Contrast enhanced endoscopic ultrasound detects early therapy response following anti-TNF-therapy in patients with ulcerative colitis.

BACKGROUND AND AIM: Though colonoscopy plays a crucial role in assessing active ulcerative colitis (aUC), its scope is limited to the mucosal surface. Endoscopic ultrasound (EUS) coupled with contrast-enhancement (dCEUS) can precisely quantify bowel wall thickness and microvascular circulation, potentially enabling the quantitative evaluation of inflammation.We conducted a prospective, longitudinal study to assess therapy response using dCEUS in aUC patients undergoing treatment with adalimumab (ADA) or infliximab (IFX). METHODS: 30 ADA- and 15 IFX-treated aUC patients were examined at baseline and at 2, 6, 14 weeks of therapy and 48 weeks of follow-up. Bowel wall thickness (BWT) was measured by EUS in the rectum. Vascularity was quantified by dCEUS using Rise Time (RT) and Time To Peak (TTP). Therapy response was defined after 14 weeks using the Mayo Score. RESULTS: Patients with aUC displayed a mean BWT of 3.9±0.9 mm. In case of response to ADA/IFX a significant reduction in BWT was observed after 2 weeks (p=0.04), whereas non-responders displayed no significant changes. The TTP was notably accelerated at baseline and significantly normalised by week 2 in responders (p=0.001), while non-responders exhibited no significant alterations (p=0.9). At week 2, the endoscopic Mayo score did not exhibit any changes, thus failing to predict treatment responses. CONCLUSION: dCEUS enables the early detection of therapy response in patients with aUC, which serves as a predictive marker for long term clinical success. Therefore, dCEUS serves as a diagnostic tool for assessing the probability of future therapy success.

New insights into neuropathology and pathogenesis of autoimmune glial fibrillary acidic protein meningoencephalomyelitis.

Anti-glial fibrillary acidic protein (GFAP) meningoencephalomyelitis (autoimmune GFAP astrocytopathy) is a new autoimmune central nervous system (CNS) disease diagnosable by the presence of anti-GFAP autoantibodies in the cerebrospinal fluid and presents as meningoencephalomyelitis in the majority of patients. Only few neuropathological reports are available and little is known about the pathogenic mechanisms. We performed a histopathological study of two autopsies and nine CNS biopsies of patients with anti-GFAP autoantibodies and found predominantly a lymphocytic and in one autopsy case a granulomatous inflammatory phenotype. Inflammatory infiltrates were composed of B and T cells, including tissue-resident memory T cells. Although obvious astrocytic damage was absent in the GFAP-staining, we found cytotoxic T cell-mediated reactions reflected by the presence of CD8+/perforin+/granzyme A/B+ cells, polarized towards astrocytes. MHC-class-I was upregulated in reactive astrocytes of all biopsies and two autopsies but not in healthy controls. Importantly, we observed a prominent immunoreactivity of astrocytes with the complement factor C4d. Finally, we provided insight into an early phase of GFAP autoimmunity in an autopsy of a pug dog encephalitis that was characterized by marked meningoencephalitis with selective astrocytic damage with loss of GFAP and AQP4 in the lesions.Our histopathological findings indicate that a cytotoxic T cell-mediated immune reaction is present in GFAP autoimmunity. Complement C4d deposition on astrocytes could either represent the cause or consequence of astrocytic reactivity. Selective astrocytic damage is prominent in the early phase of GFAP autoimmunity in a canine autopsy case, but mild or absent in subacute and chronic stages in human disease, probably due to the high regeneration potential of astrocytes. The lymphocytic and granulomatous phenotypes might reflect different stages of lesion development or patient-specific modifications of the immune response. Future studies will be necessary to investigate possible implications of pathological subtypes for clinical disease course and therapeutic strategies.

Aß38 and Aß43 do not differentiate between Alzheimer's disease and cerebral amyloid angiopathy.

