Immune cell distribution and DNA methylation signatures differ between tumor and stroma enriched compartment in pancreatic ductal adenocarcinoma.

The presence of abundant tumor stroma is a prominent characteristic of pancreatic ductal adenocarcinomas (PDAC) that potentially influences disease progression and therapy response. This study aims to investigate immune cell infiltration and epigenetic profiles in tumor cell enriched ("Tumor") and stroma cell enriched ("Stroma") regions within human PDAC tissue samples. By comparing those regions, we identified 25,410 differentially methylated positions (DMPs) distributed across 6,963 unique genes. Pathway enrichment analysis using the top 2,000 DMPs that were either hyper- or hypomethylated indicated that immune response pathways and the estrogen receptor pathway are epigenetically dysregulated in Tumor and Stroma regions, respectively. In terms of immune cell infiltration, we observed overall low levels of T cells in both regions. In Tumor regions however, occurrence of tumor-associated macrophages (TAMs) was higher than in Stroma regions (p = 0.02) concomitant with a dualistic distribution that stratifies PDAC patients into those with high and low TAM infiltration. By categorizing TAM levels into quartiles, our analysis revealed that PDAC patients with more than 1,515 TAMs per mm² exhibited significantly shorter overall survival (p = 0.036). Our data suggest that variations in inflammatory characteristics between the Tumor and Stroma defined compartments of PDAC may primarily stem from the presence of macrophages rather than lymphocytes. The abundance of TAMs within regions enriched with tumor cells correlates with patient survival, underscoring the potential significance of exploring therapeutic interventions targeting TAMs. Furthermore, directing attention towards the estrogen receptor pathway may represent a promising strategy to address the stroma cell component within the PDAC tumor microenvironment.

Correlation of histologic, imaging, and artificial intelligence features in NAFLD patients, derived from Gd-EOB-DTPA-enhanced MRI: a proof-of-concept study.

OBJECTIVE: To compare unsupervised deep clustering (UDC) to fat fraction (FF) and relative liver enhancement (RLE) on Gd-EOB-DTPA-enhanced MRI to distinguish simple steatosis from non-alcoholic steatohepatitis (NASH), using histology as the gold standard. MATERIALS AND METHODS: A derivation group of 46 non-alcoholic fatty liver disease (NAFLD) patients underwent 3-T MRI. Histology assessed steatosis, inflammation, ballooning, and fibrosis. UDC was trained to group different texture patterns from MR data into 10 distinct clusters per sequence on unenhanced T1- and Gd-EOB-DTPA-enhanced T1-weighted hepatobiliary phase (T1-Gd-EOB-DTPA-HBP), then on T1 in- and opposed-phase images. RLE and FF were quantified on identical sequences. Differences of these parameters between NASH and simple steatosis were evaluated with χ2- and t-tests, respectively. Linear regression and Random Forest classifier were performed to identify associations between histological NAFLD features, RLE, FF, and UDC patterns, and then determine predictors able to distinguish simple steatosis from NASH. ROC curves assessed diagnostic performance of UDC, RLE, and FF. Finally, we tested these parameters on 30 validation cohorts. RESULTS: For the derivation group, UDC-derived features from unenhanced and T1-Gd-EOB-DTPA-HBP, plus from T1 in- and opposed-phase, distinguished NASH from simple steatosis (p ≤ 0.001 and p = 0.02, respectively) with 85% and 80% accuracy, respectively, while RLE and FF distinguished NASH from simple steatosis (p ≤ 0.001 and p = 0.004, respectively), with 83% and 78% accuracy, respectively. On multivariate regression analysis, RLE and FF correlated only with fibrosis (p = 0.040) and steatosis (p ≤ 0.001), respectively. Conversely, UDC features, using Random Forest classifier predictors, correlated with all histologic NAFLD components. The validation group confirmed these results for both approaches. CONCLUSION: UDC, RLE, and FF could independently separate NASH from simple steatosis. UDC may predict all histologic NAFLD components. CLINICAL RELEVANCE STATEMENT: Using gadoxetic acid-enhanced MR, fat fraction (FF > 5%) can diagnose NAFLD, and relative liver enhancement can distinguish NASH from simple steatosis. Adding AI may let us non-invasively estimate the histologic components, i.e., fat, ballooning, inflammation, and fibrosis, the latter the main prognosticator. KEY POINTS: • Unsupervised deep clustering (UDC) and MR-based parameters (FF and RLE) could independently distinguish simple steatosis from NASH in the derivation group. • On multivariate analysis, RLE could predict only fibrosis, and FF could predict only steatosis; however, UDC could predict all histologic NAFLD components in the derivation group. • The validation cohort confirmed the findings for the derivation group.

