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Gastrointestinal (GI) cancers such as colorectal cancer (CRC), gastric cancer (GC), esophageal cancer (EG), pancreatic duct adenocarcinoma (PDAC) or hepatocellular cancer (HCC) belong to the most commonly diagnosed types of cancer and are among the most frequent causes of cancer related death worldwide. Most types of GI cancer develop in a stepwise fashion with the occurrence of various driver mutations during tumor progression. Understanding the precise function of mutations driving GI cancer development has been regarded as a prerequisite for an improved clinical management of GI malignancies. During recent years, CRISPR/Cas9 has developed into a powerful tool for genome editing in cancer research by knocking in and knocking out even multiple genes at the same time. Within this review, we discuss recent applications for CRISPR/Cas9-based genome editing in GI cancer research including CRC, GC, EG, PDAC and HCC. These applications include functional studies of candidate genes in cancer cell lines or organoids in vitro as well as in murine cancer models in vivo, library screening for the identification of previously unknown driver mutations and even gene therapy of GI cancers.

e16592 Background: Until 2018 sorafenib was the only first-line systemic treatment option for advanced hepatocellular carcinoma (HCC). Ten years later a new agent, called lenvatinib, showed equal efficiency in comparison to sorafenib. Currently sorafenib and lenvatinib are options for first line treatment with regorafenib, cabozantinib and ramucirumab (if alpha fetoprotein > 400 ng / ml) as second-line treatment choices. The first published results from Imbrave150-trial shows promising effects regarding the combination of atezolizumab and bevacizumab for first line treatment in advanced HCC. This highlights the question for reasonable sequence therapies. Yet, it has so not been demonstrated, if lenvatinib is an option for a treatment line beyond the second. Methods: We identified 5 patients with advanced HCC from our hospital, who had been treated with local ablative procedures as well as at least three different systemic therapies with still excellent clinical performance status before receiving lenvatinib. Retrospective analysis was performed to examine clinical and radiologicresponse rates as well as side effects. Results: 3/5 patients achieved regression in at least third treatment lines. One patient showed stable disease, and only one of these five patients showed a mixed response. The toxicity profile was heterogeneous. One patient developed a mild hand-foot syndrome. Another one evolved hypertension. Developing of proteinuria led to stop treatment in one patient. One patient showed psychiatric disorder, which was possibly associated with lenvatinib. Response was independent of the underlying cause of HCC. Conclusions: Lenvatinib is a promising agent for treating advanced HCC in a later treatment line and revealed a tolerable toxicity profile.

Molecular mechanisms specific to colitis-associated cancers have been poorly characterized. Using comparative whole-genome expression profiling, we observed differential expression of epiregulin (EREG) in mouse models of colitis-associated, but not sporadic, colorectal cancer. Similarly, EREG expression was significantly upregulated in cohorts of patients with colitis-associated cancer. Furthermore, tumor-associated fibroblasts were identified as a major source of EREG in colitis-associated neoplasms. Functional studies showed that Ereg-deficient mice, although more prone to colitis, were strongly protected from colitis-associated tumors. Serial endoscopic studies revealed that EREG promoted tumor growth rather than initiation. Additionally, we demonstrated that fibroblast-derived EREG requires ERK activation to induce proliferation of intestinal epithelial cells (IEC) and tumor development in vivo. To demonstrate the functional relevance of EREG-producing tumor-associated fibroblasts, we developed a novel system for adoptive transfer of these cells via mini-endoscopic local injection. It was found that transfer of EREG-producing, but not Ereg-deficient, fibroblasts from tumors significantly augmented growth of colitis-associated neoplasms in vivo. In conclusion, our data indicate that EREG and tumor-associated fibroblasts play a crucial role in controlling tumor growth in colitis-associated neoplasms.

Inflammation drives expression of VEGFR2, which is expressed on and drives growth of tumor cells in colitis-associated cancer., Whereas the inhibition of vascular endothelial growth factor (VEGF) has shown promising results in sporadic colon cancer, the role of VEGF signaling in colitis-associated cancer (CAC) has not been addressed. We found that, unlike sporadic colorectal cancer and control patients, patients with CAC show activated VEGFR2 on intestinal epithelial cells (IECs). We then explored the function of VEGFR2 in a murine model of colitis-associated colon cancer characterized by increased VEGFR2 expression. Epithelial cells in tumor tissue expressed VEGFR2 and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation. Blockade of VEGF function via soluble decoy receptors suppressed tumor development, inhibited tumor angiogenesis, and blocked tumor cell proliferation. Functional studies revealed that chronic inflammation leads to an up-regulation of VEGFR2 on IECs. Studies in conditional STAT3 mutant mice showed that VEGFR signaling requires STAT3 to promote epithelial cell proliferation and tumor growth in vivo. Thus, VEGFR-signaling acts as a direct growth factor for tumor cells in CAC, providing a molecular link between inflammation and the development of colon cancer.

