Strain specific differences in vitamin D3 response: impact on gut homeostasis.

Vitamin D3 regulates a variety of biological processes irrespective of its well-known importance for calcium metabolism. Epidemiological and animal studies indicate a role in immune regulation, intestinal barrier function and microbiome diversity. Here, we analyzed the impact of different vitamin D3- containing diets on C57BL/6 and BALB/c mice, with a particular focus on gut homeostasis and also investigated effects on immune cells in vitro. Weak regulatory effects were detected on murine T cells. By trend, the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 suppressed IFN, GM-CSF and IL-10 cytokine secretion in T cells of C57BL/6 but not BALB/c mice, respectively. Using different vitamin D3-fortified diets, we found a tissue-specific enrichment of mainly CD11b+ myeloid cells but not T cells in both mouse strains e.g. in spleen and Peyer's Patches. Mucin Reg3γ and Batf expression, as well as important proteins for gut homeostasis, were significantly suppressed in the small intestine of C57BL76 but not BALB/c mice fed with a high-vitamin D3 containing diet. Differences between both mouse stains were not completely explained by differences in vitamin D3 receptor expression which was strongly expressed in epithelial cells of both strains. Finally, we analyzed gut microbiome and again an impact of vitamin D3 was detected in C57BL76 but not BALB/c. Our data suggest strain-specific differences in vitamin D3 responsiveness under steady state conditions which may have important implications when choosing a murine disease model to study vitamin D3 effects.

Intestinal IgA-positive plasma cells are highly sensitive indicators of alloreaction early after allogeneic transplantation and associate with both graft-versus-host disease and relapse-related mortality.

Intestinal immunoglobulin A (IgA) is strongly involved in microbiota homeostasis. Since microbiota disruption is a major risk factor of acute graft-versus-host disease (GvHD), we addressed the kinetics of intestinal IgA-positive (IgA+) plasma cells by immunohistology in a series of 430 intestinal biopsies obtained at a median of 1,5 months after allogeneic stem cell transplantation (allo-SCT) from 115 patients (pts) at our center. IgA+ plasma cells were located in the subepithelial lamina propria and suppressed in the presence of histological aGvHD (GvHD Lerner stage 0: 131+/-8 IgA+ plasma cells/mm2; stage 1-2: 108+/-8 IgA+ plasma cells/mm2; stage 3-4: 89+/-16 IgA+ plasma cells/mm2; P=0.004). Overall, pts with IgA+ plasma cells below median had an increased treatment related mortality (P=0.04). Time courses suggested a gradual recovery of IgA+ plasma cells after day 100 in the absence but not in the presence of GvHD. Vice versa IgA+ plasma cells above median early after allo-SCT were predictive of relapse and relapse-related mortality (RRM): pts with low IgA+ cells had a 15% RRM at 2 and at 5 years, while pts with high IgA+ cells had a 31% RRM at 2 years and more than 46% at 5 years; multivariate analysis indicated high IgA+ plasma cells in biopsies (hazard ratio =2.7; 95% confidence interval: 1.04-7.00) as independent predictors of RRM, whereas Lerner stage and disease stage themselves did not affect RRM. In contrast, IgA serum levels at the time of biopsy were not predictive for RRM. In summary, our data indicate that IgA+ cells are highly sensitive indicators of alloreaction early after allo-SCT showing association with TRM but also allowing prediction of relapse independently from the presence of overt GvHD.

Round-Robin test for the histological diagnosis of acute colonic Graft-versus-Host disease validating established histological criteria and grading systems.

Histomorpholgy is one of the mainstays of acute Graft-versus-host disease (GvHD) diagnosis. However, concerns about reproducibility and the most appropriate grading system question its usefulness. Our aim was to assess histomorphological parameters and previously reported grading systems for GvHD regarding reproducibility and validity. Moreover, we propose that sum scores, derived by combining separately scored morphological parameters into a total score, might provide a simplified but equally effective means to grade GvHD. A total of 123 colon biopsies were assessed across four pathologists for intestinal GvHD using a Round-Robin test and results were correlated with clinical findings. Interobserver reproducibility was high for histological parameters that were evaluated as indicators of acute GvHD. Published grading systems were moderately reproducible (ICC 0.679-0.769) while simplified sum scores, in comparison, showed better interrater reliability (ICC 0.818-0.896). All grading systems and sum scores were associated with clinical signs of GvHD and in part with therapy response and survival. However, they were not able to stratify patients according to the clinical severity of GvHD. In a hot-spot analysis 1 crypt apoptotic body (CAB) in 10 crypts was a reasonable cut-off value for minimal diagnostic criteria of GvHD. In conclusion, histology can contribute to the diagnosis of GvHD and is reproducible. Published grading systems are able to reflect clinical findings as are simplified sum scores, which showed improved reproducibility and might be easier to handle as they are based on adding up histological parameters rather than transferring histological findings into a separate grading system. Sum scores will have to be further tested in a prospective setting.

