Definitive evidence for Club cells as progenitors for mutant Kras/Trp53-deficient lung cancer.

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.

Immunohistochemical markers for histopathological diagnosis and differentiation of acute cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication following stem-cell or solid-organ transplantation. Accurate diagnosis of cutaneous GvHD is challenging, given that drug eruptions and viral rashes may present with similar clinical/histological manifestations. Specific markers are not available. We performed the histological examination of biopsy samples from acute GvHD (aGvHD; n = 54), Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN; n = 27), maculopapular drug eruption (MDE; n = 26) and healthy controls (n = 26). Samples of aGvHD showed a decrease in Langerhans cells (LC, p = 0.0001) and an increase in macrophages (MΦ, p = 0.0001) compared to healthy skin. Compared to SJS/TEN, MDE and healthy skin, aGvHD biopsies contained greater numbers of CD4+ and CD8+ T cells. The majority of CD4+ T-helper cells were localized in the upper dermis, whereas cytotoxic CD8+ T cells were found in the epidermis. Increased numbers of CD56+ natural killer (NK) cells in the upper dermis of aGvHD skin (p = 0.007) were not observed in controls or SJS/TEN and MDE. There were no differences in elafin staining between aGvHD and the latter two conditions. Acute GvHD appears to have a distinct inflammatory cell profile (T cells/NK cells) that may aid establishing in a more accurate diagnosis, especially when used to rule out differential diagnoses such as SJS/TEN or MDE.

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Dermatopathic lymphadenopathy with Langerhans cell chimerism in graft-versus-host disease of the skin.

Dermatopathic lymphadenopathy (DL) is well-known in inflammatory skin disease; however, it has not been reported in graft-versus-host disease (GvHD) after allogeneic stem cell transplantation. Here, we report 2 cases of DL in patients with acute GvHD of the skin and demonstrate complete donor chimerism of Langerhans cells within the lymph nodes.

Hypoxia-Associated Marker CA IX Does Not Predict the Response of Locally Advanced Rectal Cancers to Neoadjuvant Chemoradiotherapy.

Hypoxia-associated proteome changes have been shown to be associated with resistance to chemo- and radiotherapy. Our study evaluated the role of the hypoxia-inducible (HIF)-1 target gene carbonic anhydrase (CA) IX in the prediction of the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer (stages II and III). A total of 29 pretreatment biopsy specimens were stained for CA IX by immunohistochemistry, converted to digital images and evaluated in a quantitative fashion using image analysis software. Contrary to our expectations, a trend towards a correlation between better tumor regression (>50%) and higher expression of CA IX (p=0.056) was found. CA IX was also present more frequently in pathological tumor stage T1 (pT1) tumors (p=0.048). Conversely, no association with lymph node metastasis was identified. In conclusion, as a single marker, CA IX expression is not able to identify a hypoxia-related treatment resistant phenotype in rectal cancer.

NIH Consensus development project on criteria for clinical trials in chronic graft-versus-host disease: II. The 2014 Pathology Working Group Report.

The 2005 National Institute of Health (NIH) Consensus Conference outlined histopathological diagnostic criteria for the major organ systems affected by both acute and chronic graft-versus-host disease (GVHD). The 2014 Consensus Conference led to this updated document with new information from histopathological studies of GVHD in the gut, liver, skin, and oral mucosa and an expanded discussion of GVHD in the lungs and kidneys. The recommendations for final histological diagnostic categories have been simplified from 4 categories to 3: no GVHD, possible GVHD, and likely GVHD, based on better reproducibility achieved by combining the previous categories of "consistent with GVHD" and "definite GVHD" into the single category of "likely GVHD." Issues remain in the histopathological characterization of GVHD, particularly with respect to the threshold of histological changes required for diagnostic certainty. Guidance is provided for the incorporation of biopsy information into prospective clinical studies of GVHD, particularly with respect to biomarker validation.

Donor and host B cell-derived IL-10 contributes to suppression of graft-versus-host disease.

