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Histopathology plays a fundamental role in the diagnosis and subtyping of solid tumors and has become a cornerstone of modern precision oncology. Histopathological evaluation is typically performed manually by expert pathologists due to the complexity of visual data. However, in the last ten years, new artificial intelligence (AI) methods have made it possible to train computers to perform visual tasks with high performance, reaching similar levels as experts in some applications. In cancer histopathology, these AI tools could help automate repetitive tasks, making more efficient use of pathologists’ time. In research studies, AI methods have been shown to have an astounding ability to predict genetic alterations and identify prognostic and predictive biomarkers directly from routine tissue slides. Here, we give an overview of these recent applications of AI in computational pathology, focusing on new tools for cancer research that could be pivotal in identifying clinical biomarkers for better treatment decisions.

The journal of pathology (2022). doi:10.1002/path.5898, Published by Wiley, Bognor Regis [u.a.]

Different immunohistochemical programmed death-ligand 1 (PD-L1) assays and scorings have been reported to yield variable results in triple-negative breast cancer (TNBC). We compared the analytical concordance and reproducibility of four clinically relevant PD-L1 assays assessing immune cell (IC) score, tumor proportion score (TPS), and combined positive score (CPS) in TNBC. Primary TNBC resection specimens (n = 104) were stained for PD-L1 using VENTANA SP142, VENTANA SP263, DAKO 22C3, and DAKO 28–8. PD-L1 expression was scored according to guidelines on virtual whole slide images by four trained readers. The mean PD-L1 positivity at IC-score ≥1% and CPS ≥1 ranged between 53% and 75% with the highest positivity for SP263 and comparable levels for 22C3, 28–8, and SP142. Inter-assay agreement was good between 28–8 and 22C3 across all scores and cut-offs (kappa 0.68–0.74) and for both assays with SP142 at IC-score ≥1% and CPS ≥1 (kappa 0.61–0.67). The agreement between SP263 and all other assays was substantially lower for all scores. Inter-reader agreement for each assay was good to excellent for IC-score ≥1% (kappa 0.73–0.78) and CPS ≥1 (kappa 0.68–0.74), fair to good for CPS ≥10 (kappa 0.52–0.67) and TPS ≥1% (kappa 0.53–0.72). The percentage of overlapping cases in the positive/negative category was >90% between IC-score ≥1% and CPS ≥1 but below when comparing IC-score ≥1% with CPS ≥10. We demonstrate an overall good inter-reader agreement for all PD-L1 assays in TNBC along with assay specific differences in positivity and concordances, which may aid to select the right test strategy in routine diagnostics., Highlights • Different PD-L1 IHC assays and scorings may show variable results in TNBC. • Overall good assay concordance between SP142, 22C3, and 28–8 at IC-score 1%. • Overall good assay concordance between SP142, 22C3, and 28–8 at CPS 1. • SP142 is less optimal for CPS assessment at higher cut-offs. • SP263 assay is not interchangeable with the other three PD-L1 assays.

The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address this important issue, we analyzed the impact of heme iron (0.25 µmol/g diet) on the intestinal microbiota, gut inflammation and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 µmol/g diet) was used as reference. 16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.

Artificial Intelligence (AI) has a multitude of applications in cancer research and oncology. However, the training of AI systems is impeded by the limited availability of large datasets due to data protection requirements and other regulatory obstacles. Federated and swarm learning represent possible solutions to this problem by collaboratively training AI models while avoiding data transfer. However, in these decentralized methods, weight updates are still transferred to the aggregation server for merging the models. This leaves the possibility for a breach of data privacy, for example by model inversion or membership inference attacks by untrusted servers. Homomorphically encrypted federated learning (HEFL) is a solution to this problem because only encrypted weights are transferred, and model updates are performed in the encrypted space. Here, we demonstrate the first successful implementation of HEFL in a range of clinically relevant tasks in cancer image analysis on multicentric datasets in radiology and histopathology. We show that HEFL enables the training of AI models which outperform locally trained models and perform on par with models which are centrally trained. In the future, HEFL can enable multiple institutions to co-train AI models without forsaking data governance and without ever transmitting any decryptable data to untrusted servers.One Sentence SummaryFederated learning with homomorphic encryption enables multiple parties to securely co-train artificial intelligence models in pathology and radiology, reaching state-of-the-art performance with privacy guarantees.

