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AimsThe histopathological diagnosis of low-grade dysplasia (LGD) in Barrett’s oesophagus (BO) is associated with poor interobserver agreement and guidelines dictate expert review. To facilitate nationwide expert review in the Netherlands, a web-based digital review panel has been set up, which currently consists of eight ‘core’ pathologists. The aim of this study was to evaluate if other pathologists from the Dutch BO expert centres qualify for the expert panel by assessing their performance in 80 consecutive LGD reviews submitted to the panel.MethodsPathologists independently assessed digital slides in two phases. Both phases consisted of 40 cases, with a group discussion after phase I. For all cases, a previous consensus diagnosis made by five core pathologists was available, which was used as reference. The following criteria were used: (1) percentage of ‘indefinite for dysplasia’ diagnoses, (2) percentage agreement with consensus diagnosis and (3) proportion of cases with a consensus diagnosis of dysplasia underdiagnosed as non-dysplastic. Benchmarks were based on scores of the core pathologists.ResultsAfter phase I, 1/7 pathologists met the benchmark score for all quality criteria, yet three pathologists only marginally failed the agreement with consensus diagnosis (score 68.3%, benchmark 69%). After a group discussion and phase II, 5/6 remaining aspirant panel members qualified with all scores within the benchmark range.ConclusionsThe Dutch BO review panel now consists of 14 pathologists, who—after structured assessments and group discussions—can be considered homogeneous in their review of biopsies with LGD.

Endoscopic resection (ER) is an important diagnostic step in management of patients with early Barrett’s esophagus (BE) neoplasia. Based on ER specimens, an accurate histological diagnosis can be made, which guides further treatment. Based on depth of tumor invasion, differentiation grade, lymphovascular invasion, and margin status, the risk of lymph node metastases and local recurrence is judged to be low enough to justify endoscopic management, or high enough to warrant invasive surgical esophagectomy. Adequate assessment of these histological risk factors is therefore of the utmost importance. Aim of this study was to assess pathologist concordance on these histological features on ER specimens and evaluate causes of discrepancy. Of 62 challenging ER cases, one representative H&E slide and matching desmin and endothelial marker were digitalized and independently assessed by 13 dedicated GI pathologists from 8 Dutch BE expert centers, using an online assessment module. For each histological feature, concordance and discordance were calculated. Clinically relevant discordances were observed for all criteria. Grouping depth of invasion categories according to expanded endoscopic treatment criteria (T1a and T1sm1 vs. T1sm2/3), ≥1 pathologist was discrepant in 21% of cases, increasing to 45% when grouping diagnoses according to the traditional T1a versus T1b classification. For differentiation grade, lymphovascular invasion, and margin status, discordances were substantial with 27%, 42%, and 32% of cases having ≥1 discrepant pathologist, respectively. In conclusion, histological assessment of ER specimens of early BE cancer by dedicated GI pathologists shows significant discordances for all relevant histological features. We present propositions to improve definitions of diagnostic criteria.

Fluorescence molecular endoscopy (FME) is an emerging technique in the field of gastroenterology that holds potential to improve diagnosis and guide therapy, by serving as a 'red-flag' endoscopic imaging technique. Here, we investigated the safety, feasibility and optimal method of administration of EMI-137, targeting c-Met, during FME in Barrett's Esophagus (BE) and report several outcome parameters for early phase FME studies. Methods: FME was performed in 15 Barrett's neoplasia patients. EMI-137 was administered to three cohorts of five patients: 0.13 mg/kg intravenously (IV); 0.09 mg/kg IV or topically at a dose of 200 μg/cm BE (n=1) or 100 μg/cm BE (n=4). Fluorescence was visualized in vivo, quantified in vivo using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy and correlated to histopathology and immunohistochemistry. EMI-137 localization was assessed using fluorescence microscopy. Results: FME using different IV and topical doses of EMI-137 appeared to be safe and correctly identified 16/18 lesions, although modest target-to-background ratios were observed (median range of 1.12-1.50). C-Met overexpression varied between lesions, while physiological expression in the stomach-type epithelium was observed. Microscopically, EMI-137 accumulated around the neoplastic cell membranes. We identified several outcome parameters important for the validation of EMI-137 for FME: 1) the optimal administration route; 2) optimal dose and safety; 3) in vivo FME contrast; 4) quantification of intrinsic fluorescence; 5) ex vivo correlation of fluorescence, histopathology and target expression; and 6) microscopic tracer distribution. Conclusions: C-Met targeted FME using EMI-137 may not be the ideal combination to improve BE surveillance endoscopies, however the identified outcome parameters may serve as a valuable guidance for designing and performing future early phase clinical FME studies, independent of which fluorescent tracer is investigated.

Objectives To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. Methods Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2–5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. Results Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance (R2s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. Conclusions Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. Key Points • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

Fluorescence molecular endoscopy (FME) is an emerging technique that has the potential to improve the 22% colorectal polyp detection miss-rate. We determined the optimal dose-to-imaging interval and safety of FME using EMI-137, a c-Met-targeted fluorescent peptide, in a population at high risk for colorectal cancer. Methods: We performed in vivo FME and quantification of fluorescence by multidiameter single-fiber reflectance/single-fiber fluorescence spectroscopy in 15 patients with a dysplastic colorectal adenoma. EMI-137 was intravenously administered (0.13 mg/kg) at a 1-, 2- or 3-h dose-to-imaging interval (n = 3 patients per cohort). Two cohorts were expanded to 6 patients on the basis of target-to-background ratios. Fluorescence was correlated to histopathology and c-Met expression. EMI-137 binding specificity was assessed by fluorescence microscopy and in vitro experiments. Results: FME using EMI-137 appeared to be safe and well tolerated. All dose-to-imaging intervals showed significantly higher fluorescence in the colorectal lesions than in surrounding tissue, with a target-to-background ratio of 1.53, 1.66, and 1.74 for the 1-, 2-, and 3-h cohorts, respectively, and a mean intrinsic fluorescence of 0.035 vs. 0.023 mm-1 (P < 0.0003), 0.034 vs. 0.021 mm-1 (P < 0.0001), and 0.033 vs. 0.019 mm-1 (P < 0.0001), respectively. Fluorescence correlated with histopathology on a macroscopic and microscopic level, with significant c-Met overexpression in dysplastic mucosa. In vitro, a dose-dependent specific binding was confirmed. Conclusion: FME using EMI-137 appeared to be safe and feasible within a 1- to 3-h dose-to-imaging interval. No clinically significant differences were observed among the cohorts, although a 1-h dose-to-imaging interval was preferred from a clinical perspective. Future studies will investigate EMI-137 for improved colorectal polyp detection during screening colonoscopies.

