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In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single‐cell transcriptome analysis of tumors and matched non‐cancerous tissues of twelve colorectal cancer patients. We defined patient‐overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen‐activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient‐derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen‐activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR‐BRAF‐MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non‐genetic cancer cell heterogeneity and re‐routing of trajectories as a response to targeted therapy. SYNOPSIS Colorectal cancer (CRC) cells can adopt a range of transcriptomic states. This study uses single cell RNA sequencing of primary CRC tissue and organoids to identify patient‐overarching CRC cell transcriptome clusters. RNA metabolic labelling indicates preferred CRC cell developmental trajectories. CRC cells of multiple patients clustered into six groups – termed TC1‐4, Goblet‐like, and stem‐like – characterized by differential transcriptional footprints of oncogenic signaling pathways. CRC organoid cells develop along a decreasing MAPK gradient. Experimental targeting of EGFR‐MAPK in CRC organoids re‐routes developmental trajectories. Clinically relevant inhibition of EGFR‐MAPK can result in preferential CRC cell development towards endpoints expressing high levels of stem cell markers. Graphical Abstract Colorectal cancer (CRC) cells can adopt a range of transcriptomic states. This study uses single cell RNA sequencing of primary CRC tissue and organoids to identify patient‐overarching CRC cell transcriptome clusters. RNA metabolic labelling indicates preferred CRC cell developmental trajectories.

Background This meta-analysis sought to evaluate the potential benefits and harms of laparoscopic gastrectomy with D2 lymphadenectomy for locally advanced gastric cancer versus open surgery. Methods A comprehensive search for randomized controlled studies that compared laparoscopic versus open gastrectomy with D2 lymphadenectomy for locally advanced gastric cancer published until December 31, 2018, was conducted. Operative outcomes, early postoperative outcomes, and long-term results were analyzed using a random effects model. Results Five randomized controlled trials containing a collective total of 2157 patients were included. In comparison with open surgery, laparoscopic gastrectomy for locally advanced gastric cancer showed similar risks of short-term mortality and serious adverse events within 30 days after surgery. Regarding intraoperative outcomes, operative time was increased for the laparoscopic approach, whereas the estimated intraoperative blood loss tended to be less. However, the amount of evidence was low for most outcomes. In addition, the results for the length of hospital stay and time to first flatus did not show statistically significant differences. The number of harvested lymph nodes and compliance with D2 lymphadenectomy did not significantly differ between the two groups, indicating oncological equivalence of both approaches. However, long-term oncological results could not be evaluated due to a lack of relevant data in four of the trials. Conclusion Laparoscopic gastrectomy with D2 lymphadenectomy can be performed with equivalent overall short-term morbidity and mortality versus the open approach for locally advanced gastric cancer. However, further well-designed randomized controlled trials are necessary to assess the possible advantages and risks of the laparoscopic approach as well as the long-term results.

Background Lymph node staging of ductal adenocarcinoma of the pancreatic head (PDAC) by cross-sectional imaging is limited. The aim of this study was to determine the diagnostic accuracy of expanded criteria in nodal staging in PDAC patients. Methods Sixty-six patients with histologically confirmed PDAC that underwent primary surgery were included in this retrospective IRB-approved study. Cross-sectional imaging studies (CT and/or MRI) were evaluated by a radiologist blinded to histopathology. Number and size of lymph nodes were measured (short-axis diameter) and characterized in terms of expanded morphological criteria of border contour (spiculated, lobulated, and indistinct) and texture (homogeneous or inhomogeneous). Sensitivities and specificities were calculated with histopathology as a reference standard. Results Forty-eight of 66 patients (80%) had histologically confirmed lymph node metastases (pN+). Sensitivity, specificity, and Youden’s Index for the criterion “size” were 44.2%, 82.4%, and 0.27; for “inhomogeneous signal intensity” 25.6%, 94.1%, and 0.20; and for “border contour” 62.7%, 52.9%, and 0.16, respectively. There was a significant association between the number of visible lymph nodes on preoperative CT and lymph node involvement (pN+, p = 0.031). Conclusion Lymph node staging in PDAC is mainly limited due to low sensitivity for detection of metastatic disease. Using expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivity remaining low. Combining specific criteria yields improved sensitivity with specificity and PPV remaining high.