Nature medicine 28(6), 1232-1239 (2022). doi:10.1038/s41591-022-01768-5, Published by Nature America Inc., New York, NY

Deep learning (DL) can predict microsatellite instability (MSI) from routine histopathology slides of colorectal cancer (CRC). However, it is unclear whether DL can also predict other biomarkers with high performance and whether DL predictions generalize to external patient populations. Here, we acquire CRC tissue samples from two large multi-centric studies. We systematically compare six different state-of-the-art DL architectures to predict biomarkers from pathology slides, including MSI and mutations in BRAF, KRAS, NRAS, and PIK3CA. Using a large external validation cohort to provide a realistic evaluation setting, we show that models using self-supervised, attention-based multiple-instance learning consistently outperform previous approaches while offering explainable visualizations of the indicative regions and morphologies. While the prediction of MSI and BRAF mutations reaches a clinical-grade performance, mutation prediction of PIK3CA, KRAS, and NRAS was clinically insufficient.

Background Pathological TNM staging (pTNM) is the strongest prognosticator in colorectal carcinoma (CRC) and the foundation of its post-operative clinical management. Tumours that invade pericolic/perirectal adipose tissue generally fall into the pT3 category without further subdivision. Methods The histological depth of invasion into the pericolic/perirectal fat was digitally and conventionally measured in a training cohort of 950 CRCs (Munich). We biostatistically calculated the optimal cut-off to stratify pT3 CRCs into novel pT3a (≤3 mm)/pT3b (>3 mm) subgroups, which were then validated in two independent cohorts (447 CRCs, Bayreuth/542 CRCs, Mainz). Results Compared to pT3a tumours, pT3b CRCs showed significantly worse disease-specific survival, including in pN0 vs pN+ and colonic vs. rectal cancers (DSS: P P Discussion The delineation of pT3a/pT3b subcategories of CRC based on the histological depth of adipose tissue invasion adds valuable prognostic information to the current pT3 classification and implementation into current staging practices of CRC should be considered.

Histopathology : journal of the British Division of the International Academy of Pathology 80(7), 1121-1127 (2022). doi:10.1111/his.14659, Published by Wiley-Blackwell, Oxford [u.a.]

INTRODUCTION: Immune checkpoint inhibitors (ICI) have been approved for patients with clear cell renal cell carcinoma (ccRCC), but not all patients benefit from ICI. One reason is the tumor microenvironment (TME) that has substantial influence on patient's prognosis and therapy response. Thus, we comprehensively analyzed the TME of ccRCC regarding prognostic and predictive properties. METHODS: Tumor-infiltrating CD3-positive T-cells, CD8-positive cytotoxic T-lymphocytes (CTLs), regulatory T-cells, B-cells, plasma cells, macrophages, granulocytes, programmed cell death receptor 1 (PD-1), and its ligand PD-L1 were examined in a large hospital-based series of ccRCC with long-term follow-up information (n = 756) and in another patient collective with information on response to nivolumab therapy (n = 8). Tissue microarray technique and digital image analysis were used. Relationship between immune cell infiltration and tumor characteristics, cancer-specific survival (CSS), or response to ICI was examined. RESULTS: Univariate survival analysis revealed that increased tumor-infiltrating B-cells, T-cells, and PD-1-positive cells were significantly associated with favorable CSS and high levels of intratumoral granulocytes, macrophages, cytotoxic T-cells, and PD-L1 significantly with poor CSS. High CTL or B-cell infiltration and high PD-L1 expression of ccRCC tumor cells qualified as independent prognostic biomarkers for patients' CSS. Significantly higher densities of intratumoral T-cells, CTLs, and PD-1-positive immune cells were observed in ccRCC with response to ICI compared with patients with mixed or no response (CD3: p = 0.003; CD8: p = 0.006; PD-1: p = 0.01). DISCUSSION: This study shows that subsets of tumor-infiltrating leukocytes in the TME and also PD-1/PD-L1 provide prognostic and predictive information for patients with ccRCC. Keywords: Renal cell carcinoma, Immune cell, Checkpoint inhibitor, Biomarker