Graft-versus-host disease (GvHD) is a frequent life-threatening complication following allogeneic HSC transplantation (HSCT). IL-10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL-10-deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC-mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL-10 resulted in increased allogeneic T-cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL-10 was prominently produced by host- and donor-derived CD5(int) CD1d(int) TIM-1(int) B cells in this disease, and consistent with this, allogeneic HSCT resulted in exacerbated GvHD when mice lacking IL-10 expression in B cells were used as donor or host, compared with controls. Taken together, this study demonstrates that host and donor B cell-derived IL-10 provides a unique mechanism of suppression of acute GvHD, and suggests that DCs are the targets of this B cell-mediated suppressive effect. These findings open novel therapeutic possibilities based on the use of B cells to increase the feasibility of allogeneic HSCT.

Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis.

Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4-/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4-/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4-/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.

Rectal cancer: assessment of response to neoadjuvant chemoradiation by dynamic contrast-enhanced MRI.

PURPOSE: To assess pretreatment functional and morphological tumor characteristics with magnetic resonance imaging (MRI) in advanced rectal carcinoma and to identify factors predicting response to neoadjuvant chemoradiation. MATERIALS AND METHODS: In a prospective study, 95 patients with rectal carcinoma underwent dynamic contrast-enhanced MRI before and after chemoradiation. Quantitative parameters were derived from a pharmacokinetic two-compartment model. Tumors were also characterized with regard to mucinous status at pretreatment high-resolution MRI as nonmucinous or mucinous. Response to treatment was defined as a downshift in the local tumor stage. RESULTS: The parameter k21 (contrast medium exchange rate) was higher at pretreatment MRI in nonmucinous compared with mucinous carcinomas (P < 0.001). The effect of chemoradiation on dynamic MR parameters was higher in nonmucinous carcinomas than in the mucinous subtype (P < 0.001). A higher rate of response to treatment was linked with nonmucinous morphology (P < 0.001). Multivariate analysis revealed an association between mucinous tumor morphology and poor response (odds ratio [95% confidence interval]: 0.113 [0.032-0.395], P < 0.001) as well as an association between a high 75th percentile of k21 and a higher response rate (odds ratio: 1.043 [1.001-1.086], P = 0.019). CONCLUSION: Functional and morphological parameters of pretreatment MRI can assess tumor characteristics associated with the effectiveness of chemoradiation before treatment initiation.

Histological diagnosis of acute graft-versus-host disease in different sites of the upper gastrointestinal tract with correlation to endoscopic findings.

Graft-versus-host disease (GvHD) involving the intestine is a threat to patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). We evaluated biopsies from different sites of the upper gastrointestinal tract (GIT) of 97 patients after alloHSCT. Forty-six patients with clinical symptoms consistent with upper GI GvHD revealed histological features of GvHD in the esophagus, stomach, and/or duodenum. Biopsies of the duodenum and esophagus were significantly more sensitive for signs of GvHD than those of the gastric antrum or corpus. The histological features of GvHD were significantly correlated with the endoscopic findings of ulcers, erosion, atrophy, and white plaques; however, the sensitivity and specificity of the latter were low. In univariate analysis, overall mortality was significantly associated with histological GvHD signs in all four sites. Nonrelapse mortality was associated with histologic GvHD features in the antrum only. Regarding GvHD diagnosis, biopsies of the upper gastrointestinal tract should include the duodenum and/or esophagus.

Entamoeba histolytica-associated proctitis and ileitis mimicking Crohn's disease-A case report.

We report about a proctitis and ileitis terminalis, leading to the misdiagnosis of Chron's disease, in a male patient who has sex with men. Molecular multiplex analysis identified Entamoeba histolytica as the underlying cause. We provide diagnostic images, clues and pitfalls for diagnosis of E. histolytica associated proctitis.

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.

Rectal Cancer: MR imaging of the mesorectal fascia and effect of chemoradiation on assessment of tumor involvement.