Prognostic impact of variant histologies in urothelial bladder cancer treated with radical cystectomy.

OBJECTIVES: To evaluate variant histologies (VHs) for disease-specific survival (DSS) in patients with invasive urothelial bladder cancer (BCa) undergoing radical cystectomy (RC). MATERIALS AND METHODS: We analysed a multi-institutional cohort of 1082 patients treated with upfront RC for cT1-4aN0M0 urothelial BCa at eight centres. Univariable and multivariable Cox' regression analyses were used to assess the effect of different VHs on DSS in overall cohort and three stage-based analyses. The stages were defined as 'organ-confined' (≤pT2N0), 'locally advanced' (pT3-4N0) and 'node-positive' (pTanyN1-3). RESULTS: Overall, 784 patients (72.5%) had pure urothelial carcinoma (UC), while the remaining 298 (27.5%) harboured a VH. Squamous differentiation was the most common VH, observed in 166 patients (15.3%), followed by micropapillary (40 patients [3.7%]), sarcomatoid (29 patients [2.7%]), glandular (18 patients [1.7%]), lymphoepithelioma-like (14 patients [1.3%]), small-cell (13 patients [1.2%]), clear-cell (eight patients [0.7%]), nested (seven patients [0.6%]) and plasmacytoid VH (three patients [0.3%]). The median follow-up was 2.3 years. Overall, 534 (49.4%) disease-related deaths occurred. In uni- and multivariable analyses, plasmacytoid and small-cell VHs were associated with worse DSS in the overall cohort (both P = 0.04). In univariable analyses, sarcomatoid VH was significantly associated with worse DSS, while lymphoepithelioma-like VH had favourable DSS compared to pure UC. Clear-cell (P = 0.015) and small-cell (P = 0.011) VH were associated with worse DSS in the organ-confined and node-positive cohorts, respectively. CONCLUSIONS: More than 25% of patients harboured a VH at time of RC. Compared to pure UC, clear-cell, plasmacytoid, small-cell and sarcomatoid VHs were associated with worse DSS, while lymphoepithelioma-like VH was characterized by a DSS benefit. Accurate pathological diagnosis of VHs may ensure tailored counselling to identify patients who require more intensive management.

The Search for the Optimal cut-off Value of p53-Immunohistochemistry to Predict Prognosis of Invasive Bladder Cancer: A Multi-Center, Multi-Laboratory Analysis.

Introduction: Mutations in the TP53 gene are indicative of worse outcome in bladder cancer and are usually assessed by immunohistochemistry. To define p53-overexpression, a threshold of >10% is most commonly used (cut-off1). Recently, a novel cut-off (aberrant = 0% or ≥50%) (cut-off2) showed better correlation to clinical outcome. In this study, we evaluate the association between p53-immunohistochemistry cut-offs, clinico-pathological variables and disease-specific survival (DSS). Methods: Seven-hundred-fifty chemotherapy-naïve patients who underwent radical cystectomy were included (92% muscle-invasive bladder cancer. In addition to cut-off1 and cut-off2, a third cut-off (cut-off3) was determined based on the highest Youden-index value. Cut-off values were associated with clinico-pathological variables and FGFR3 mutation status. The Kaplan-Meier method was used to estimate DSS. Results: Aberrant p53-expression was found in 489 (65%) (cut-off1) and 466 (62%) (cut-off2) tumors. Cut-off3 was determined at 25% and aberrant p53-expression in 410 cases (55%) (cutoff3). p53-expression levels were significantly associated with higher pT-stage (cut-off1/2/3: P = 0.047, P = 0.006 and P = 0.0002, respectively), higher grade (all, P < 0.0001), and FGFR3 wild-type (cut-off1: P = 0.02, cut-offs2&3: P = 0.001). Median follow-up was 5.3 years (interquartile range, 4.0-6.0 years). p53-expression was not associated with DSS for any of the three cut-offs (cut-off1/2/3: P-log-rank = 0.566, 0.77 and 0.50, respectively). If we only considered locally advanced bladder cancer, results on DSS remained non-significant. Conclusion: This multi-center, multi-laboratory study showed that, regardless of the cut-off used, p53-immunohistochemistry did not enable selection of patients with worse outcome. Our results suggest that p53-immunohistochemistry alone is not suitable to guide clinical decision making after radical cystectomy.