Differential diagnosis between Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) using cerebrospinal fluid (CSF) biomarkers is challenging. A recent study suggested that the addition of Aß38 and Aß43 to a standard AD biomarker panel (Aß40, Aß42, t-tau, p-tau) to improve the differential diagnosis. We tested this hypothesis in an independent German cohort of CAA and AD patients and controls using the same analytical techniques. We found excellent discrimination between AD and controls and between CAA and controls, but not between AD and CAA. Adding Aß38 and Aß43 to the panel did not improve the discrimination between AD and CAA.

Human APOE4 Protects High-Fat and High-Sucrose Diet Fed Targeted Replacement Mice against Fatty Liver Disease Compared to APOE3.

Recent genome- and exome-wide association studies suggest that the human APOE ε4 allele protects against non-alcoholic fatty liver disease (NAFLD), while ε3 promotes hepatic steatosis and steatohepatitis. The present study aimed at examining the APOE genotype-dependent development of fatty liver disease and its underlying mechanisms in a targeted replacement mouse model. Male mice expressing the human APOE3 or APOE4 protein isoforms on a C57BL/6J background and unmodified C57BL/6J mice were chronically fed a high-fat and high-sucrose diet to induce obesity. After 7 months, body weight gain was more pronounced in human APOE than endogenous APOE expressing mice with elevated plasma biomarkers suggesting aggravated metabolic dysfunction. APOE3 mice exhibited the highest liver weights and, compared to APOE4, massive hepatic steatosis. An untargeted quantitative proteome analysis of the liver identified a high number of proteins differentially abundant in APOE3 versus APOE4 mice. The majority of the higher abundant proteins in APOE3 mice could be grouped to inflammation and damage-associated response, and lipid storage, amongst others. Results of the targeted qRT-PCR and Western blot analyses contribute to the overall finding that APOE3 as opposed to APOE4 promotes hepatic steatosis, inflammatory- and damage-associated response signaling and fibrosis in the liver of obese mice. Our experimental data substantiate the observation of an increased NAFLD-risk associated with the human APOEε3 allele, while APOEε4 appears protective. The underlying mechanisms of the protection possibly involve a higher capacity of nonectopic lipid deposition in subcutaneous adipose tissue and lower hepatic pathogen recognition in the APOE4 mice.

Correlation of 99mTc-DPD bone scintigraphy with histological amyloid load in patients with ATTR cardiac amyloidosis.

BACKGROUND: The significance of measuring 99mTc-labelled-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) in transthyretin (ATTR) cardiac amyloidosis has not been adequately studied. This single-centre observational study evaluated the correlation between 99mTc-DPD scintigraphy and histological amyloid load in endomyocardial biopsy (EMB). METHODS: Twenty-eight patients with biopsy-proven ATTR amyloidosis and concomitantly available 99mTc-DPD scintigraphy were included. Visual Perugini scoring, and (semi-)quantitative analysis of cardiac 99mTc-DPD uptake by planar whole-body imaging and single photon emission computed tomography (SPECT/CT) using regions of interest (ROI) were performed. From this, heart-to-whole-body ratio (H/WB) and heart-to-contralateral-chest ratio (H/CL) were calculated. The histological amyloid load was quantified using two different staining methods. RESULTS: Increased cardiac tracer uptake was documented in all patients (planar: ROImean 129 ± 37 cps; SPECT/CT: ROImean 369 ± 142 cps). Histological amyloid load (19 ± 13%) significantly correlated with Perugini score (r = 0.69, p < .001) as well as with cardiac 99mTc-DPD uptake (planar: r = 0.64, p < .001; H/WB: r = 0.50, p = .014; SPECT/CT: r = 0.53, p = .008; H/CL: r = 0.43, p = .037) (results are shown for correlations with Congo Red-staining). CONCLUSION: In ATTR, cardiac 99mTc-DPD uptake significantly correlated with histological amyloid load in EMB. Further studies are needed to implement thresholds in cardiac 99mTc-DPD uptake measurements for risk stratification and guidance of therapy.

Exploring the Interplay between Nutrients, Bacteriophages, and Bacterial Lipases in Host- and Bacteria-mediated Pathogenesis.