PURPOSE: To evaluate the impact of chemoradiation on the reliability of MRI in assessing tumor involvement of the mesorectal fascia in patients with rectal cancer. MATERIALS AND METHODS: Presurgical MRI was performed in 150 patients; among them 85 had received neoadjuvant long-course chemoradiation. A standardized imaging protocol (1.5 Tesla [T] system, image voxel size 0.6 × 0.4 × 3 mm(3) ), standardized surgery, and histopathological examination were applied for the entire patient population. Images were analyzed to identify potential tumor involvement of the mesorectal fascia (minimum tumor distance to fascia ≤1 mm) and compared with histopathology as the reference standard. Results of nonirradiated and irradiated patients were compared to define the impact of chemoradiation on imaging reliability. RESULTS: In nonirradiated patients, MRI was reliable in predicting or excluding tumor involvement of the mesorectal fascia, positive predictive value 80%, negative predictive value 89%. The frequency of overestimating tumor involvement was significantly higher in irradiated patients (P = 0.005, positive predictive value 42%). CONCLUSION: Discussions about MRI assessment of tumor involvement of the mesorectal fascia as a basis for recommending neoadjuvant chemoradiation should focus on investigations that excluded irradiated patients, because MRI is less reliable after chemoradiation and tends to overestimate mesorectal tumor involvement.

Siltuximab-Related Favorable Clinical Outcome for a Patient Suffering from Idiopathic Multicentric Castleman Disease.

RationalCastleman disease is a rare lymphoproliferative disorder that can be subdivided into unicentric and multicentric forms, the latter of which causes a spectrum of serious medical conditions. Here, we present a case of idiopathic multicentric Castleman disease in the eighth decade of life. Patient Concerns. First hospitalized due to unexplained progressive anemia, the patient was readmitted to the hospital 18 months later with suspected lymphoma. Clinical examination revealed a progressive lymphadenopathy. Diagnoses. Histopathologic lymph node features, anemia, elevated CRP and IL6 levels, splenomegaly, and hypoalbuminemia indicated multicentric Castleman (MCD) disease. Interventions. The patient was treated intravenously with a dose of 11 mg/kg siltuximab every 3 weeks. Outcomes. Timely correct diagnosis through the stringent use of consensus diagnostic criteria and sufficient siltuximab therapy has considerably promoted favorable clinical outcomes in a patient suffering from MCD.

Veno-occlusive disease of the lung after allogeneic haematopoietic stem-cell transplantation: An autopsy study.

Pulmonary veno-occlusive disease (pVOD) is a potentially life-threatening sequela of allogeneic haematopoietic stem-cell transplantation (alloHSCT). We conducted a morphometric evaluation of autopsy lung tissue to determine the incidence of pVOD and its association with donor type, conditioning regime, hepatic sinusoidal obstruction syndrome (hSOS), survival time, and graft versus host disease (GvHD). The degree of occlusion of pulmonary veins in 78 autopsy cases after alloHSCT and 12 control cases was assigned to one of the following categories: none, minor thickening of the intima (up to 33% narrowing), moderate pVOD wherein about half of the lumen (34-66%) is occluded, or advanced pVOD with near total or total (67-100%) obliteration of the lumen. Minor thickening of the intima was found in all patients after alloHSCT (median: 66% of the vessels) and it was found to a lesser extent in the control cases (median: 12%). Moderate to advanced pVOD was seen in 95% of the cases, but only in a minority of the veins and venules (median: 6% of the veins and venules). PVOD was not significantly correlated with other histopathological findings within the lungs, including acute pneumonia, desquamative pneumonia, acute respiratory distress syndrome, organising and non-specific pneumonia, and bronchiolitis obliterans or acute lung disease. PVOD was significantly associated with a conditioning regimen including cyclophosphamide, fludarabine, or antithymocyte globulin and the duration of survival after alloHSCT. It was not associated with acute or chronic GvHD, other intestinal lung diseases, hSOS, or donor characteristics. PVOD was found in most patients after they underwent alloHSCT, although it mainly involved only a minority of the vessels. It was associated with the conditioning regime and the duration of survival after alloHSCT.

Prevalence of corneal findings and their interrelation with hematological findings in monoclonal gammopathy.