Background Different tumor-specific prognostic factors have been identified in recent years for patients who undergo surgery due to pancreatic head cancer, but the results often were inconsistent. Furthermore, the impact of postoperative complications on patient long-term survival has not been described. Methods The long-term outcome of 428 patients who underwent resection of pancreatic head cancer at our center during a 17-year period was evaluated. Perioperative details, including postoperative complications, as well as the follow-up of all patients who left the hospital postoperatively were collected in a prospective database. Univariate and multivariate models were used to identify potential prognostic factors and to evaluate the impact of postoperative complications on long-term survival. Results The median survival was 15.5 months with a postoperative complication rate (grade I–IV) of 32.7%. Independent prognostic significance was detected for grading ( P  < 0.001), R status ( P  = 0.001), and lymph node status ( P  = 0.003). The occurrence of severe postoperative complications (grade III–IV) was associated with a significantly shortened survival (16.5 vs. 12.4 months; P  = 0.002) and was identified as an independent prognostic factor ( P  = 0.002). Conclusions This large study demonstrates that severe postoperative complications have a strong impact on the long-term survival of patients with pancreatic head cancer comparable to tumor characteristics, such as lymph node status, grading, or R status. As a result, the improvement of surgical procedures in specialized centers might lead to a survival benefit in these patients.

Crohn’s disease (CD) is associated with changes in the microbiome. The role of these changes and their precise association with disease course and activity remain ambiguous. In this prospective single-center study, the mucosal microbiome of surgical CD and non-CD patients was compared at the time of surgery. Microbial analyses were individually performed for ileal and colonic tissue samples obtained during surgery using 16S-rRNA-gene amplicon sequencing. Three groups out of the 46 included patients were formed: 1) a study group of CD of patients who received ileocecal resection due to CD involvement (CD study, n=10); 2) a control group of non-CD of patients who received intestinal resection due to indications other than CD (non-CD control, n=27); and 3) a second control group of CD who underwent resection of the intestine not affected by CD (CD non-affected control, n=9). Species richness and Shannon diversity were not different between all formed groups and regions analyzed (p>0.05). Several significant taxonomic differences were seen at the phylum-, order-, and genus-levels between the formed groups, such as a decrease of Firmicutes (phylum-level) and an increase of Bacteroides and Escherichia/Shigella/Pseudescherichia (genus-level) in CD study – colon vs. the non-CD control – colon (p ≤ 0.05). The CD non-affected control presented the largest amount of differentially abundant taxa in comparison to the other groups. These results underline that CD is accompanied by changes in affected and non-affected intestinal regions compared to non-CD controls. This study contributes the mucosal microbiome of a well-defined subset of surgical CD patients without confounding aspects of the fecal microbiome or regional microbial differences to the existing literature.

Despite the success of elastography in grading hepatic fibrosis by stiffness related noninvasive markers the relationship between viscoelastic constants in the liver and tissue structure remains unclear. We therefore studied the mechanical properties of 16 human liver specimens with different degrees of fibrosis, inflammation and steatosis by wideband magnetic resonance elastography (MRE) and static indentation experiments providing the specimens׳ static Young׳s modulus (E), dynamic storage modulus (G′) and dynamic loss modulus (G″). A frequency-independent shear modulus μ and a powerlaw exponent α were obtained by fitting G′ and G″ using the two-parameter sprinpot model. The mechanical parameters were compared to the specimens׳ histology derived parameters such as degree of Fibrosis (F), inflammation score and fat score, amount of hydroxyproline (HYP) used for quantification of collagen, blood markers and presurgery in vivo function tests. The frequency averaged parameters G′, G″ and μ were significantly correlated with F (G′: R=0.762, G″: R=0.830; μ: R=0.744; all P<0.01) and HYP (G′: R=0.712; G″: R=0.720; μ: R=0.731; all P<0.01). The powerlaw exponent α displayed an inverse correlation with F (R=–0.590, P=0.034) and a trend of inverse correlation with HYP (R=–0.470, P=0.089). The static Young׳s modulus E was less correlated with F (R=0.587, P=0.022) and not sensitive to HYP. Although inflammation was highly correlated with F (R=0.773, P<0.001), no interaction was discernable between inflammation and mechanical parameters measured in this study. Other histological and blood markers as well as liver function test were correlated with neither F nor the measured mechanical parameters. In conclusion, viscoelastic constants measured by wideband MRE are highly sensitive to histologically proven fibrosis. Our results suggest that, in addition to the amount of connective tissue, subtle structural changes of the viscoelastic matrix determine the sensitivity of mechanical tissue properties to hepatic fibrosis.