Meeting actual benchmarks for short- and long-term outcomes after cytoreductive surgery for peritoneal surface malignancy at a newly established academic treatment center.

BACKGROUND: Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy is a curative treatment for selected patients with peritoneal surface malignancy. Reaching actual outcomes benchmarks is challenging given the complex nature of peritoneal surface malignancy surgery. The aim of this study was to assess how the benchmarks for morbidity and oncologic outcome can be reached at a newly established program for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. METHODS: Building on existing institutional experience in complex abdominal surgery and interdisciplinary ovarian cancer treatment, a peritoneal surface malignancy center for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy was established at the Medical University of Vienna using a structured mentoring process. This is a retrospective analysis of the first 100 consecutive patients. Morbidity and mortality were assessed using the Clavien-Dindo classification, and oncologic outcomes using overall survival. RESULTS: Major morbidity and mortality were 26% and 3%, and median overall survival was 49.0 months. In patients with colorectal peritoneal metastases, the median overall survival was 35.1 months (all colorectal peritoneal metastases patients) and 48.8 months in the subgroup with Peritoneal Surface Disease Severity Score ≤3. No median overall survival could be calculated in patients with low-grade appendiceal mucinous neoplasms, appendiceal adenocarcinoma, or peritoneal mesothelioma due to >50% of patients being alive at the end of follow-up. CONCLUSION: We show that the current morbidity and oncological outcomes benchmarks can be reached within the first 100 cases of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy at a newly established peritoneal surface malignancy center. Previous institutional experience in complex abdominal surgery and a structured mentoring process are key factors in achieving this goal.

Evaluation of Intestinal Fibrosis with 68Ga-FAPI PET/MR Enterography in Crohn Disease.

Background In Crohn disease, differentiation between active intestinal inflammation and fibrosis has implications for treatment, but current imaging modalities are not reliably accurate. Purpose To evaluate the predictive value of gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor (FAPI) PET/MR enterography for the assessment of bowel wall fibrosis in Crohn disease. Materials and Methods In this prospective single-center study, consecutive participants with Crohn disease and obstructive symptoms underwent preoperative 68Ga-FAPI PET/MR enterography from May 2021 to January 2022. Histopathologic analysis of resected bowel segments was performed to grade active inflammation (A0-A2) and fibrosis (F0-F2), which served as the reference standard. The fibroblast activation protein (FAP) expression in bowel wall layers was analyzed immunohistochemically for each layer. 68Ga-FAPI-derived maximum standardized uptake value (SUVmax) was compared with histopathologic results by using mixed-model analysis of variance and Bonferroni-corrected post hoc tests. Results In 14 participants (mean age, 45 years ± 9 [SD]; 10 men), fibrosis was diagnosed histopathologically in 28 of 51 bowel segments (grade F1, n = 14; grade F2, n = 14). Mean SUVmax was higher in segments with fibrosis than without (7.6 vs 2.0; P < .001). In severe fibrosis, mean SUVmax was higher than in mild to moderate fibrosis (8.9 ± 0.9 vs 6.2 ± 0.9; P = .045). Bowel segments with isolated active inflammation had lower mean 68Ga-FAPI uptake than segments with combined active inflammation and fibrosis (SUVmax, 3.2 ± 0.4 vs 8.1 ± 0.1; P = .005). With an SUVmax cutoff value of 3.5, the area under the receiver operating characteristic curve for the prediction of fibrosis was 0.94 (95% CI: 0.9, 1.0), with sensitivity of 26 of 28 segments (93%) and specificity of five of six segments (83%). 68Ga-FAPI-derived SUVmax correlated with FAP expression across all bowel layers (R2 = 0.50, P < .001). Conclusion Higher gallium 68 fibroblast activation protein inhibitor uptake at PET/MR enterography was associated with histopathologically assessed bowel wall fibrosis in participants with Crohn disease, suggesting diagnostic potential for treatment decisions. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by O'Shea in this issue.