Objective Neoadjuvant chemoradiotherapy followed by surgery establishes a considerable pathologic complete response (pCR) in EC. The aim was to determine site of residual tumor and its prognostic impact. Summary background data High rates of residual tumor in the adventitial region even inside the radiation fields will influence current decision-making. Methods We evaluated resection specimens with marked target fields from 151 consecutive EC patients treated with carboplatin/paclitaxel and 41.4Gy between 2009 and 2018. Results In radically resected (R0) specimens 19.8% (27/136) had a pCR (ypT0N0) and 14% nearly no response (tumor regression grade: tumor regression grade 4-5). Residual tumor commonly extended in or restricted to the adventitia (43.1%; 47/109), whereas 7.3% was in the mucosa (ypT1a), 16.5% in the submucosa (ypT1b) and 6.4% only in lymph nodes (ypT0N+). Macroscopic residues in R0-specimens of partial responders (tumor regression grade 2-3: N = 90) were found in- and outside the gross tumor volume (GTV) in 33.3% and 8.9%, and only microscopic in- and outside the clinical target volume in 58.9% and 1.1%, respectively. Residual nodal disease was observed proximally and distally to the clinical target volume in 2 and 5 patients, respectively. Disease Free Survival decreased significantly if macroscopic tumor was outside the GTV and in ypT2-4aN+. Conclusions After neoadjuvant chemoradiotherapy, pCR and ypT1aN0 were seen in a limited number of R0 resected specimens (19.8% and 7.3%, respectively), whereas 6.4% had only nodal disease (yT0N+). Disease Free Survival decreased significantly if macroscopic residue was outside the GTV and in responders with only nodal disease. Therefore, we should be cautious in applying wait and see strategies.

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2-3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 +/- 2.5 (mean SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.

Clinical significance of the pT4 category in colon cancer is increasing with several therapeutic implications. The aim of this study was to evaluate variability in diagnosing pT4a colon cancer. Twelve pathologists classified 66 preselected scanned Hematoxylin/Eosin-stained slides with tumor cells at a distance of 25–1500 μm (n = 22), 0–25 μm (n = 22), or on (n = 22) the peritoneal surface. Inter- and intraobserver variability were calculated using Kappa statistics. For interlaboratory variability, pathology reports of pT3 and pT4a colon cancer were extracted from the Dutch Pathology Registry between 2012 and 2015. The proportion of pT4a (pT4a/(pT3+pT4a)) was compared between 33 laboratories. Potential risk of understaging was assessed by determining the average number of blocks taken from pT3 and pT4a N0-2M0 tumors with metachronous peritoneal metastasis. Interobserver variability among 12 pathologists was 0.50 (95%CI 0.41–0.60; moderate agreement). Intraobserver variability (8 pathologists) was 0.71 (substantial agreement). A total of 7745 reports with pT3 or pT4aN0-2M0 colon cancer from 33 laboratories were included for interlaboratory analysis. Median percentage of pT4a was 15.5% (range 3.2–24.6%). After adjustment for case mix, 8 labs diagnosed pT4a significantly less or more frequently than the median lab. Metachronous peritoneal metastases were histologically verified in 170 of 6629 pT3 and in 129 of 1116 pT4a tumors, with a mean number of blocks of 4.03(SD 1.51) and 4.78 (SD 1.76) taken from the primary tumors, respectively (p

Background: Diagnostic screening of premalignant esophageal lesions is hampered by the absence of biomarkers indicative of metaplastic and/or malignant transformation. The aim of this exploratory study was to investigate the potential use of miRNAs as biomarkers capable of identifying patients with (pre)malignant lesions: Barrett’s esophagus (BE) metaplasia, high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). Methods: A total of 69 patients were included in the study. Six serum samples from each of four study groups, i.e., patients with normal squamous epithelium (SE), BE, HGD and EAC, were profiled using the Nanostring miRNA analysis platform. Differential miRNA expression patterns then were validated in 69 patient samples using qRT-PCR. Results: miRNA expression profiling revealed seven miRNAs with a 2-fold change in expression level. Validation by qRT-PCR confirmed that serum miR-320e levels were significantly decreased in the BE group compared to the SE (P≤0.001, AUC 0.790) and HGD groups (P≤0.005, AUC 0.786). Serum miR-199a-3p levels were significantly decreased in the BE group compared to the SE group (P≤0.001), area under the curve (AUC) of 0.813. Conclusions: The results of this study suggest that decreased serum miRNA levels of miR-199a-3p and miR-320e could help to identify patients with BE and HGD.

AIMS: Interobserver agreement for dysplasia in Barrett's oesophagus (BO) is low, and guidelines advise expert review of dysplastic cases. The aim of this study was to assess the added value of p53 immunohistochemistry (IHC) for the homogeneity within a group of dedicated gastrointestinal (GI) pathologists.METHODS AND RESULTS: Sixty-single haematoxylin and eosin (HE) slide referral BO cases [20 low-grade dysplasia (LGD); 20 high-grade dysplasia (HGD); and 20 non-dysplastic BO reference cases] were digitalised and independently assessed twice in random order by 10 dedicated GI pathologists. After a 'wash-out' period, cases were reassessed with the addition of a corresponding p53 IHC slide. Outcomes were: (i) proportion of 'indefinite for dysplasia' (IND) diagnoses; (ii) interobserver agreement; and (iii) diagnostic accuracy as compared with a consensus 'gold standard' diagnosis defined at an earlier stage by five core expert BO pathologists after their assessment of this case set. Addition of p53 IHC decreased the mean proportion of IND diagnoses from 10 of 60 to eight of 60 (P = 0.071). Mean interobserver agreement increased significantly from 0.45 to 0.57 (P = 0.0021). The mean diagnostic accuracy increased significantly from 72% to 82% (P = 0.0072) after p53 IHC addition.CONCLUSION: Addition of p53 IHC significantly improves the histological assessment of BO biopsies, even within a group of dedicated GI pathologists. It decreases the proportion of IND diagnoses, and increases interobserver agreement and diagnostic accuracy. This justifies the use of accessory p53 IHC within our upcoming national digital review panel for BO biopsy cases.

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( < 3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.

