional lymph node metastases (LNM) in endoscopically resected pT1 colorectal cancer (CRC) is crucial in treatment stratification for subsequent radical surgery. Several miRNAs have been linked to CRC invasion and metastasis, including the oncogenic miR-17/92 cluster, and expression levels might have predictive value in the risk assessment of early metastatic progression in CRC. We performed global miRNA microarray using tissue samples from the invasive front of pT1 CRC and investigated associations of the miR-17/92 cluster and presence of LNM. In total, 56 matched pT1 CRCs were thoroughly clinicopathologically characterized, and miRNA microarrays were performed on invasive front tissue samples. Global miRNA intensities were screened using paired t-tests between pT1pN+ and pT1pN0. Associations between miR-17/92 and histopathological features were analyzed using general linear models and tumor cell adjusted expression intensities. miR-17-3p and miR-92a were significantly higher expressed in the invasive front of tumors with LNM compared to those without, corresponding to 1.53-fold higher expression of miR-17-3p (95%CI: 1.04-2.24, P = .030) and 1.28-fold higher expression of miR-92a (95%CI: 1.01-1.68, P = .042). An inverse association between miR-19a and presence of high-grade tumor budding was observed (1.55-fold, 95%CI: 1.13-2.12, P = .008). We provide evidence for associations between early regional LNM and high expression levels of the miR-17/92 cluster members: miR-17-3p and miR-92a, in the invasive front of CRC. Our results support a role for the miR-17/92 cluster in early metastatic progression of CRC and calls for further investigation.

Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer.

INTRODUCTION: miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC. MATERIAL AND METHODS: Two parallel studies on archived formalin-fixed paraffin-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors was investigated in FFPE tissue from invasive border and central tumor zones from 47 early metastatic CRCs matched with 47 non-metastatic CRCs. Intra-tumor expression gradients were analyzed on both cohorts. RESULTS: Mean intra-tumor coefficient of variation in the heterogeneity cohort was 38.5% (range: 33.1-49.0%) only slightly less than variation between patients (45.1%, range 37.0-49.5%). We demonstrated systematic expression gradients between tumor zones equal to a 3.23 (p=0.003) and 1.36 (p=0.014) fold lower expression in invasive areas for miR-200c, 1.52 (p<0.001) and 1.27 (p=0.021) fold lower expression in invasive areas for miR-92a. For miR-21 we found a 1.75 (p<0.001) and 1.21 (p=0.064) fold higher expression in invasive areas compared to luminal and central zones, respectively. No significant difference in expression levels between metastatic and non-metastatic tumors was demonstrated, nor a difference in intra-tumor gradients between metastatic and non-metastatic tumors. CONCLUSION: This study provides evidence for moderate intra-tumor and inter-patient heterogeneities of three well-described potential prognostic markers in CRC. We demonstrate intra-tumor expression gradients indicating a differentiated expression of the target miRNAs between functional tumor zones, but the potential role as markers of early metastatic disease is still not fully clarified.

Intra-tumor heterogeneity of microRNA-92a, microRNA-375 and microRNA-424 in colorectal cancer.

UNLABELLED: Various microRNAs (miRNAs) have been investigated in order to improve diagnostics and risk assessment in colorectal cancer (CRC). To clarify the potential of miRNA profiling in CRC, knowledge of intra-tumor heterogeneity in expression levels is crucial. The study aim was to estimate the intra-tumor variance of three selected miRNAs: miR-92a, miR-375 and miR-424 in CRC tissue. MATERIAL AND METHODS: A retrospective study on archived formalin-fixed paraffin embedded tissue from 9 patients with CRC. miRNA tissue expression levels were analyzed by qRT-PCR on tissue representing luminal, central and invasive border zones. Variance components were estimated based on ∆∆Cp values using mixed modeling and presented as coefficients of variation (CV). RESULTS: Intra-tumor variance was approximately half of the variance observed between patients with a mean intra-tumor CV of 56.4% (range 33.1-77.1%) and a mean inter-patient CV of 101.7% (range 48.8-152.7%). Furthermore we found a significant systematic difference in expression levels between tumor zones for miR-92a and miR-375 with a luminal-invasive difference equal to 0.60 Cp (95% CI: 0.30-0.89, p=0.0003) for miR-92a and a luminal-invasive difference equal to 0.78 Cp (95% CI: 0.10-1.46, p=0.027) for miR-375. Conclusion While the intra-tumor variance of miR-92a, miR-375 and miR-424 is substantial, it only constitutes approximately 30% of the total variation. Functional deregulation between tumor zones might contribute to variations in measured expression levels, and thus knowledge of specific intra-tumor expression patterns is crucial in tissue sampling for research as well as in future diagnostics.

[Eosinophilic ascites is a rare presentationpart of eosinophilic gastroenteritis]. / Eosinofil ascites er en sjælden præsentation af eosinofil gastroenteritis.

An otherwise healthy 49-year-old male was admitted due to ascites and obstipation. He had no history of atopy. He went through an extensive diagnostic workup including laparascopy and bone marrow biopsy. All the tests came out normal except for a large number of eosinophils in the blood and the ascites. The diagnosis of idiopatic eosinophilic ascites was made. After drainage spontaneous remission was achieved. Eosinophilic ascites is a rare disorder of unknown aethiology and is a part of the syndrome called eosinophilic gastroenteritis. In symptomatic patients the choice of treatment is prednisone.