PURPOSE: To determine prevalence of paraproteinemic keratopathy (PPK) among patients with monoclonal gammopathy (MG). To evaluate interrelation between corneal and hematological parameters in patients with PPK. METHODS: Fifty-one patients with monoclonal gammopathy of undetermined significance (n = 19), smoldering multiple myeloma (n = 5) or multiple myeloma (n = 27) were prospectively included in this study. Best-corrected visual acuity, slit-lamp biomicroscopy, Scheimpflug tomography, in-vivo confocal laser scanning microscopy, optical coherence tomography and complete hematological workup were assessed. RESULTS: We identified n = 19 patients with bilateral corneal opacities compatible with PPK. PPK was newly diagnosed in 13 (29%) of 45 patients with a primary hematological diagnosis and in n = 6 patients without previous hematological diagnosis. The most common form was a discreet stromal flake-like PPK (n = 14 of 19). The median level of M-protein (p = 0.59), IgA (p = 0.53), IgG (p = 0.79) and IgM (p = 0.59) did not differ significantly between the patients with and without PPK. The median level of the FLC κ in serum of patients with kappa-restricted plasma cell dyscrasia was 209 mg/l in patients with PPK compared to 38.1 mg/l in patients without PPK (p = 0.18). Median level of FLC lambda in serum of patients with lambda-restricted plasma cell dyscrasia was lower in patients with PPK compared to patients without PPK (p = 0.02). CONCLUSION: The PPK was mostly discreet, but its prevalence (29%) was higher than expected. Median level of the monoclonal paraprotein was not significantly higher in patients with PPK compared to patients without PPK. Our results suggest a lack of correlation between morphology and severity of the ocular findings and severity of the monoclonal gammopathy. TRIAL REGISTRATION: German Clinical Trial Register: DRKS00023893.

Ablation of c-FLIP in hepatocytes enhances death-receptor mediated apoptosis and toxic liver injury in vivo.

BACKGROUND & AIMS: Apoptosis is crucially involved in acute and chronic liver injury, including viral, cholestatic, toxic, and metabolic liver disease. Additionally, dysregulation of apoptosis signaling pathways has been implicated in hepatocarcinogenesis. The most prominent members of the apoptosis-mediating tumor necrosis factor receptor superfamily are the TNF-R1 (CD120a) and the CD95 (Apo-1/Fas) receptor. Although extensively studied, the intracellular signaling events in hepatocytes are only incompletely understood. METHODS: To examine the role of the caspase-8 homolog cellular FLICE-inhibitory protein (c-FLIP) in liver injury, we generated mice with hepatocyte specific deletion of c-FLIP. Three models of acute liver injury were employed: the agonistic anti-CD95 antibody Jo2, d-galactosamine and LPS (GalN/LPS), and concanavalin A. RESULTS: Conditional ablation of c-FLIP in hepatocytes augmented liver injury and cell death in all three models of liver injury. CD95- and GalN/LPS-induced liver injury was ameliorated by a pancaspase inhibitor, while ConA-induced injury was unaffected by caspase inhibition. Augmented activation of the MAPK JNK was observed in parallel to liver injury in c-FLIP knockout mice in all injury models; however, inhibition of JNK only affected TNF- and ConA-mediated injury. CONCLUSIONS: In summary, c-FLIP is a central regulator of cell death in hepatocytes, involving increased activation of caspases and the MAPK JNK.

Evaluation of novel LCI CAD EYE system for real time detection of colon polyps.

BACKGROUND: Linked color imaging (LCI) has been shown to be effective in multiple randomized controlled trials for enhanced colorectal polyp detection. Recently, artificial intelligence (AI) with deep learning through convolutional neural networks has dramatically improved and is increasingly recognized as a promising new technique for enhancing colorectal polyp detection. AIM: This study aims to evaluate a newly developed computer-aided detection (CAD) system in combination with LCI for colorectal polyp detection. METHODS: First, a convolutional neural network was trained for colorectal polyp detection in combination with the LCI technique using a dataset of anonymized endoscopy videos. For validation, 240 polyps within fully recorded endoscopy videos in LCI mode, covering the entire spectrum of adenomatous histology, were used. Sensitivity (true-positive rate per lesion) and false-positive frames in a full procedure were assessed. RESULTS: The new CAD system used in LCI mode could process at least 60 frames per second, allowing for real-time video analysis. Sensitivity (true-positive rate per lesion) was 100%, with no lesion being missed. The calculated false-positive frame rate was 0.001%. Among the 240 polyps, 34 were sessile serrated lesions. The detection rate for sessile serrated lesions with the CAD system used in LCI mode was 100%. CONCLUSIONS: The new CAD system used in LCI mode achieved a 100% sensitivity per lesion and a negligible false-positive frame rate. Note that the new CAD system used in LCI mode also specifically allowed for detection of serrated lesions in all cases. Accordingly, the AI algorithm introduced here for the first time has the potential to dramatically improve the quality of colonoscopy.