Quantitative fluorescence endoscopy (QFE) is a new technique that can visualise and quantify fluorescently tagged tumour tissue. In 25 patients with locally advanced rectal cancer (LARC), we evaluated QFE targeting vascular endothelial growth factor A (VEGFA) to detect residual tumour after neoadjuvant chemoradiotherapy (nCRT). QFE detected significantly higher fluorescence in tumour compared with normal rectal tissue and fibrosis, and improved prediction of final pathology results in 16% of patients compared with standard MRI and white-light endoscopy. QFE is a promising technique to aid clinical response assessment in patients with LARC and warrants further validation in larger clinical trials. ClinicalTrials.gov (NCT01972373). Patients with LARC receive nCRT followed by surgery to achieve local disease control. Interestingly, 15%–27% of patients have a pathological complete response, that is, no residual cancer cells are found in the surgical specimen.1–3 There is an increasing interest in identifying patients with a clinical complete response before surgery, as non-operative management for these patients is associated with high survival rates, reduced morbidity and improved functional outcomes.4–8 However, assessing tumour response after nCRT is challenging. White-light endoscopy provides only morphological information, while MRI cannot always distinguish viable tumour from fibrosis.9–11 QFE is a novel endoscopy technique that visualises and quantitatively measures the presence of targeted fluorescence tracers in tissue. We hypothesised that VEGFA-targeted QFE can be of additional value in restaging patients with LARC. In untreated patients, QFE showed clearly enhanced fluorescence in all rectal tumours compared with normal rectal tissue (figure 1A). The tumour-to-normal ratio of 3.1 (figure 1B) signifies QFE can be used to localise rectal cancer. Figure 1 (A) Representative fluorescence images of the quantitative fluorescence endoscopy (QFE) procedure in untreated rectal cancer. From left to right: a high-definition white-light video endoscope image; a white-light image from the QFE fibreoptic, followed by the corresponding near-infrared …

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24− EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.

The incidence of oesophageal adenocarcinoma (EAC) is steeply rising. Early lesion detection is a critical factor for improving disease prognosis. We developed and investigated wide-field near-infrared fluorescence molecular endoscopy (NIR-FME), using systemic and topical administration of a fluorescence-labelled antibody against vascular endothelial growth factor (VEGFA). Fourteen patients with Barrett’s oesophagus (BE) underwent endoscopic mucosal resection (EMR) combined with NIR-MFE. From a total of 20 confirmed aberrant lesions identified with NIR-FME, 4 lesions were missed by high-definition (HD) narrowband imaging (NBI) and white-light endoscopy (WLE). This overall 25% detection enhancement advocates NIR-FME as a promising ‘red-flag’ technique for improving early oesophageal lesion detection. ClinicalTrials.gov ID NCT02129933. Worldwide, >450 000 people are diagnosed with oesophageal cancer each year. In the western world, at least 80% of these cancers concern EAC.1–4 Late-stage disease detection challenges the efficacy of therapies, resulting in 400 000 deaths each year. Therefore, there is a pressing need for new endoscopic techniques that enable early EAC detection. Endoscopic imaging of targeted fluorescent agents directed against cancer-specific cellular and subcellular moieties, that is, fluorescence molecular endoscopy (FME), has been heralded as a novel method for oesophageal dysplasia and cancer detection.5 FME enables surface and subsurface visualisation of cancer-specific pathophysiology, going beyond the surface-only morphology and tissue discolourations visible with HD-WLE.6–10 In this study, we investigated for the first time real-time NIR-FME of EAC and dysplasia. A pilot study in patients with BE was based on a novel custom-built NIR fluorescence endoscope, paired to a HD-WL clinical scope (online supplementary figure 1) and employed the anti-VEGFA antibody bevacizumab, labelled with the NIR-fluorescent 800CW. The potential of VEGFA as an early discriminating target in BE was first confirmed by immunohistochemistry, demonstrating that all dysplastic tissues expressed VEGFA (online supplementary figure 2). ### Supplementary file 1 [SP1.pdf] The study furthermore compared systemic versus topical tracer administration during clinical inspection of …

Background: Dysplasia assessment of Barrett's esophagus biopsies is associated with low observer agreement; guidelines advise expert review. We have developed a web-based review panel for dysplastic Barrett's esophagus biopsies.Objective: The purpose of this study was to test if 10 gastrointestinal pathologists working at Dutch Barrett's esophagus expert centres met pre-set benchmark scores for quality criteria.Methods: Ten gastrointestinal pathologists twice assessed 60 digitalized Barrett's esophagus cases, enriched for dysplasia; then randomised (7520 assessments). We tested predefined benchmark quality criteria: (a) percentage of 'indefinite for dysplasia' diagnoses, benchmark score ≤14% for all cases, ≤16% for dysplastic subset, (b) intra-observer agreement; benchmark score ≥0.66/≥0.39, (c) percentage agreement with 'gold standard diagnosis'; benchmark score ≥82%/≥73%, (d) proportion of cases with high-grade dysplasia underdiagnosed as non-dysplastic Barrett's esophagus; benchmark score ≤1/78 (≤1.28%) assessments for dysplastic subset.Results: Gastrointestinal pathologists had seven years' Barrett's esophagus-experience, handling seven Barrett's esophagus-cases weekly. Three met stringent benchmark scores; all cases and dysplastic subset, three met extended benchmark scores. Four pathologists lacked one quality criterion to meet benchmark scores.Conclusion: Predefined benchmark scores for expert assessment of Barrett's esophagus dysplasia biopsies are stringent and met by some gastrointestinal pathologists. The majority of assessors however, only showed limited deviation from benchmark scores. We expect further training with group discussions will lead to adherence of all participating gastrointestinal pathologists to quality criteria, and therefore eligible to join the review panel.

Contains fulltext : 170986pub.pdf (Publisher’s version ) (Closed access) Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)-targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. METHODS: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples-48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps-and tissue derived from patients with Lynch syndrome-78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116(luc) tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116(luc) tumors (2-5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. RESULTS: Immunohistochemistry showed high VEGF-A expression in 79%-96% and high EGFR expression in 51%-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. CONCLUSION: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red-flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

Background. Circumferential resection margins (CRM) for esophageal cancer (EC), defined by the College of American Pathologists (CAP; >0 mm) or the Royal College of Pathologists (RCP; >1 mm) as tumor-free (R0), are based on a surgery-alone approach. We evaluated the usefulness of both definitions in current practice with neoadjuvant chemoradiotherapy (nCRT).Methods. CRMs were measured in 209 patients (104 with nCRT) with locally advanced EC after transthoracic esophagectomy. Local recurrence and cancer related death were scored as events. Patients were followed for at least 2 years or until death. Prognostic factors (P Results. Independent prognostic factors (P Conclusions. nCRT affected the CRM cutoff values. After nCRT, the CRM R0 according to the CAP was only prognostic for 2-year LRFS. However, in the surgery-alone group, it was prognostic for both the 2-year DFS and LRFS.