The interpretation of digitized histopathology images has been transformed thanks to artificial intelligence (AI). End-to-end AI algorithms can infer high-level features directly from raw image data, extending the capabilities of human experts. In particular, AI can predict tumor subtypes, genetic mutations and gene expression directly from hematoxylin and eosin (H&E) stained pathology slides. However, existing end-to-end AI workflows are poorly standardized and not easily adaptable to new tasks. Here, we introduce DeepMed, a Python library for predicting any high-level attribute directly from histopathological whole slide images alone, or from images coupled with additional meta-data (https://github.com/KatherLab/deepmed). Unlike earlier computational pipelines, DeepMed is highly developer-friendly: its structure is modular and separates preprocessing, training, deployment, statistics, and visualization in such a way that any one of these processes can be altered without affecting the others. Also, DeepMed scales easily from local use on laptop computers to multi-GPU clusters in cloud computing services and therefore can be used for teaching, prototyping and for large-scale applications. Finally, DeepMed is user-friendly and allows researchers to easily test multiple hypotheses in a single dataset (via cross-validation) or in multiple datasets (via external validation). Here, we demonstrate and document DeepMed’s abilities to predict molecular alterations, histopathological subtypes and molecular features from routine histopathology images, using a large benchmark dataset which we release publicly. In summary, DeepMed is a fully integrated and broadly applicable end-to-end AI pipeline for the biomedical research community.

Background: Colorectal mixed adenoneuroendocrine carcinomas (MANECs) are clinically highly aggressive neoplasms. MANECs are composed of variable adenocarcinoma components combined with morphologically distinct neuroendocrine carcinoma components, which are confirmed by synaptophysin immunohistochemistry, the gold standard marker of a neuroendocrine differentiation. However, the biological behavior of adenocarcinomas that express synaptophysin but do not show a typical neuroendocrine morphology remains unclear. Methods: We investigated synaptophysin expression in 1002 conventional colorectal adenocarcinomas and correlated the results with clinicopathological characteristics and patient survival and compared the survival characteristics of synaptophysin expression groups to MANECs. Results: Synaptophysin expression in conventional colorectal adenocarcinomas was associated with a shortened disease-free survival (p = 0.037), but not with overall survival or disease-specific survival (DSS) in univariate analyses and without any survival impact in multivariate analyses. Patients with “true” MANECs, on the other hand, showed a significantly shorter survival than all conventional adenocarcinomas with or without synaptophysin expression in uni- and multivariate analyses (e.g., multivariate DSS: p &lt, 0.001, HR: 5.20). Conclusions: Our study demonstrates that synaptophysin expression in conventional colorectal adenocarcinomas, in contrast to MANECs, is not associated with a significantly poorer clinical outcome when compared to adenocarcinomas without synaptophysin expression. Furthermore, our data suggest that conventional adenocarcinomas with a diffuse synaptophysin expression should not be classified as MANECs, also strongly arguing that synaptophysin testing should be reserved for carcinomas with an H&amp, E morphology suggestive of a neuroendocrine differentiation.

Background Immunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin–eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters. Methods We evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups. Results CDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor. Conclusion CDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors.

Epidermal growth factor receptor (EGFR) as a prevalent oncogene regulates proliferation, apoptosis and differentiation and thereby contributes to carcinogenesis. Even though, the documentation on its clinical relevance is surprisingly heterogeneous in the scientific literature. Here, we systematically investigated the correlation of mRNA to survival time and pathological parameters by analyzing 30 datasets in silico. Furthermore, the prognostic value of membrane-bound, cytoplasmic (mcEGFR) and nuclear expression (nEGFR) of EGFR was experimentally analyzed by immunohistochemical staining of 502 biopsies from 27 tumor types. We found that protein expression of EGFR showed better prognostic efficiency compared to mRNA, and that mcEGFR expression was positively correlated with nEGFR expression (p < 0.001). Unexpectedly, both mcEGFR and nEGFR expression were associated with low T stage (p < 0.001 and p = 0.004; respectively). Moreover, positive mcEGFR was significantly related to high differentiation (p = 0.027). No significant correlation was found with any other pathological parameters. Collectively, our results imply that the oncogenic function of EGFR may be more related to nascent stages of carcinogenesis than to advanced and progressive tumors, which may as well explain at least partially the occurrence of secondary resistance against EGFR-directed therapy.