BACKGROUND AND AIMS: Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. MATERIALS AND METHODS: 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. RESULTS: Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1ß, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. CONCLUSION: The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.

Exploration of the tumor biological significance of PCLO in gastric cancer - Results from a large Central European cohort.

INTRODUCTION: A recent multiregional whole exome sequencing of 48 tumour samples from 9 gastric adenocarcinomas discovered PCLO mutations in 23 (47.9%) tumour samples. Based on that unexpected high prevalence of PCLO mutations, we hypothesized a tumour biological significance of PCLO in gastric cancer (GC). METHODS: Tumour samples (whole tissue sections) obtained from 466 patients resected for therapy-naive GC were stained with an anti-PCLO antibody. The Histoscore for tumour cells and the presence of immunostaining of stromal cells and tumour vessels was docu-mented for each case. An algorithm for PCLO immunopositivity was formed and correlat-ed with clinicopathological patient characteristics. RESULTS: 175 GCs were classified as PCLO-positive within tumour cells, and 291 as negative. Stromal cells were positive for PCLO in 106 cases and tumour vessels in 84. PCLO positive GCs more often showed an intestinal phenotype, a lower T-category and were more commonly associated with Helicobacter pylori-infection. A separate analysis of PCLO ex-pression in intestinal and diffuse type GCs, respectively, showed no significant correla-tions. Patients with PCLO negative/low tumour cells showed a shortened overall (14.0±1.4 vs. 16.0±1.8 months) and tumour specific survival (15.0±1.6 months vs. 17.9±3.6). Compar-ison of PCLO's genotype with its phenotype in 48 tumour samples obtained from nine cases showed no direct correlations with missense mutations. DISCUSSION/CONCLUSION: Our data provide evidence that PCLO is differentially expressed in GC and might delay tumour progression.

Deep learning trained on lymph node status predicts outcome from gastric cancer histopathology: a retrospective multicentric study.

AIM: Gastric cancer (GC) is a tumour entity with highly variant outcomes. Lymph node metastasis is a prognostically adverse biomarker. We hypothesised that GC primary tissue contains information that is predictive of lymph node status and patient prognosis and that this information can be extracted using deep learning (DL). METHODS: Using three patient cohorts comprising 1146 patients, we trained and validated a DL system to predict lymph node status directly from haematoxylin and eosin-stained GC tissue sections. We investigated the concordance between the DL-based prediction from the primary tumour slides (aiN score) and the histopathological lymph node status (pN). Furthermore, we assessed the prognostic value of the aiN score alone and when combined with the pN status. RESULTS: The aiN score predicted the pN status reaching area under the receiver operating characteristic curves of 0.71 in the training cohort and 0.69 and 0.65 in the two test cohorts. In a multivariate Cox analysis, the aiN score was an independent predictor of patient survival with hazard ratios of 1.5 in the training cohort and of 1.3 and 2.2 in the two test cohorts. A combination of the aiN score and the pN status prognostically stratified patients by survival with p-values <0.05 in logrank tests. CONCLUSION: GC primary tumour tissue contains additional prognostic information that is accessible using the aiN score. In combination with the pN status, this can be used for personalised management of GC patients after prospective validation.

MDM2 amplification is rare in gastric cancer.

The MDM2 proto-oncogene (MDM2) is a primary negative regulator of p53. The latter is frequently mutated in gastric cancer (GC). In the present study, we aimed to validate gene amplification, protein expression, and the putative tumor biological function of MDM2 in a well-characterized Western GC cohort. MDM2 amplification and protein expression were studied in a cohort of 327 GCs by fluorescence in situ hybridization (FISH) and immunohistochemistry. Gene amplification and protein expression were correlated with diverse clinicopathological patient characteristics including patient outcome. Immunohistochemically, 97 GCs (29.7%) were categorized as MDM2 positive and 230 GCs (70.3%) as negative. An amplification of MDM2 was found in 11 (3.4%) cases without evidence of intratumoral heterogeneity. Nine of these eleven (81.8%) cases showed MDM2 protein expression. MDM2 amplification correlated significantly with MDM2 protein expression (p < 0.001). On a case-by-case analysis, MDM2-amplified cases showed varied histological phenotypes and were most commonly microsatellite stable; EBV, HER2, and MET negative; and FGFR2 positive. A single case harbored both, MDM2 amplification and TP53 mutation. MDM2 amplification and MDM2 expression, respectively, did not correlate with overall or tumor-specific survival. Our targeted analysis of MDM2 in a well-characterized cohort of GC patients showed that MDM2 amplification is rare, of no specific histological phenotype, and may not be always mutually exclusive with TP53 mutations. Given the low number of cases, currently, no diagnostic or therapeutic recommendation related to MDM2 amplification can be given for GC of Western origin.