Circulating caspase-cleaved cytokeratin 18 correlates with tumour burden and response to therapy in patients with colorectal cancer liver metastasis.

BACKGROUND AND AIMS: Systemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. MATERIALS AND METHODS: Fifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. RESULTS: CRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). CONCLUSIONS: Apoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.

Using Artificial Intelligence to Find the Optimal Margin Width in Hepatectomy for Colorectal Cancer Liver Metastases.

Importance: In patients with resectable colorectal cancer liver metastases (CRLM), the choice of surgical technique and resection margin are the only variables that are under the surgeon's direct control and may influence oncologic outcomes. There is currently no consensus on the optimal margin width. Objective: To determine the optimal margin width in CRLM by using artificial intelligence-based techniques developed by the Massachusetts Institute of Technology and to assess whether optimal margin width should be individualized based on patient characteristics. Design, Setting, and Participants: The internal cohort of the study included patients who underwent curative-intent surgery for KRAS-variant CRLM between January 1, 2000, and December 31, 2017, at Johns Hopkins Hospital, Baltimore, Maryland, Memorial Sloan Kettering Cancer Center, New York, New York, and Charité-University of Berlin, Berlin, Germany. Patients from institutions in France, Norway, the US, Austria, Argentina, and Japan were retrospectively identified from institutional databases and formed the external cohort of the study. Data were analyzed from April 15, 2019, to November 11, 2021. Exposures: Hepatectomy. Main Outcomes and Measures: Patients with KRAS-variant CRLM who underwent surgery between 2000 and 2017 at 3 tertiary centers formed the internal cohort (training and testing). In the training cohort, an artificial intelligence-based technique called optimal policy trees (OPTs) was used by building on random forest (RF) predictive models to infer the margin width associated with the maximal decrease in death probability for a given patient (ie, optimal margin width). The RF component was validated by calculating its area under the curve (AUC) in the testing cohort, whereas the OPT component was validated by a game theory-based approach called Shapley additive explanations (SHAP). Patients from international institutions formed an external validation cohort, and a new RF model was trained to externally validate the OPT-based optimal margin values. Results: This cohort study included a total of 1843 patients (internal cohort, 965; external cohort, 878). The internal cohort included 386 patients (median [IQR] age, 58.3 [49.0-68.7] years; 200 men [51.8%]) with KRAS-variant tumors. The AUC of the RF counterfactual model was 0.76 in both the internal training and testing cohorts, which is the highest ever reported. The recommended optimal margin widths for patient subgroups A, B, C, and D were 6, 7, 12, and 7 mm, respectively. The SHAP analysis largely confirmed this by suggesting 6 to 7 mm for subgroup A, 7 mm for subgroup B, 7 to 8 mm for subgroup C, and 7 mm for subgroup D. The external cohort included 375 patients (median [IQR] age, 61.0 [53.0-70.0] years; 218 men [58.1%]) with KRAS-variant tumors. The new RF model had an AUC of 0.78, which allowed for a reliable external validation of the OPT-based optimal margin. The external validation was successful as it confirmed the association of the optimal margin width of 7 mm with a considerable prolongation of survival in the external cohort. Conclusions and Relevance: This cohort study used artificial intelligence-based methodologies to provide a possible resolution to the long-standing debate on optimal margin width in CRLM.

Tumor cell budding in preoperative biopsies of esophageal and gastroesophageal junction carcinoma independently predicts survival in a grade-dependent manner.