Background and purpose: Neoadjuvant chemoradiotherapy (nCRT) improves survival in esophageal cancer (EC) patients, but the response to treatment is heterogeneous and little is known regarding prognostic and predictive markers in these patients. CD44, SOX2 and SHH have been implicated in resistance to CRT, possibly through an association with a cancer stem cell phenotype.Material and methods: 101 EC patients treated with nCRT and surgery were included. Sufficient pretreatment biopsy material was present in 71 patients, of which 53 patients were non-complete responders on nCRT (nCR). Protein expression was examined using immunohistochemistry (IHC). Prognostic factors were determined using Cox regression analysis for disease free survival (DFS) and cause specific survival (CSS) in the complete cohort, the pre-treatment biopsies group and post-treatment nCR group.Results: Low CD44 expression in the nCR group was an independent prognostic factor for both DFS and CSS (DFS HR 2.81, p = 0.002 and CSS HR 3.48, p = 0.002). Absent SOX2 expression in pretreatment biopsies was related to systemic recurrence (p = 0.029) while low,SHH in pretreatment biopsies was an independent prognostic factor for a poor DES (HR 2.27, p = 0.036). No relation between marker expression and response to nCRT was observed.Conclusions: Low expression of CD44 and SHH are associated with a poor survival outcome in EC patients treated with nCRT. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Metastatic rectal cancer patients could benefit from novel therapeutic approaches. The signaling network formed by chemokines and their receptors can promote metastasis and resistance to current anticancer treatments. This study assessed the expression of chemokine receptor 4 (CXCR4) and its ligand CXCL12 immuhistochemically in stage IV rectal tumors. Paraffin-embedded primary tumor collected before and after local radiotherapy and systemic treatment with bevacizumab, oxaliplatin and capecitabine was analyzed. Receptor and ligand expression was assessed in the cytoplasm and nucleus of tumor, stromal and normal rectal crypt cells. Baseline expression of CXCR4 and CXCL12 was correlated with patients' pathologic response to treatment. At diagnosis (n=46), 89% of the rectal tumors expressed cytoplasmic CXCR4 and 81% CXCL12. Nuclear CXCR4 expression in tumor cells was present in 30% and nuclear CXCL12 expression in 35% of the tumors. After radiochemotherapy and administration of bevacizumab, nuclear CXCL12 expression was observed in 79% of residual tumors, as compared to 31% of the paired tumor samples expressing nuclear CXCL12 before treatment (P=0.001). There were no differences in CXCR4 or CXCL12 expression at baseline between the patients who had (n=9) and did not have (n=30) a pathologic complete response. Our results show that CXCR4 and CXCL12 are extensively expressed in primary rectal tumors of patients presenting with metastatic disease, while radiochemotherapy and bevacizumab further upregulate CXCL12 expression. These data indicate the importance of the CXCR4/CXCL12 axis in rectal tumor biology, and may suggest the CXCR4/CXCL12 receptor-ligand pair as a potential therapeutic target in metastatic rectal cancer.

Background: Barrett's oesophagus can progress towards oesophageal adenocarcinoma through a metaplasia-dysplasia-carcinoma sequence, but the underlying mechanisms are poorly understood. The transcription factor GATA6 is known to be involved in columnar differentiation and proliferation, and GATA6 gene amplification was recently linked with poor survival in oesophageal adenocarcinoma.Aim: To study the expression of GATA6 during Barrett's oesophagus development and malignant transformation. To determine the prognostic value of GATA6 in oesophageal adenocarcinoma.Methods: Two retrospective cohorts were derived from the pathological archive of the University Medical Center Groningen. The first cohort contained 130 tissue samples of normal squamous epithelium, metaplasia, dysplasia and oesophageal adenocarcinoma. The second cohort consisted of a tissue microarray containing tissue from 92 oesophageal adenocarcinoma patients. Immunohistochemistry was used to examine GATA6 protein expression and to correlate GATA6 expression in oesophageal adenocarcinoma with overall and disease-free survival.Results: The percentage of GATA6-positive cells was low in squamous epithelium (10%) but increased progressively in Barrett's oesophagus (30%, P Conclusion: GATA6 expression is progressively increased during Barrett's oesophagus development and its malignant transformation. However, no prognostic value of GATA6 expression could be found in oesophageal adenocarcinoma. (C) 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Background. It has been suggested that markers associated with cancer stem cells (CSC) may play a role in esophageal cancer. Our aim was to investigate the expression pattern of proposed CSC markers ALDH1, Axin2, BMI1, CD44, and SOX2 in esophageal adenocarcinoma (EAC) and to relate their expression to survival.Methods. In this study we included 94 EAC patients and examined the expression of the above-mentioned markers by using immunohistochemistry on tissue microarrays. Expression was scored as positive or negative or categorized as low or high in terms of an immunoreactivity score (IRS). Expression rates were related to clinicopathologic characteristics and overall and disease-free survival (DFS).Results. In a multivariate analysis, negative expression of CD44 and of SOX2 were both significant prognostic factors for DFS [hazard ratio (HR), 1.73; 95 % confidence interval (CI), 1.00-2.96; P = 0.046 and HR, 2.06; 95 % CI 1.14-3.70 P = 0.016). When CD44 and SOX2 expression were analyzed together, negative SOX2 expression was an independent prognostic factor for DFS (HR, 1.91; 95 % CI 1.05-3.46; P = 0.034). Low IRS scores for ALDH1 or Axin2 were associated with a reduced median survival (12.8 vs. 28.7 and 12.1 vs. 25.5 months, respectively). However, these markers and BMI1 were not prognostic factors for survival.Conclusions. Loss of CD44 expression and loss of SOX2 expression are prognostic factors of poor survival in EAC patients. This suggests a role of these proteins in EAC that requires further investigation.

Purpose/Objective(s): The aim of this study was to analyze the accuracy of gross tumor volume (GTV) delineation and clinical target volume (CTV) margins for neoadjuvant chemoradiation therapy (neo-CRT) in esophageal carcinoma at pathologic examination and to determine the impact on survival.Methods and Materials: The study population consisted of 63 esophageal cancer patients treated with neo-CRT. GTV and CTV borders were demarcated in situ during surgery on the esophagus, using anatomical reference points to provide accurate information regarding tumor location at pathologic evaluation. To identify prognostic factors for disease-free survival (DFS) and overall survival (OS), a Cox regression analysis was performed.Results: After resection, macroscopic residual tumor was found outside the GTV in 7 patients (11%). Microscopic residual tumor was located outside the CTV in 9 patients (14%). The median follow-up was 15.6 months. With multivariate analysis, only microscopic tumor outside the CTV (hazard ratio [HR], 4.96; 95% confidence interval [CI], 1.03-15.36), and perineural growth (HR, 5.77; 95% CI, 1.27-26.13) were identified as independent prognostic factors for OS. The 1-year OS was 20% for patients with tumor outside the CTV and 86% for those without (PConclusions: Microscopic tumor outside the CTV is associated with markedly worse OS after neo-CRT. This may either stress the importance of accurate tumor delineation or reflect aggressive tumor behavior requiring new adjuvant treatment modalities. (C) 2014 Elsevier Inc.