The 2019 World Health Organization (WHO) classification of colorectal carcinoma (CRC) profoundly reclassified CRC subtypes and introduces tumor budding as a second major grading criterion, while condensing conventional grade into a 2-tiered system. So far it remains largely unexplored how these parameters interact with each other and whether they truly have an independent impact on patient prognosis. We reclassified a large single-center cohort of 1004 CRCs spanning 2 decades for adjusted WHO grade (low vs. high), tumor budding (Bd1/Bd2/Bd3), and CRC subtype (adenocarcinoma not otherwise specified, micropapillary, mucinous, serrated, medullary, adenoma-like, signet-ring cell, mixed adenoneuroendocrine carcinoma/neuroendocrine carcinoma, undifferentiated) according to the criteria of the 2019 WHO classification. We investigated the interaction of these parameters, their connection to stage/microsatellite status, and their significance for patient survival in the different subgroups. Specific subtypes other than adenocarcinoma not otherwise specified represented one third of all CRCs and were unevenly distributed throughout stage and microsatellite subgroups. Subtypes, WHO grade and tumor budding profoundly impacted all survival parameters (P

ObjectiveBleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive.DesignWe used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis.ResultsErosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevatedPAD4expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding.ConclusionOur findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.

Background Muscle-invasive bladder cancer (MIBC) is the second most common genitourinary malignancy, and is associated with high morbidity and mortality. Recently, molecular subtypes of MIBC have been identified, which have important clinical implications. Objective In the current study, we tried to predict the molecular subtype of MIBC samples from conventional histomorphology alone using deep learning. Design, setting, and participants Two cohorts of patients with MIBC were used: (1) The Cancer Genome Atlas Urothelial Bladder Carcinoma dataset including 407 patients and (2) our own cohort including 16 patients with treatment-naive, primary resected MIBC. This resulted in a total of 423 digital whole slide images of tumor tissue to train, validate, and test the deep learning algorithm to predict the molecular subtype. Outcome measurements and statistical analysis Various accuracy measurements including the area under the receiver operating characteristic curves were used to evaluate the deep learning model. A sliding window approach to visualize classification was used. Class activation maps were used to identify image features that are most relevant to call a specific class. Results and limitations The deep learning model showed great performance in the prediction of the molecular subtype of MIBC patients from hematoxylin and eosin (HE) slides alone—similar to or better than pathology experts. Using different visualization techniques, we identified new histopathological features that were most relevant to our model. Conclusions Deep learning can be used to predict important molecular features in MIBC patients from HE slides alone, potentially improving the clinical management of this disease significantly. Patient summary In patients with bladder cancer, a computer program found changes in the appearance of tumor tissue under the microscope and used these to predict genetic alterations. This could potentially benefit patients.

Simple Summary The immunohistochemical analysis of Special AT-rich sequence-binding protein 2 (SATB2) is increasingly being used to detect colorectal differentiation. Our study aimed to investigate SATB2 expression levels and the prognostic relevance of SATB2 loss in colorectal carcinoma (CRC), especially in comparison with CDX2, the standard marker of colorectal differentiation. We tested SATB2 expression in 1039 CRCs and identified SATB2 as a strong prognosticator in the overall cohort as well as in specific subcohorts, including high-risk subgroups. Compared to CDX2, SATB2 showed a higher prognostic power but was lost at a much higher frequency, generally rendering SATB2 as the less sensitive marker for colorectal differentiation compared to CDX2. Abstract Background: Special AT-rich sequence-binding protein 2 (SATB2) has emerged as an alternative immunohistochemical marker to CDX2 for colorectal differentiation. However, the distribution and prognostic relevance of SATB2 expression in colorectal carcinoma (CRC) have to be further elucidated. Methods: SATB2 expression was analysed in 1039 CRCs and correlated with clinicopathological and morphological factors, CDX2 expression as well as survival parameters within the overall cohort and in clinicopathological subgroups. Results: SATB2 loss was a strong prognosticator in univariate analyses of the overall cohort (p < 0.001 for all survival comparisons) and in numerous subcohorts including high-risk scenarios (UICC stage III/high tumour budding). SATB2 retained its prognostic relevance in multivariate analyses of these high-risk scenarios (e.g., UICC stage III: DSS: p = 0.007, HR: 1.95), but not in the overall cohort (DSS: p = 0.1, HR: 1.25). SATB2 loss was more frequent than CDX2 loss (22.2% vs. 10.2%, p < 0.001) and of higher prognostic relevance with only moderate overlap between SATB2/CDX2 expression groups. Conclusions: SATB2 loss is able to identify especially aggressive CRCs in high-risk subgroups. While SATB2 is the prognostically superior immunohistochemical parameter compared to CDX2 in univariate analyses, it appears to be the less sensitive marker for colorectal differentiation as it is lost more frequently.