BACKGROUND: Tumor budding is a prognostic factor in biopsies of different tumor entities. Recent evidence suggests that this also applies to esophageal squamous cell carcinomas. Since esophageal cancer is diagnosed by biopsy, the aim of this study was to investigate whether tumor budding in pretherapeutic biopsies of a mixed tumor population of the esophagus and gastroesophageal junction might predict survival. METHODS: In this retrospective analysis, samples of 78 patients were analyzed (55 adenocarcinomas, 17 squamous cell carcinomas, 5 adenosquamous carcinomas, 1 carcinosarcoma). In addition to preoperative biopsies, budding foci in corresponding resection specimens were assessed and related to overall and relapse-free survival. RESULTS: The main finding was that the number of budding foci in preoperative biopsies predicted overall survival independent of the patient's age and disease stage in a grade-specific (P = .009) manner. In patients with grade 2 tumors, each additional budding focus was associated with an increased chance of death by a factor of 1.28 (hazard ratio 95% confidence interval 1.06-1.55, P = .011). There was no significant association between survival and the number of budding foci in patients with grade 3 tumors, and no budding was observed in grade 1 tumors. Budding foci in resection specimens also showed a certain association with survival, but to a lesser degree. CONCLUSION: Budding foci in preoperative biopsies might serve to improve prognostic accuracy in esophageal carcinomas.

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment.

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.

Mutant KRAS as a prognostic biomarker after hepatectomy for rectal cancer metastases: Does the primary disease site matter?

BACKGROUND: The prognostic implication of mutant KRAS (mKRAS) among patients with primary disease in the rectum remains unknown. METHODS: From 2000 to 2018, patients undergoing hepatectomy for colorectal liver metastases at 10 collaborating international institutions with documented KRAS status were surveyed. RESULTS: A total of 834 (65.8%) patients with primary colon cancer and 434 (34.2%) patients with primary rectal cancer were included. In patients with primary colon cancer, mKRAS served as a reliable prognostic biomarker of poor overall survival (OS) (hazard ratio [HR]: 1.58, 95% CI 1.28-1.95) in the multivariable analysis. Although a trend towards significance was noted, mKRAS was not found to be an independent predictor of OS in patients with primary rectal tumors (HR 1.34, 95% CI 0.98-1.80). For colon cancer, the specific codon impacted in mKRAS appears to reflect underlying disease biology and oncologic outcomes, with codon 13 being associated with particularly poor OS in patients with left-sided tumors (codon 12, HR 1.56, 95% CI 1.22-1.99; codon 13, HR 2.10 95% CI 1.43-3.08;). Stratifying the rectal patient population by codon mutation did not confer prognostic significance following hepatectomy. CONCLUSIONS: While the left-sided colonic disease is frequently grouped with rectal disease, our analysis suggests that there exist fundamental biologic differences that drive disparate outcomes. Although there was a trend toward significance of KRAS mutations for patients with primary rectal cancers, it failed to achieve statistical significance.

Delayed antiretroviral therapy in HIV-infected individuals leads to irreversible depletion of skin- and mucosa-resident memory T cells.

People living with HIV (PLWH) are at increased risk for developing skin and mucosal malignancies despite systemic reconstitution of CD4+ T cells upon antiretroviral therapy (ART). The underlying mechanism of chronic tissue-related immunodeficiency in HIV is unclear. We found that skin CD4+ tissue-resident memory T (Trm) cells were depleted after HIV infection and replenished only upon early ART initiation. TCR clonal analysis following early ART suggested a systemic origin for reconstituting CD4+ Trm cells. Single-cell RNA sequencing in PLWH that received late ART treatment revealed a loss of CXCR3+ Trm cells and a tolerogenic skin immune environment. Human papilloma virus-induced precancerous lesion biopsies showed reduced CXCR3+ Trm cell frequencies in the mucosa in PLWH versus HIV- individuals. These results reveal an irreversible loss of CXCR3+ Trm cells confined to skin and mucosa in PLWH who received late ART treatment, which may be a precipitating factor in the development of HPV-related cancer.

Mucosal Biofilms Are an Endoscopic Feature of Irritable Bowel Syndrome and Ulcerative Colitis.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. METHODS: The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning-based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. RESULTS: Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. CONCLUSIONS: The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.