Ulcerative colitis (UC) is an inflammatory disease of the colon. Involvement of the small bowel is limited to backwash ileitis or pouch-related conditions. Here, we report two men with UC who presented with small bowel inflammation and even perforation, within 1 month after subtotal colectomy. Endoscopy showed diffuse enteritis. Histology showed marked apoptosis of epithelial cells in both cases. One patient responded to steroids and the other to a calcineurin inhibitor. Both patients had no evidence of Crohn's disease in the small intestine before this event. Several more cases of small intestinal lesions in patients with well-established UC have been reported. The majority typically presented shortly after colectomy and responded well to steroids. The pathogenesis of this enteritis is unknown, but seems to be distinct from Crohn's disease and may be associated with UC and colectomy.

Background Lymph node status is the most important predictive factor in colorectal carcinoma. Recurrences occur in 20% of the patients without lymph node metastases. The sentinel lymph node (SLN) biopsy is a tool to facilitate identification of micrometastatic disease and aberrant lymphatic drainage. We studied the feasibility of in vivo SLN detection in a multi-centre setting and evaluated nodal micro-staging using immunohistochemistry (IHC).Materials and Methods Sub-serosal injection with Patent Blue dye was used in the SLN procedure in 69 patients operated for localized colon cancer in six Dutch hospitals. Each SLN was examined with routine haematoxylin-eosin staining. In tumour-negative SLNs, we performed CK7/8 or 18 IHC.Results The procedure was successful in 67 of 69 patients (97%). The SLN was negative in 43 patients. In three cases, it was false negative, resulting in a negative predictive value of 93% and an accuracy of 96%. In 24 of 27 patients with lymph node metastases in a successful SLN procedure, the SLN was positive (sensitivity 89%). In 15 patients, the SLN was the only positive node (21%). In nine patients, we only found micrometastases or isolated tumour cells, resulting in 18% upstaging. Aberrant lymphatic drainage was seen in three patients (4%).Conclusions The SLN procedure in localized colon carcinoma is reliable in a multi-centre setting. It is helpful to identify patients who would be classified as stage II with conventional staging (18%) and who might benefit from adjuvant treatment.

ADAM metallopeptidase domain 17 (ADAM17) is responsible for processing large numbers of proteins. Recently, 1 family involving 2 patients with a homozygous mutation in ADAM17 were described, presenting with skin lesions and diarrhea. In this report, we describe a second family confirming the existence of this syndrome. The proband presented with severe diarrhea, skin rash, and recurrent sepsis, eventually leading to her death at the age of 10 months. We performed exome sequencing and detailed pathological and immunological investigations. We identified a novel homozygous frameshift mutation in ADAM17 (NM_003183.4:c.308dupA) leading to a premature stop codon. CD4(+) and CD8(+) T-cell stimulation assays showed severely diminished tumor necrosis factor-α and interleukin-2 production. Skin biopsies indicated a focal neutrophilic infiltrate and spongiotic dermatitis. Interestingly, the patient developed unexplained systolic hypertension and nonspecific hepatitis with apoptosis. This report provides evidence for an important role of ADAM17 in human immunological response and underscores its multiorgan involvement.

OBJECTIVE: Despite the use of immunosuppressive drugs, recurrent and de novo inflammatory bowel disease (IBD) can develop after orthotopic liver transplantation (OLT). Cytomegalovirus (CMV) infection has been suggested to play a role in the pathogenesis of IBD. The aim of this study was to investigate the role of CMV infection in the development of IBD after OLT.MATERIAL AND METHODS: All 84 patients who underwent transplantation for primary sclerosing cholangitis (PSC) or autoimmune hepatitis (AIH) in our center between May 1987 and June 2002 and who survived the first year after transplantation were included in the study. Diagnosis of active CMV infection was made using the pp65-antigenemia assay.RESULTS: Thirty-one of the 84 patients (37%) had IBD prior to OLT. Eighteen patients (21%) experienced IBD after OLT, either as flare-up (n=12) or de novo (n=6), at a median of 1.4 years (range 0.3 to 6.3) after OLT. Forty-eight percent of all patients experienced CMV infection after OLT, at a median of 27 days (range 8 to 193). CMV infection was primary in half the patients. At 1, 3, and 5 years after OLT, active IBD-free survival without CMV infection was 91, 88, and 88%, respectively. With CMV infection these figures were 93, 82, and 67%. De novo IBD was seen only in those who had experienced a CMV infection (p=0.02).CONCLUSIONS: In patients transplanted for end-stage PSC or AIH, active IBD, especially de novo IBD, occurred more often in patients who experienced CMV infection in the postoperative period. This finding supports a pathogenic role for CMV in the development of IBD.

In 36 consecutive patients with a foregut carcinoid with extensive local tumor growth and liver metastases with a carcinoid syndrome, six patients had complaints of postprandial abdominal pain and attacks of subileus based on segmental intestinal ischemia. A diagnosis of abdominal angina was supported by a positive response to nitroglycerin in two and ischemia of the ileum demonstrated by angiography in two other patients. Complaints were reduced in all patients after surgery. Histopathology of the resected small bowel specimens showed elastic vascular sclerosis in three patients and ischemic changes in three other patients, confirming the clinical diagnosis. Resection of ischemic bowel can provide relief in patients with segmental intestinal ischemia caused by carcinoid-induced vascular sclerosis.

Patients with a history of sporadic adenomas have increased epithelial cell proliferative activity, an intermediate risk marker for colorectal cancer. Reduction of proliferation by dietary intervention may reflect a decreased colorectal cancer risk. To evaluate whether calcium or resistant starch could reduce proliferative activity throughout the colon, we performed a randomized controlled trial in 111 sporadic adenoma patients. Patients received two placebos, I g of calcium + placebo, or 30 g of amylomaize (19 g of resistant starch) + placebo. After 2 mo, biopsies were collected from the cecum, transverse and sigmoid colon, and rectum during colonoscopy. Epithelial cell proliferation was determined by dividing the number of 5-bromo-2-deoxyuridine-labeled nuclei by the total number of nuclei x 100 (labeling index, LI). LI of luminal, mid, and basal compartments was determined. Twenty-five patients dropped out. In the remaining 86 patients (28 treated with placebo, 30 with calcium + placebo, and 28 with resistant starch + placebo), no difference was observed in total LI, the LI of the three compartments, or the crypt length in the four areas of the colorectum. Colonic epithelial cell proliferative activity throughout the colon of sporadic adenoma patients is not affected by supplementation with 1 g of calcium or 19 g of resistant starch.