Prognostic markers in invasive bladder cancer: FGFR3 mutation status versus P53 and KI-67 expression: a multi-center, multi-laboratory analysis in 1058 radical cystectomy patients.

OBJECTIVES: To determine the association between the FGFR3 mutation status and immuno-histochemistry (IHC) markers (p53 and Ki-67) in invasive bladder cancer (BC), and to analyze their prognostic value in a multicenter, multi-laboratory radical cystectomy (RC) cohort. PATIENTS AND METHODS: We included 1058 cN0M0, chemotherapy-naive BC patients who underwent RC with pelvic lymph-node dissection at 8 hospitals. The specimens were reviewed by uro-pathologists. Mutations in the FGFR3 gene were examined using PCR-SNaPshot; p53 and Ki-67 expression were determined by standard IHC. FGFR3 mutation status as well as p53 (cut-off>10%) and Ki-67 (cut-off>20%) expression were correlated to clinicopathological parameters and disease specific survival (DSS). RESULTS: pT-stage was

GPR Expression in Intestinal Biopsies From SCT Patients Is Upregulated in GvHD and Is Suppressed by Broad-Spectrum Antibiotics.

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients' gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response.

An investigation of the diagnostic, predictive, and prognostic impacts of three colonic biopsy grading systems for acute graft versus host disease.

Acute graft versus host disease (aGvHD) is an important, life-threatening complication after allogeneic hematopoietic stem cell transplantation (alloHSCT). To investigate the value of multiple simultaneous colon biopsies in improving diagnostic accuracy in patients with aGvHD, we retrospectively analyzed 157 patients after alloHSCT. The biopsies were evaluated individually using three established histological grading systems (Lerner, Sale, and Melson). The maximum, minimum, median, and mean histological aGvHD grades were calculated for each patient, and the results were correlated with the Glucksberg grade of clinical manifestation of GvHD, steroid therapy status, and outcome. We found that multiple colon biopsies enhanced diagnostic sensitivity. Moreover, higher histological grades correlated with steroid therapy initiation and refractoriness; the latter particularly occurred when advanced damage was present in all samples and healthy colon mucosa was reduced or absent. On multivariate analysis, the minimal Lerner and Glucksberg grades for intestinal aGvHD were significantly associated with steroid treatment failure. Ninety-nine patients died. The median survival was 285 days after the biopsies were taken. Fifteen patients died from relapse of their underling disorder and 84 from other causes, mostly infection (53 patients) and GvHD (14 patients). Multivariate analysis revealed a significant association between none-relapse mortality and the mean Lerner grade, minimum Melson grade, Glucksberg organ stage, and platelet counts. Thus, we found the Lerner system to be superior to the other grading methods in most instances and histologic evaluation of multiple simultaneously obtained biopsies from the colon to result in a higher diagnostic yield, which helps plan systemic steroid treatment while predicting treatment response and outcome.

FGFR3 Mutation Status and FGFR3 Expression in a Large Bladder Cancer Cohort Treated by Radical Cystectomy: Implications for Anti-FGFR3 Treatment?†.

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. PATIENT SUMMARY: Oncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

Non-invasive diagnosis of acute intestinal graft-versus-host disease by a new scoring system using ultrasound morphology, compound elastography, and contrast-enhanced ultrasound.

Acute gastrointestinal (GI) graft-versus-host disease (GvHD) is a life-threating complication in patients after allogeneic stem cell transplantation (ASCT). In 60 sonographic analyses, a novel scoring system for non-invasive diagnosis of severe GI GvHD was developed. The score comprised morphological and vascular changes using B-mode and color-coded Doppler sonography, changes of mural stiffness using compound elastography, and dynamic microvascularisation using contrast-enhanced ultrasound (CEUS). Furthermore, inflammatory parameters such as CRP, Calprotectin, and regenerating islet-derived protein 3α (Reg3α) were obtained. ROC curve analysis of our novel GvHD sum score revealed an area under the curve of 1.0 (95% CI: 0.99-1.00) in diagnosing GI GvHD and 0.88 (95% CI: 0.79-0.96) for severe GI GvHD. A sum score above 5 correlated with GI GvHD with a sensitivity of 97.6% (41/42) and a specificity of 94.4% (17/18) and score values above 10 with severe GI GvHD with a sensitivity of 91.7% (11/12) and specificity of 79.2% (38/48). The additional use of inflammatory parameters did not improve the predictive power. CEUS is a promising, non-invasive tool for the diagnosis of acute GI GvHD. Together with further descriptive parameters for inflammatory processes, it gains significant diagnostic accuracy in identifying patients with severe stages of acute intestinal GvHD.