Autoimmune hepatitis-is histology conclusive?

Liver biopsy is an essential and necessary element in the diagnosis and management of autoimmune hepatitis, and is of very special importance in the scoring system for diagnosis. Histopathology shows moderate to severe inflammatory infiltrates with abundant plasma cells in the enlarged portal tracts with interface hepatitis and moderate to severe necroinflammatory lesions in the lobules with lymphoplasmacytic reaction. Regeneration develops with rosette formation and regenerative nodules. One important issue is the differentiation between acute onset of autoimmune hepatitis and a flare up of chronic disease; this cannot be diagnosed in some cases clinically and therefore requires a biopsy to evaluate the stage of the disease. There are some variants of the disease with cholestatic features such as autoantibody negative autoimmune hepatitis and giant cell hepatitis as well as overlap syndromes with primary biliary cholangitis and primary sclerosing cholangitis. Clinically, three types of autoimmune hepatitis are differentiated according to autoantibody formation and the clinical picture, however, histopathologically there is no difference between these three types. Differential diagnosis of autoimmune hepatitis includes drug-induced liver injury with minocycline, alpha methyldopa, nitrofurantoin and checkpoint inhibitors such as infliximab. Wilson´s disease is also an important differential diagnosis especially in young adults. A liver biopsy is mandatory to confirm the diagnosis of autoimmune hepatitis but histopathology alone is not conclusive.

The food additive EDTA aggravates colitis and colon carcinogenesis in mouse models.

Inflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10-/- model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.

The Prognostic Impact of Primary Tumor Site Differs According to the KRAS Mutational Status: A Study By the International Genetic Consortium for Colorectal Liver Metastasis.

OBJECTIVE: To examine the prognostic impact of tumor laterality in colon cancer liver metastases (CLM) after stratifying by Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutational status. BACKGROUND: Although some studies have demonstrated that patients with CLM from a right sided (RS) primary cancer fare worse, others have found equivocal outcomes of patients with CLM with RS versus left-sided (LS) primary tumors. Importantly, recent evidence from unresectable metastatic CRC suggests that tumor laterality impacts prognosis only in those with wild-type tumors. METHODS: Patients with rectal or transverse colon tumors and those with unknown KRAS mutational status were excluded from analysis. The prognostic impact of RS versus LS primary CRC was determined after stratifying by KRAS mutational status. RESULTS: 277 patients had a RS (38.6%) and 441 (61.4%) had a LS tumor. Approximately one-third of tumors (28.1%) harbored KRAS mutations. In the entire cohort, RS was associated with worse 5-year overall survival (OS) compared with LS (39.4% vs 50.8%, P = 0.03) and remained significantly associated with worse OS in the multivariable analysis (hazard ratio 1.45, P = 0.04). In wild-type patients, a worse 5-year OS associated with a RS tumor was evident in univariable analysis (43.7% vs 55.5%, P = 0.02) and persisted in multivariable analysis (hazard ratio 1.49, P = 0.01). In contrast, among patients with KRAS mutated tumors, tumor laterality had no impact on 5-year OS, even in the univariable analysis (32.8% vs 34.0%, P = 0.38). CONCLUSIONS: This study demonstrated, for the first time, that the prognostic impact of primary tumor side differs according to KRAS mutational status. RS tumors were associated with worse survival only in patients with wild-type tumors.

Placental growth factor levels neither reflect severity of portal hypertension nor portal-hypertensive gastropathy in patients with advanced chronic liver disease.