PURPOSE: Mutations in K-ras andTP53 genes are common in colorectal cancer. They affect biologic behavior and might influence chemotherapy susceptibility in these tumors. We investigated whether the survival of patients with Dukes C colon cancer treated with adjuvant chemotherapy is influenced by K-ras andTP53 mutations. METHODS: Mutation screening of the hot spots of the K-ras gene and of the evolutionarily conserved regions of theTP53 gene was performed by denaturing gradient gel electrophoresis technique in formalin-fixed paraffin-embedded specimens of 55 consecutive patients with Dukes C colon cancer treated with adjuvant 5-fluorouracil-based chemotherapy. The median follow-up was 47 (range, 32–66) months. RESULTS: Alterations in the mutation hot spots of K-ras were found at codon 12 (n=11) and 13 (n=4) in 15 of the 55 carcinomas (27 percent). No mutation was found at codon 61. Mutations of a probably causative nature in the evolutionarily conserved regions (exons 5–8) of theTP53 gene were found in 24 tumors (44 percent). K-ras andTP53 mutations were found equally in the group with recurrent disease (7/26 (26 percent) and 12/27 (44 percent), respectively) and in the group without recurrences (8/28 (24 percent) and 12/28 (43 percent), respectively). Cancer-specific survival did not differ significantly between patients with K-ras orTP53 or both mutated and nonmutated tumors, respectively (log-rank test: K-ras, P=0.72 andTP53, P=0.77; K-ras andTP53, P=0.8). Also, potentially aggressive K-ras codon 12 and 13 mutations had the same survival as tumors without these mutations (log-rank test;P=0.73). CONCLUSIONS: Patients with K-ras orTP53 or both mutated Dukes C colon tumors have the same survival as nonmutated tumors when treated with adjuvant chemotherapy. These data suggest that mutations in K-ras orTP53 alone are not prognostic indicators in patients with Dukes C colon cancer receiving adjuvant 5-Fluorouracil-based therapy.

Anthranoid laxatives are widely used laxatives of natural origin. Because of their chemical structure they are carried unabsorbed to the large bowel, where metabolism to the active aglycones takes place. These aglycones exert their laxative effect by damaging epithelial cells, which leads directly and indirectly to changes in absorption, secretion and motility. Damaged epithelial cells can be found as apoptotic bodies in the pigmented colonic mucosa, characteristic for pseudomelanosis coli. Pseudomelanosis coli is a condition caused by chronic (ab)use of anthranoid laxatives and has recently been associated with an increased risk of colorectal carcinoma. In vitro and animal studies have shown a potential role of anthranoid laxatives in both the initiation and promotion of tumorigenesis. Studies in humans have also suggested tumour promoting activities for these laxatives. Although the short-term use of these substances is generally safe, long-term use cannot be recommended.

To determine whether the expression of transforming growth factor alpha (TGF-alpha), its receptor (epidermal growth factor receptor [EGFr]), p53 nuclear protein, and proliferation influences prognosis of patients with liver metastases, a study was performed in 45 liver metastases and 33 corresponding primary colorectal carcinomas in patients referred for liver surgery. The expression of TGF-alpha, EGFr, p53 nuclear protein, and proliferation rate was correlated with clinicopathological characteristics and survival after partial liver resection. In liver metastases, TGF-alpha expression was low in 42%, intermediate in 35%, and high in 23%. TGF-alpha expression was higher in liver metastases derived from lymph node-positive primary carcinomas, in synchronous and in irresectable liver metastases compared with those derived from lymph node-negative primary carcinomas, metachronous, and resectable liver metastases. Nuclear p53 expression was found in 83% of primary tumors and 71% of liver metastases. p53 expression did not correlate with the various clinicopathological characteristics. Ki67 expression was not associated with clinicopathological characteristics in primary and metastatic tumors. In the 38 patients in whom a partial liver resection was performed, median survival was 25 months in patients with a higher TGF-alpha expression in the metastasis than in the primary tumor and 60 months in patients with comparable or lower TGF-alpha expression in the metastasis than in the primary tumor (P = .036). Median survival after liver resection was 21 months in patients with p53-negative liver metastases and 58 months in patients with p53-positive metastases (P = .043). By multivariate analysis, p53 and EGFr expression on liver metastases were the best predictors of disease-free survival after partial liver resection, with relative risks of 2.38 and 3.33, respectively. In patients with colorectal liver metastases, referred for liver surgery, a higher TGF-alpha expression is associated with unfavorable tumor characteristics, whereas p53 and absence of EGFr expression is associated with a better survival after partial liver resection.

Between November 1987 and January 1996 pelvic exenteration for primary rectal cancer was carried out in 11 male patients. Two underwent a primary resection with subtotal cystectomy. In the remaining nine patients, the treatment commenced with a staging laparotomy and the fashioning of an end colostomy of the descending colon, followed by preoperative radiotherapy (50-56 Gy in 5 weeks). Total exenteration with uretero-ileo-cutaneostomy was carried out 4-6 weeks later. In two patients, the exenteration was performed despite the detection of hepatic metastases during the second laparotomy. The pathological staging was T4 in six and T3 in five patients. The immediate postoperative course was uneventful in eight patients and their hospital stay averaged 20 days. Serious postoperative complications prolonged the hospital stay of three patients. Three died during the first 6 months. Recurrent disease caused the death of three of the eight remaining patients: one locoregional (7.5 years after surgery), one from pre-existing hepatic metastases (18 months after surgery) and one from the combination of locoregional and distant recurrence (15 months after surgery). Four patients are on follow-up without evidence of disease, 96, 43, 23 and 22 months after surgery. One patient is alive 20 months after exenteration, with two pulmonary metastases having been recently excised. We conclude that, notwithstanding the morbidity rate, total pelvic exenteration is an acceptable option for advanced primary rectal cancer in male patients. Preoperative radiotherapy should be administered for cancers seated in the lower two-thirds of the rectum and perioperative chemotherapy should be considered seriously.