Autopsy rates in Western countries consistently decline to an average of <5%, although clinical autopsies represent a reasonable tool for quality control in hospitals, medically and economically. Comparing pre- and postmortal diagnoses, diagnostic discrepancies as uncovered by clinical autopsies supply crucial information on how to improve clinical treatment. The study aimed at analyzing current diagnostic discrepancy rates, investigating their influencing factors and identifying risk profiles of patients that could be affected by a diagnostic discrepancy. Of all adult autopsy cases of the Charité Institute of Pathology from the years 1988, 1993, 1998, 2003 and 2008, the pre- and postmortal diagnoses and all demographic data were analyzed retrospectively. Based on power analysis, 1,800 cases were randomly selected to perform discrepancy classification (class I-VI) according to modified Goldman criteria. The rate of discrepancies in major diagnoses (class I) was 10.7% (95% CI: 7.7%-14.7%) in 2008 representing a reduction by 15.1%. Subgroup analysis revealed several influencing factors to significantly correlate with the discrepancy rate. Cardiovascular diseases had the highest frequency among class-I-discrepancies. Comparing the 1988-data of East- and West-Berlin, no significant differences were found in diagnostic discrepancies despite an autopsy rate differing by nearly 50%. A risk profile analysis visualized by intuitive heatmaps revealed a significantly high discrepancy rate in patients treated in low or intermediate care units at community hospitals. In this collective, patients with genitourinary/renal or infectious diseases were at particularly high risk. This is the current largest and most comprehensive study on diagnostic discrepancies worldwide. Our well-powered analysis revealed a significant rate of class-I-discrepancies indicating that autopsies are still of value. The identified risk profiles may aid both pathologists and clinicians to identify patients at increased risk for a discrepant diagnosis and possibly suboptimal treatment intra vitam.

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract.

Chemoresistance in pancreatic ductal adenocarcinoma (PDAC) frequently contributes to failure of systemic therapy. While the radiosensitizing properties of 5-fluorouracil (FU) are well known, it is unknown whether ionizing radiation (IR) sensitizes towards FU cytotoxicity. Here, we hypothesize that upregulation of thymidine phosphorylase (TP) by IR reverses FU chemoresistance in PDAC cells. The FU resistant variant of the human PDAC cell line AsPC-1 (FU-R) was used to determine the sensitizing effects of IR. Proliferation rates of FU sensitive parental (FU-S) and FU-R cells were determined by WST-1 assays after low (0.05 Gy) and intermediate dose (2.0 Gy) IR followed by FU treatment. TP protein expression in PDAC cells before and after IR was assessed by Western blot. To analyze the specificity of the FU sensitizing effect, TP was ablated by siRNA. FU-R cells showed a 2.7-fold increase of the half maximal inhibitory concentration, compared to FU-S parental cells. Further, FU-R cells showed a concomitant IR resistance towards both doses applied. When challenging both cell lines with FU after IR, FU-R cells had lower proliferation rates than FU-S cells, suggesting a reversal of chemoresistance by IR. This FU sensitizing effect was abolished when TP was blocked by anti-TP siRNA before IR. An increase of TP protein expression was seen after both IR doses. Our results suggest a TP dependent reversal of FU-chemoresistance in PDAC cells that is triggered by IR. Thus, induction of TP expression by low dose IR may be a therapeutic approach to potentially overcome FU chemoresistance in PDAC.

Background Several lines of evidence indicate that Sirt1, a class III histone deacetylase (HDAC) is implicated in the initiation and progression of malignancies and thus gained attraction as druggable target. Since data on the role of Sirt1 in pancreatic ductal adenocarcinoma (PDAC) are sparse, we investigated the expression profile and prognostic significance of Sirt1 in vivo as well as cellular effects of Sirt1 inhibition in vitro . Methods Sirt1 expression was analyzed by immunohistochemistry in a large cohort of PDACs and correlated with clinicopathological and survival data. Furthermore, we investigated the impact of overexpression and small molecule inhibition on Sirt1 in pancreatic cancer cell culture models including combinatorial treatment with chemotherapy and EGFR-inhibition. Cellular events were measured quantitatively in real time and corroborated by conventional readouts including FACS analysis and MTT assays. Results We detected nuclear Sirt1 expression in 36 (27.9%) of 129 PDACs. SIRT1 expression was significantly higher in poorly differentiated carcinomas. Strong SIRT1 expression was a significant predictor of poor survival both in univariate (p = 0.002) and multivariate (HR 1.65, p = 0.045) analysis. Accordingly, overexpression of Sirt1 led to increased cell viability, while small molecule inhibition led to a growth arrest in pancreatic cancer cells and impaired cell survival. This effect was even more pronounced in combinatorial regimens with gefitinib, but not in combination with gemcitabine. Conclusions Sirt1 is an independent prognosticator in PDACs and plays an important role in pancreatic cancer cell growth, which can be levered out by small molecule inhibition. Our data warrant further studies on SIRT1 as a novel chemotherapeutic target in PDAC.