BACKGROUND & AIMS: Experimental data indicates that placental growth factor (PLGF) is involved in the pathophysiology of portal hypertension (PH) due to advanced chronic liver disease (ACLD). We investigated serum levels of PLGF and its "scavenger", the receptor soluble fms-like tyrosine kinase-1 (sFLT1, or sVEGFR1), in ACLD patients with different severity of PH and portal-hypertensive gastropathy (PHG). METHODS: PLGF and sVEGFR1 were measured in ACLD patients with hepatic venous pressure gradient (HVPG) ≥6 mmHg (n = 241) and endoscopic evaluation of PHG (n = 216). Patients with pre-/posthepatic PH, TIPS, liver transplantation and hepatocellular carcinoma were excluded. RESULTS: Thirty-two (13%) patients had HVPG 6-9 mmHg, 128 (53%) 10-19 mmHg and 81 (34%) ≥20 mmHg; 141 (59%) had decompensated ACLD (dACLD). PLGF (median 17.2 vs. 20.8 vs. 22.4 pg/mL; p = 0.002), sVEGFR1 (median 96.0 vs. 104.8 vs. 119.3 pg/mL; p < 0.001) levels increased across HVPG strata, while PLGF/sVEGFR1 ratios remained similar (0.19 vs. 0.20 vs. 0.18 pg/mL; p = 0.140). The correlation between PLGF and HVPG was weak (Rho = 0.190,95%CI 0.06-0.31; p = 0.003), and the PLGF/sVEGFR1 ratio did not correlate with HVPG (p = 0.331). The area-under-the-receiver operating characteristics (AUROC) for PLGF to detect clinically significant PH (CSPH;i.e. HVPG ≥ 10 mmHg) yielded only 0.688 (0.60-0.78; p < 0.001). When compared to ACLD patients without PHG, PLGF levels (20 without vs. 21.4 with mild vs. 17.1 pg/mL with severe PHG, respectively; p = 0.005) and PLGF/sVEGFR1 ratios (0.20 vs. 0.19 vs. 0.17; p = 0.076) did not increase with mild and severe PHG. CONCLUSION: While PLGF levels tended to increase with severity of PH, the PLGF/sVEGFR1 ratio remained stable across HVPG strata. Neither PLGF nor the PLGF/sVEGFR1 ratio had diagnostic value for prediction of CSPH. The severity of PHG was also not associated with stepwise increases in PLGF levels or PLGF/sVEGFR1 ratio.

Apparent Intracellular Helicobacter pylori Detected by Immunohistochemistry: The Missing Link in Eradication Failure.

BACKGROUND: Helicobacter pylori is primarily an extracellularly living bacterium. However, seemingly intracellular occurrence can often be detected by immunohistochemical stains. Considering antimicrobial resistance, we investigated the impact of the apparent intracellular H. pylori (aiHp) on treatment failure of first-line triple therapies. METHODS: Gastric biopsies of 814 patients infected with H. pylori naive to treatment were analyzed before and after eradication therapy by immunohistochemistry. Of these, 373 received treatment consisting of amoxicillin, clarithromycin, and proton pump inhibitor (AC/PPI). Availability of polymerase chain reaction-based clarithromycin susceptibility test results from pretreatment gastric biopsies was a precondition for matching 52 aiHp to 52 non-aiHp cases within the AC/PPI group. RESULTS: AiHp were detected mostly in low counts predominantly in corpus biopsies, rarely in antrum biopsies (95.2% vs 24.6%); they were found in 497 (61%) of all patients and in 192 of 373 patients (51.5%) in the AC/PPI group. The eradication rate in aiHp versus non-aiHp cases was 44.4% versus 72.9% in the entire sample and 45.3% versus 66.8% in the AC/PPI group. Among the 104 paired patients, respective values were 46.2% versus 78.8%; in clarithromycin-susceptible cases, 60.6% versus 91.9%. Both aiHp and resistance to clarithromycin proved to be highly significant (P ≤ .001) and independent predictors of eradication failure. Twelve of 13 aiHp cases with a clarithromycin-sensitive strain who failed eradication developed resistance to the antibiotic. CONCLUSIONS: AiHp found by immunohistochemical staining especially in corpus biopsies proved to be a risk factor for failure of first-line triple therapies; occurrence of aiHp should be considered with regard to therapy options.

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.

Comparison of the diagnostic quality of aspiration and core-biopsy needles for transjugular liver biopsy.