136 Background: The main objective of this study was to develop and validate a method to reconstruct the gross and clinical tumor volume (GTV and CTV) on the esophageal specimen in order to facilitate a good pathologic examination of the original tumor area after neo-adjuvant chemoradiation (CRT). Methods: The GTV and CTV borders of 25 patients were defined by a radiation oncologist on the planning CT in relation to 5 anatomical reference points. After CRT, the GTV and CTV borders were marked in vivo on the esophagus during surgical resection. Finally, the pathologist evaluated the presence of macroscopic and microscopic tumor in- and outside the GTV and CTV. The radiation tumor response was scored according to the standardized 5-tier Mandard classification. Radiation induced side effects were scored as well. Results: The Mandard classification could be scored on basis of the GTV alone in 68% of the cases (N=17). For the other patients (N=8), the GTV and the CTV should both be incorporated for correct evaluation of the tumor response. Five patients (20%) showed complete tumor response (Mandard 1), 68% (N=17) showed partial response (Mandard 2-3) and 12% (N=3) showed hardly any response (Mandard 4-5). In the partial responders, macroscopic tumor was found within the delineated GTV and microscopic tumor remained within the CTV both in 100% of the cases. In two patients (40%) with hardly any response, microscopic tumor was also found outside the CTV. This might be caused by tumor growth during the neo-adjuvant treatment or by geographical miss. Nine patients turned out to have positive lymph nodes. On average 18 (range 8-30) lymph nodes were evaluated per patients. Giant cell reactions, lymphocyte infiltration, and fibrosis, which indicate tumor regression were seen in the CTV and GTV, and were most pronounced in the GTV. Conclusions: This study suggested that demarcation of the GTV and CTV on the esophagus in vivo is important for standardized pathologic evaluation of the esophagus after neo-adjuvant chemoradiation. Furthermore using this method we determined microscopic tumor outside the CTV in 40% of the cases (N=2) of the bad responders (Mandard 4-5), illustrating the importance of our method in this patient category.

Microvillus inclusion disease (MVID) is a rare congenital enteropathy associated with brush border atrophy and reduced expression of enzymes at the enterocytes' apical surface. MVID is associated with mutations in the MYO5B gene, which is expressed in all epithelial tissues. Whether organs other than the intestine are affected in MVID is unclear. We report 2 patients with MVID that developed renal Fanconi syndrome while receiving total parenteral nutrition. Renal Fanconi syndrome has been correlated to apical plasma membrane defects in kidney proximal tubular epithelial cells. The aim of the present study was to determine whether MYO5B mutations in these patients correlate with similar apical plasma membrane defects in renal tubular epithelial cells as observed in the intestine.: Biopsies from kidney, duodenum, ileum, jejunum, and colon of 2 patients with MVID carrying MYO5B mutations and of age-matched controls were fixed in paraffin and analyzed with immunohistochemistry and transmission electron microscopy.: Structural defects of the brush border and apical recycling endosome organization are observed in enterocytes of all of the segments of the small intestine and colon. MYO5B mutations in patients with MVID with renal Fanconi syndrome do not correlate with aberrant apical plasma membrane morphology or altered apical recycling endosome organization in renal tubular epithelial cells.: MYO5B mutations have divergent effects on the apical membrane system in kidney and intestinal epithelial cells. Epithelial defects presented in MVID are therefore likely triggered by intestine-specific factors, the identification of which may provide new targets and open avenues for the development of alternative therapeutic strategies to combat this devastating disease.

Background Bi-allelic germline mutations of one of the DNA mismatch repair genes, so far predominantly found in PMS2 , cause constitutional MMR-deficiency syndrome. This rare disorder is characterised by paediatric intestinal cancer and other malignancies. We report the clinical, immunohistochemical and genetic characterisation of four families with bi-allelic germline PMS2 mutations. We present an overview of the published gastrointestinal manifestations of CMMR-D syndrome and propose recommendations for gastro-intestinal screening. Methods and Results The first proband developed a cerebral angiosarcoma at age 2 and two colorectal adenomas at age 7. Genetic testing identified a complete PMS2 gene deletion and a frameshift c.736_741delinsTGTGTGTGAAG (p.Pro246CysfsX3) mutation. In the second family, both the proband and her brother had multiple intestinal adenomas, initially wrongly diagnosed as familial adenomatous polyposis. A splice site c.2174+1G>A, and a missense c.137G>T (p.Ser46Ile) mutation in PMS2 were identified. The third patient was diagnosed with multiple colorectal adenomas at age 11; he developed a high-grade dysplastic colorectal adenocarcinoma at age 21. Two intragenic PMS2 deletions were found. The fourth proband developed a cerebral anaplastic ganglioma at age 9 and a high-grade colerectal dysplastic adenoma at age 10 and carries a homozygous c.2174+1G>A mutation. Tumours of all patients showed microsatellite instability and/or loss of PMS2 expression. Conclusions Our findings show the association between bi-allelic germline PMS2 mutations and severe childhood-onset gastrointestinal manifestations, and support the notion that patients with early-onset gastrointestinal adenomas and cancer should be investigated for CMMR-D syndrome. We recommend yearly follow-up with colonoscopy from age 6 and simultaneous video-capsule small bowel enteroscopy from age 8.

PURPOSE: The impact of the reported number of lymph nodes at pathologic examination of colon specimens on survival was studied.METHODS: The data of 2,281 patients with localized colon cancer were retrospectively reviewed. The effect of tumor characteristics and surgical and pathologic factors on the number of lymph nodes and examined lymph node numbers on nodal status and survival were analyzed.RESULTS: The number of examined nodes increased with T stage, left-sided tumors, and mucinous morphology, but decreased with age. The proportion of node-positive patients increased with a larger number of nodes. A high number of examined nodes and high T stage affected nodal status. The five-year overall survival was 51.3 percent for node-positive patients vs. 68.2 percent for node-negative patients. Node-negative patients had a significantly higher five-year crude and relative survival when more lymph nodes were examined. This was not found for the node-positive group and for all patients combined.CONCLUSIONS: T stage, localization, and patient age were predictive for the number of nodes examined. A higher number of examined nodes was associated with an increase in node positivity. The survival benefit can be explained by stage migration. Eventually this may lead to an overall survival benefit, as more patients are classified as node-positive, and therefore will receive adjuvant therapy.

togenous metastases, most often in lungs, brain, and bones. We describe a patient with metastases of endometrial cancer in the small bowel. An 85−year−old female patient was ad− mitted with severe anemia and melena. She had endometrial cancer (Figo stage IIIB), 2 years previously, for which she un− derwent an operation and received adju− vant radiotherapy. She was admitted with severe anemia (Hb 3.8 mmol/L) and melena. Upper endoscopy did not reveal any abnormalities. Colonoscopy was nor− mal. Video capsule endoscopy was carried out. With this examination, two tumors were discovered with signs of recent hemor− rhage (adherent clot) (l" Figure 1). Dur− ing laparotomy, a large tumor in the small bowel was encountered. A large part of the small bowel was resected. An end−to− end enteroenterostomy was carried out. Histological examination showed a carci− nosarcoma with extensive angioinvasive growth. The immunohistochemical pic− ture was identical to the endometrial tu− mor, which was removed in 2004. As well as features of a carcinoma, signs of a sarcoma were also seen, compatible with tumor differentiation (l" Figure 2 a and l" 2 b). On the basis of the initial stage of the en− dometrial carcinoma (Figo IIIB), this pa− tient was at risk for developing metasta− ses. In this group of patients, the majority develops metastases within 3 years, most often in the peritoneal cavity, lungs, liver, bones, or brain. Intestinal metastases of endometrial can− cer have been described in the literature, but are rarely seen. Biegel et al. describe bleeding colonic metastases in a patient with endometrial cancer [1]. Metastases in the small bowel have also been de− scribed [2], sometimes requiring segmen− tal small−bowel resection because of bleeding complications [3]. The small−bowel metastases caused mas− sive bleeding and were diagnosed by means of videocapsule endoscopy. With the introduction of this diagnostic tool, access to the small bowel and diagnostic yield have improved significantly.