Aim: Chronic inflammation severely increases the risk for tumor development as seen in patients with inflammatory bowel disease. Especially, pro-inflammatory cytokines such as IL-6 and TNFα released by immune cells promote tumor growth through direct and indirect effects on tumor cells. The molecular mechanisms regulating the tumor-promoting cytokine release from immune cells are still poorly understood. Recent data suggest a role for hypoxia and the activation of hypoxia-inducible factors (HIFs) as important for immune cell activation. In this study, we analyzed the functional relevance of the transcription factor HIF1 in immune cells for inflammation-associated tumor development. Material and methods: In order to analyze the role of hypoxia and HIF activation, wildtype mice were exposed to azoxymethane and dextran sodium sulfate (AOM+DSS) for the induction of colitis-associated tumor development. HIF1 activation in AOM+DSS induced tumors was evaluated using western blot analysis and immunohistochemistry (IHC) of HIF1α and qPCR of HIF1 target genes. IHC double staining for HIF1α and various immune cell markers was performed for the analysis of cell specific HIF1 activation. Subsequently, HIF1 α-fl/fl mice were crossbred with LysM-cre mice to generate mice with a deficiency of HIF1 in myeloid cells (HIF1 αΔMC) and exposed to the AOM+DSS model. Tumor growth was analyzed using mini-endoscopy at repeated time points. Immune infiltration and cytokine expression were compared between HIF1αΔMC and HIF1α-fl/fl mice. Results:Western blot analysis revealed a strong accumulation of HIF1α in AOM+DSS induced tumors of wildtype mice accompanied by an upregulation of HIF1 target genes. IHC showed a significant upregulation of HIF1 α in tumor infiltrating macrophages in comparison to other immune cell subtypes. In order to analyze the functional role of HIF1 in macrophages, HIF1αΔMCmice were exposed to the AOM+DSS model in comparison to HIF1α-fl/fl mice. Whereas intestinal inflammation and early tumor induction were comparable in HIF1αΔMC and HIF1α-fl/fl mice, tumor growth was attenuated in HIF1αΔMC mice upon endoscopy. IHC and qPCR showed a similar infiltration of HIF1αΔMC and HIF1α-fl/fl tumors with immune cells. However, the expression of tumorpromoting cytokines including TNFα and IL-6 was reduced in HIF1αΔMC tumors. Conclusion: In summary, our data propose an important role for hypoxia and HIF activation in myeloid cells during colitis-associated tumor development. Especially, HIF1 activation in macrophages seems to contribute to the expression of tumor promoting cytokines. In this regard, therapeutic strategies targeting hypoxia dependent pathways in immune cells could provide new options for the prevention of colitis-associated cancer in patients with inflammatory bowel disease.

with ploidy status and the effect of ploidy status on survival in patients with CRC-IBD are not well defined. Aim: To evaluate the association between DNA content in colon adenocarcinoma cells and clinicohistological risk factors as well as survival in patients with CRC-IBD. Method: Ploidy status of colon adenocarcinoma cells in 50 patients with CRCIBD (44 CRC in ulcerative colitis, 6 CRC in Crohn`s colitis) selected by matching the Norwegian Cancer Registry with IBD cohorts of three university hospitals in Oslo was measured by high-resolution image cytometry. Clinicohistological factors as previously described in this cohort were included. The association of clinicohistological factors and ploidy status were analyzed by non-parametric tests (Mann-Whitney) and the effect of ploidy status on survival by an adjusted Cox regression model. Results: 18/50 (36 %) patients with CRC-IBD showed euploid (13 diploid, 5 tetraploid) and 32/50 (64%) patients aneuploid tumors. Fifteen patients died of CRC related causes, four patients of CRC unrelated causes, four of unknown cause and 27 patients were alive at the end of the study. Adjusted for age at diagnosis of CRC and stage of CRC (stage 1,2,3 versus stage 4), patients with aneuploid CRC showed poorer overall survival compared to patients with euploid CRC (OR=3.275, 95%CI: 1.07-10.04, p=0.038), and, as a tendency, poorer CRC-specific survival (OR=3.252, 95%CI: 0.86-12.27,p=0.082).Median age at diagnosis of IBDwas higher in euploid compared to aneuploid CRC-IBD patients (30y and 24y, respectively, p=0.045). Duration and type of IBD, active colonic inflammation and extent of inflammation at diagnosis of CRC, medication (5ASA compounds), concomitant primary sclerosing cholangitis, localization of the tumor in the colon (right versus left), the extent of neoplasia in the colon (multifocal versus unifocal), type and differentiation grade of adenocarcinomas were not associated with ploidy status, nor did they influence the effect of the ploidy status on survival. Conclusion: DNA content in colon adenocarcinomas measured by image cytometry is an independent predictor of prognosis in patients with CRC-IBD in our cohort. Patients with aneuploid CRC-IBD showed poorer survival than patients with diploid CRC. We could not identify clinical or histological risk factors for aneuploid CRC in our material.