Background: In the treatment of clear cell renal cell carcinoma (ccRCC), nivolumab is an established component of the first-line therapy with a favorable impact on progression free survival and overall survival. However, treatment-related adverse effects occur and, to date, there is no approved predictive biomarker for patient stratification. Thus, the aim of this study was to establish an ex vivo tissue slice culture model of ccRCC and to elucidate the impact of nivolumab on tumor infiltrating immune cells. Methods: Fresh tumor tissue of ccRCC was treated with the immune checkpoint inhibitor nivolumab using ex vivo tissue slice culture (TSC). After cultivation, tissue slices were formalin-fixed, immunohistochemically stained and analyzed via digital image analysis. Results: The TSC model was shown to be suitable for ex vivo pharmacological experiments on intratumoral immune cells in ccRCC. PD1 expression on tumor infiltrating immune cells was dose-dependently reduced after nivolumab treatment (p &lt, 0.01), whereas density and proliferation of tumor infiltrating T-cells and cytotoxic T-cells were inter-individually altered with a remarkable variability. Tumor cell proliferation was not affected by nivolumab. Conclusions: This study could demonstrate nivolumab-dependent effects on PD1 expression and tumor infiltrating T-cells in TSC of ccRCC. This is in line with results from other scientific studies about changes in immune cell proliferation in peripheral blood in response to nivolumab. Thus, TSC of ccRCC could be a further step to personalized medicine in terms of testing the response of individual patients to nivolumab.

The appendix gives rise to goblet cell carcinoids, which represent special carcinomas with distinct biological and histological features. Their genetic background and molecular relationship to colorectal adenocarcinoma is largely unknown. We therefore performed a next-generation sequencing analysis of 25 appendiceal carcinomas including 11 goblet cell carcinoids, 7 adenocarcinomas ex-goblet cell carcinoid, and 7 primary colorectal-type adenocarcinomas, using a modified Colorectal Cancer specific Panel comprising 32 genes linked to colorectal and neuroendocrine tumorigenesis. The mutational profiles of these neoplasms were compared with those of conventional adenocarcinomas, mixed adenoneuroendocrine carcinomas, and neuroendocrine carcinomas of the colorectum. In addition, a large-scale pan-cancer sequencing panel covering 409 genes was applied to selected cases of goblet cell carcinoid/adenocarcinoma ex-goblet cell carcinoid (n=2, respectively). Mutations in colorectal cancer-related genes (eg, TP53, KRAS, APC) were rare to absent in both, goblet cell carcinoids and adenocarcinomas ex-goblet cell carcinoid, but frequent in primary colorectal-type adenocarcinomas of the appendix. Additional large-scale sequencing of selected goblet cell carcinoids and adenocarcinomas ex-goblet cell carcinoid revealed mutations in Wnt-signaling-associated genes (USP9X, NOTCH1, CTNNA1, CTNNB1, TRRAP). These data suggest that appendiceal goblet cell carcinoids and adenocarcinomas ex-goblet cell carcinoid constitute a morphomolecular entity, histologically and genetically distinct from appendiceal colorectal-type adenocarcinomas and its colorectal counterparts. Altered Wnt-signaling associated genes, apart from APC, may act as potential drivers of these neoplasms. The absence of KRAS/NRAS mutations might render some of these tumors eligible for anti-EGFR directed therapy regimens.Modern Pathology advance online publication, 12 January 2018; doi:10.1038/modpathol.2017.184.