BACKGROUND: Liver biopsy remains essential for the diagnostic work-up of patients with liver disease. AIMS: To evaluate aspiration vs. core-biopsy needles for transjugular liver biopsy (TJLB) in patients undergoing hepatic venous pressure gradient (HVPG) measurements. METHODS: 84 patients undergoing TJLB between 06/2017 and 12/2018 were prospectively included. Liver biopsy specimens were systematically evaluated for quantitative and qualitative criteria such as number of portal tracts, sample length and fragmentation. RESULTS: In direct comparison of paired TJLB specimens (n=35), core-biopsy samples were significantly longer (median 12 vs. 9mm, p=0.012), tended to contain more portal tracts (median 8 vs. 6, p=0.064) and were less fragmented (p<0.001), which resulted in better confidence for liver fibrosis assessment (p=0.035). However, a superior quality in terms of less fragmentation of core-biopsy specimens (p<0.05) was only confirmed in patients with HVPG ≥10mmHg or liver stiffness measurement >40kPa. In contrast, the aspiration needle provided significantly longer samples in patients with HVPG <10mmHg (median 21 vs. 12mm, p=0.007) or with liver stiffness measurement <20kPa (median 21 vs. 11mm, p=0.025). CONCLUSION: In patients with HVPG ≥10mmHg, we recommend to performed TJLB using core-biopsy needles, while the aspiration needle provides high quality liver biopsy specimens in patients with HVPG <10mmHg.

Soluble guanylyl cyclase stimulation and phosphodiesterase-5 inhibition improve portal hypertension and reduce liver fibrosis in bile duct-ligated rats.

BACKGROUND: In cirrhosis, the nitric oxide-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway is impaired, which contributes to increased intrahepatic vascular resistance (IHVR) and fibrogenesis. We investigated if sGC stimulation (riociguat (RIO)), sGC activation (cinaciguat (CINA)) or phosphodiesterase (PDE)-5 inhibition (tadalafil (TADA)) improves portal hypertension (PHT) and liver fibrosis. METHODS: Fifty male Sprague-Dawley rats underwent bile-duct ligation (BDL) or sham operation. RIO (0.5 mg/kg), CINA (1 mg/kg), TADA (1.5 mg/kg) or vehicle (VEH) was administered from weeks 2 to 4 after BDL. At week 4, invasive haemodynamic measurements were performed, and liver fibrosis was assessed by histology (chromotrope-aniline blue (CAB), Picro-Sirius red (PSR)) and hepatic hydroxyproline content. RESULTS: Cirrhotic bile duct-ligated rats presented with PHT (13.1 ± 1.0 mmHg) and increased IHVR (4.9 ± 0.5 mmHg⋅min/mL). Both RIO (10.0 ± 0.7 mmHg, p = 0.021) and TADA (10.3 ± 0.9 mmHg, p = 0.050) decreased portal pressure by reducing IHVR (RIO: -41%, p = 0.005; TADA: -21%, p = 0.199) while not impacting heart rate, mean arterial pressure and portosystemic shunting. Hepatic cGMP levels increased upon RIO (+239%, p = 0.006) and TADA (+32%, p = 0.073) therapy. In contrast, CINA dosed at 1 mg/kg caused weight loss, arterial hypotension and hyperlactataemia in bile duct-ligated rats. Liver fibrosis area was significantly decreased by RIO (CAB: -48%, p = 0.011; PSR: -27%, p = 0.121) and TADA (CAB: -21%, p = 0.342; PSR: -52%, p = 0.013) compared to VEH-treated bile duct-ligated rats. Hepatic hydroxyproline content was reduced by RIO (from 503 ± 20 to 350 ± 30 µg/g, p = 0.003) and TADA (282 ± 50 µg/g, p = 0.003), in line with a reduction of the hepatic stellate cell activation markers smooth-muscle actin and phosphorylated moesin. Liver transaminases decreased under RIO (AST: -36%; ALT: -32%) and TADA (AST: -24%; ALT: -27%) treatment. Hepatic interleukin 6 gene expression was reduced in the RIO group (-56%, p = 0.053). CONCLUSION: In a rodent model of biliary cirrhosis, the sGC stimulator RIO and the PDE-5 inhibitor TADA improved PHT. The decrease of sinusoidal vascular resistance was paralleled by a reduction in liver fibrosis and hepatic inflammation, while systemic haemodynamics were not affected.