OBJECTIVE: It has been suggested that KRAS and TP53 mutated tumors might influence the phenotypic behavior of left- and right-sided colon tumors. We investigated the incidence of these mutations in left- and right-sided colon tumors and their possible influence on survival in a homogeneous group of patients with Dukes' C colon cancers.METHODS: The primary tumors of 55 patients with a sporadic Dukes' C colon cancer, all treated with adjuvant chemotherapy were analyzed for the presence of KRAS and TP53 mutations. Mutation detection of the KRAS and TP53 genes was performed on paraffin-embedded tumor material, using denaturating gradient gel electrophoresis. The 5-yr survival rates of KRAS and TP53 mutated tumors were analyzed regarding right-sided tumors (defined as tumors up to the splenic flexure) and left-sided tumors (defined as tumors from the splenic flexure to the rectosigmoid peritoneal reflection).RESULTS: KRAS mutations occurred more frequently in the right colon compared to the left colon (R = 38% (10/26); L = 10% (3/29); chi2 test: p = 0.014). KRAS mutations did not influence survival in patients with right-sided colon tumors. Patients with KRAS mutation-negative tumors in the right colon, however, had a significantly worse survival than patients with left-sided KRAS mutation-negative tumors (5-yr survival; R: 34% vs L: 65%, log-rank test: p = 0.007). TP53 mutations of a possible causative nature were found in 24 tumors (44%). Neither the incidence (R = 42% (11/26); L = 45% (13/29)) nor the survival of TP53 mutated tumors differed significantly between left- and right-sided tumors. Furthermore, survival of patients with TP53 mutation-negative tumors did not differ significantly between left- and right-sided tumors.CONCLUSIONS: There seems to be no difference in survival rate between patients with KRAS mutated and KRAS negative Dukes' C colon tumors; however, KRAS mutations are more frequently found in the right colon compared to the left colon. TP53 mutations do not have predominance for either side of the colon, and there are no differences in survival in patients with left-sided versus right-sided tumors. Patients with KRAS-nonmutated tumors in the right colon did have a worse survival compared to those with such tumors in the left colon. This suggests that other genetic factors may play a role in tumor genesis in this subgroup of patients.

26 Background: Barrett’s metaplasia is characterized by the presence of a columnar metaplastic epithelium in the esophagus. Barrett’s metaplasia can show malignant progression towards esophageal adenocarcinoma by a metaplasia-dysplasia-carcinoma sequence. The underlying mechanisms of Barrett’s metaplasia development and malignant progression are poorly understood. The transcription factor GATA-6 is known to be involved in columnar differentiation and carcinogenesis. GATA6 gene amplification was recently linked with aggressiveness in esophageal adenocarcinoma. Here, we studied GATA6 protein expression in normal squamous, metaplastic, dysplastic and esophageal adenocarcinoma tissues in order to identify a possible role of GATA-6 in the development and malignant progression of Barrett’s metaplasia. Methods: Samples of squamous epithelium (N=37), Barrett’s non-intestinal metaplasia (N=16), Barrett’s intestinal metaplasia (N=29), high-grade dysplasia (N= 39), in situ esophageal adenocarcinoma (N=29) and an esophageal adenocarcinoma tissue microarray (N=95) were stained with a polyclonal antibody against GATA6. Staining intensity was categorized as absent, weak, normal or strong. Scoring was performed by two independent observers and validated by a pathologist. Results: GATA6 expression was absent in squamous epithelium but expressed in all samples of Barrett’s metaplasia, preferentially in the lower half of the crypt. Expansion of GATA6-positive cells throughout the crypt was observed in high-grade dysplasia. While all cases of in situ esophageal adenocarcinoma showed GATA6 expression we observed complete loss of GATA6 expression in 17% of esophageal adenocarcinoma samples. Conclusions: GATA6 is absent in squamous epithelium but its expression increases along the metaplasia-dysplasia carcinoma sequence. GATA6 expression remains predominantly present in esophageal adenocarcinoma, however, its expression is lost in a subset of samples. Analysis of the relation between GATA6 expression and clinicopathological characteristics in esophageal adenocarcinoma is pending.

Purpose: We investigated the role of Tamm-Horsfall protein in interstitial cystitis.Materials and Methods: Urinary Tamm-Horsfall protein excretion was analyzed in interstitial cystitis patients and controls, and bladder biopsy specimens were stained immunohistochemically for Tamm-Horsfall protein.Results: Urinary Tamm-Horsfall protein levels in 28 women with interstitial cystitis were statistically significantly greater than those in 25 female controls. No positive staining for Tamm-Horsfall protein was demonstrated in the bladder tissue from 10 interstitial cystitis cases.Conclusions: The results support the notion that interstitial cystitis may have a systemic etiology. In addition, this assay might have clinical value in the diagnosis of interstitial cystitis.

Fourteen HBsAg-positive patients received a liver transplant in Groningen. Two were HBeAg-positive and 12 HBeAg-negative. No anti-HBs immunoglobulin was given at the time. Both HBeAg-positive and 9 of 12 of the HBeAg-negative patients became HBsAg-positive again after transplantation. Virus titers were tested in eight patients. Two HBeAg-negative patients were HBV-DNA-negative at transplantation and are still HBV-DNA-negative one-and-half-years after transplantation, both by the branched DNA hybridization technique and by PCR (cut-off values 0.7 x 10(6) and 10(3) HBV genomes/ml, respectively). One patient who had a low HBV-DNA titer at transplantation remained PCR-positive thereafter, but became HBsAg-negative. All other patients were HBV-DNA-positive and had a recurrence that rapidly led to high HBV titers. The liver histology was characterized by fibrosis and cirrhosis, centrilobular cholestasis and high expression of HBsAg and HBcAg, but with little inflammatory infiltrate. We conclude from these results that without anti-HBs immunoglobulin prophylaxis there is a high rate of HBV recurrence after transplantation. The current policy is that patients who test negative in the HBV-DNA dot-blot assay (< 10(7) genomes/ml) are transplantation candidates and are treated with high-dose anti-HBs immunoglobulin after transplantation. HBV-DNA-positive patients (> 10(7) genomes/ml) remain poor candidates for liver transplantation, even with anti-HBs immunoprophylaxis.