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1−/−) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1−/− mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1−/− animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1−/−/MGMT−/− double knockout (DKO) mice developed more, but much smaller tumors than MGMT−/− animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1−/− cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

// Sebastian Foersch 1 , Mario Schindeldecker 1, 4 , Martina Keith 2, 3 , Katrin E. Tagscherer 1 , Aurelie Fernandez 1 , Philipp J. Stenzel 1 , Sascha Pahernik 5, 6 , Markus Hohenfellner 5 , Peter Schirmacher 3 , Wilfried Roth 1, 2 and Stephan Macher-Goeppinger 1, 2, 3, 4 1 Institute of Pathology, University Medical Center Mainz, Mainz, Germany 2 Molecular Tumor Pathology, German Cancer Research Center, Heidelberg, Germany 3 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany 4 Tissue Bank, University Medical Center Mainz, Mainz, Germany 5 Department of Urology, University Hospital Heidelberg, Heidelberg, Germany 6 Department of Urology, Nuremberg General Hospital, Paracelsus Medical University, Nuremberg, Germany Correspondence to: Stephan Macher-Goeppinger, email: stephan.m-g@unimedizin-mainz.de Keywords: renal cell carcinoma, kidney, androgen receptor, treatment, prognostic marker Received: March 02, 2017 Accepted: August 26, 2017 Published: September 11, 2017 ABSTRACT Background: Despite rapid discoveries in molecular biology of renal cell carcinoma (RCC) and advances in systemic targeted therapies, development of new diagnostic and therapeutic strategies is urgently needed. The androgen receptor (AR) has been shown to hold prognostic and predicitve value in several malignancies. Here, we studied a possible association between AR expression and prognosis in patients with RCCs. Results: Low AR expression levels were associated with occurrence of distant metastasis and higher tumor stage in papillary and clear-cell RCCs. Importantly, multivariate Cox regression analyses revealed that AR is an independent prognostic factor for cancer-specific survival. Materials and Methods: The expression of AR was measured by immunohistochemistry and assessed by digital image analysis using a tissue microarray containing tumor tissue of a large and well-documented series of RCC patients with long-term follow-up information. Chi-squared tests, Kaplan-Meier curves and Cox regression models were used to investigate the possible relationship between AR expression and clinico-pathological characteristics and patient survival. Conclusions: Patients affected by AR-positive tumors exhibit a favorable prognosis by multiple Cox regression, while loss of AR expression is related to aggressive disease. Therefore, assessing AR expression offers valuable prognostic information that could improve treatment selection for metastatic disease. Moreover, our findings highlight a potential therapeutic use of AR pharmaceuticals in patients with RCCs.

Crohn's disease and ulcerative colitis are both associated with an increased risk of inflammation-associated colorectal carcinoma. Colitis-associated cancer (CAC) is one of the most important causes for morbidity and mortality in patients with inflammatory bowel diseases (IBD). Colitis-associated neoplasia distinctly differs from sporadic colorectal cancer in its biology and the underlying mechanisms. This review discusses the molecular mechanisms of CAC and summarizes the most important genetic alterations and signaling pathways involved in inflammatory carcinogenesis. Then, clinical translation is evaluated by discussing new endoscopic techniques and their contribution to surveillance and early detection of CAC. Last, we briefly address different types of concepts for prevention (i.e., anti-inflammatory therapeutics) and treatment (i.e., surgical intervention) of CAC and give an outlook on this important aspect of IBD.

Growing evidence proposes an important role for pro-inflammatory cytokines during tumor development. Several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (IL-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC). Increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients. According to experimental studies, these effects are mediated through IL-6 trans-signaling promoting tumor cell proliferation and inhibiting apoptosis through gp130 activation on tumor cells with subsequent signaling through Janus kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3). During recent years, several therapeutics targeting the IL-6/STAT3 pathway have been developed and pose a promising strategy for the treatment of CRC. This review discusses the molecular mechanisms and possible therapeutic targets involved in IL-6 signaling in CRC.

Growing evidence proposes an important role for pro-inflammatory cytokines during tumor development. Several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (IL-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC). Increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients. According to experimental studies, these effects are mediated through IL-6 trans-signaling promoting tumor cell proliferation and inhibiting apoptosis through gp130 activation on tumor cells with subsequent signaling through Janus kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3).During recent years, several therapeutics targeting the IL-6/STAT3 pathway have been developed and pose a promising strategy for the treatment of CRC. This review discusses the molecular mechanisms and possible therapeutic targets involved in IL-6 signaling in CRC.

Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC.Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluorescent signal. From all tumour sites examined with CLE in vivo, targeted specimens were obtained for histology, immunohistochemistry (IHC) and fluorescence microscopy.A VEGF-specific signal was visualised in vivo in 13/15 APCmin mice and in 9/10 xenograft tumours. CLE enabled the cytoplasmatic distribution of VEGF to be displayed due to its subcellular resolution. In human tissue, a VEGF-specific signal was observed in 12/13 malignant specimens and in 10/11 samples from healthy mucosa from the patients (p0.03). CLE findings correlated well with ex vivo microscopy.In vivo molecular imaging with specific targeting of VEGF is possible in murine tumours, human xenografts and tissue specimens using CLE. CLE with similar probes can be performed in human colonoscopy. Therefore-from a technical point of view-in vivo molecular imaging is transferable to stratification of patients with CRC during endoscopy even today. CLE could contribute to the identification of lesions at risk and potentially predict response to targeted treatment.

Background & Aims Epidermal growth factor receptor (EGFR) is a therapeutic target for colorectal cancer (CRC). However, technical challenges have limited in vivo imaging of EGFR in CRCs. Confocal laser endomicroscopy (CLE) enables accurate microscopic visualization of CRC in patients during endoscopy. We evaluated the ability to use CLE in vivo for instantaneous molecular imaging of EGFR in CRC models. Methods Tumors were grown in mice (n = 68) from human CRC cell lines that expressed high (SW480 cells) or low (SW620 cells) levels of EGFR. Tumors were visualized in vivo with a handheld CLE probe after injection of fluorescently labeled EGFR antibodies. EGFR-specific fluorescence was graded from 0 to 3+. Neoplastic and non-neoplastic specimens from human colorectal mucosa were examined. In vivo findings were correlated with histopathology, immunohistochemistry, and fluorescence microscopy analyses. Results CLE analysis of cell cultures confirmed the different expression levels of EGFR between cell lines. In living animals, CLE differentiated EGFR expression levels between tumor cell limes (mean fluorescence, 1.92 ± 0.22 [SW480] and 0.59 ± 0.21 [SW620], P = .0004). CLE analysis of EGFR expression in human specimens allowed distinction of neoplastic from non-neoplastic tissues (mean fluorescence, 2.0 ± 0.37 vs 0.25 ± 0.16, respectively, P = .0035). Conclusions CLE can be used for in vivo, molecular analysis of CRC and to differentiate EGFR expression patterns in xenograft tumors and human tissue samples. Because CLE can be performed during endoscopy, in vivo molecular imaging might be used in diagnosis of CRC and to predict response to targeted therapies.

SummaryThe intestinal epithelium constitutes an efficient barrier against the microbial flora. Here, we demonstrate an unexpected function of IL-33 as a regulator of epithelial barrier functions. Mice lacking IL-33 showed decreased Paneth cell numbers and lethal systemic infection in response to Salmonella typhimurium. IL-33 was produced upon microbial challenge by a distinct population of pericryptal fibroblasts neighboring the intestinal stem cell niche. IL-33 programmed the differentiation of epithelial progenitors toward secretory IEC including Paneth and goblet cells. Finally, IL-33 suppressed Notch signaling in epithelial cells and induced expression of transcription factors governing differentiation into secretory IEC. In summary, we demonstrate that gut pericryptal fibroblasts release IL-33 to translate bacterial infection into an epithelial response to promote antimicrobial defense.

Inflammatory bowel diseases (IBD) are accompanied by an increased risk of developing colitis-associated carcinoma (CAC). These tumors are one of the most important causes of morbidity and mortality in patients with IBD and distinctly differ from sporadic colorectal cancer in their biology and underlying mechanisms. First, this review discusses risk factors for the development of CAC and summarizes some of the most important genetic alterations and molecular pathways involved in inflammatory carcinogenesis. Then, new endoscopic techniques, such as chromoendoscopy and confocal laser endomicroscopy, and their contribution to surveillance and early detection of CAC are presented. Last, we briefly address different types of concepts for prevention (i.e. anti-inflammatory agents) and treatment (i.e. surgical resection) of CAC and give an outlook on this important aspect of IBD.