Surgical approach for convexity meningiomas: An analysis of the preoperative clinical signs, radiological features and surgical outcomes of these tumors.

BACKGROUND: Convexity meningiomas (CM) can be successfully treated with neurosurgery. However, clinical complications due to CM have been reported. Moreover, systematic investigations of CM with respect to all relevant clinical factors are currently lacking. METHODS: We performed a systematic investigation in 210 patients with supratentorial CM considering all relevant clinical and radiological factors, with a follow-up time of 19.5 years. RESULTS: Among 812 patients with intracranial meningiomas treated in our department (2003-2020), 28.2 % of intracranial meningiomas were located over the supratentorial convexity, and the patients had a median age of 62 years (95 % CI:59-64). The median follow-up was 30.4 months (95 % CI:21.6-37.1). Tumor-related symptoms were observed in 88.1 % of patients. The most common preoperative symptom was headache (28.1 %), followed by seizure (19.5 %). Symptomatic patients had significantly higher tumor volumes than asymptomatic patients (p = 0.0003; 24.5 cm3 and 6.98 cm3, respectively). Complete tumor resection was achieved in 92.9 % of patients. The most common postoperative complication was bleeding (7.1 %) in the approach area. Of all bleedings, only three were intracerebral hemorrhages and did not require surgical intervention. The second most common complication was postoperative seizure (4.7 %). The multiple logistic regression analyses showed that tumor volume (OR:1.007; 95 % CI:1.001-1.013; p = 0.02) and brain infiltration by the tumor (OR:1.961; 95 % CI:1.028-3.741; p = 0.04) had a significant impact on postoperative complications. The postoperative and final KPS scores significantly improved (p < 0.001). The tumor recurrence rate was 6.2 %, with a median time of 38 months. No surgery-related deaths occurred. CONCLUSION: A large tumor volume and brain infiltration by the tumor were significant factors for postoperative complications. The clinical conditions significantly improved postoperatively and further during the follow-up period.

Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma.

Introduction: Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages. Methods: Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations. Results: Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells. Conclusion: Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer.

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

Prevalence of amyloid in ligamentum flavum of patients with lumbar spinal stenosis.

BACKGROUND: Transthyretin (ATTR) amyloidosis is often diagnosed in an advanced stage, when irreversible cardiac damage has occurred. Lumbar spinal stenosis (LSS) may precede cardiac ATTR amyloidosis by many years, offering the opportunity to detect ATTR already at the time of LSS surgery. We prospectively assessed the prevalence of ATTR in the ligamentum flavum by tissue biopsy in patients aged >50 years undergoing surgery for LSS. METHODS: Ligamentum flavum thickness was assessed pre-operatively on axial T2 magnetic resonance imaging (MRI) slices. Tissue samples from ligamentum flavum were screened centrally by Congo red staining and immunohistochemistry (IHC). RESULTS: Amyloid in the ligamentum flavum was detected in 74/94 patients (78.7%). IHC revealed ATTR in 61 (64.9%), whereas amyloid subtyping was inconclusive in 13 (13.8%). Mean thickness of ligamentum flavum was significantly higher at all levels in patients with amyloid (p < .05). Patients with amyloid deposits were older (73.1 ± 9.2 vs. 64.6 ± 10.1 years, p = .01). No differences in sex, comorbidities, previous surgery for carpal tunnel syndrome or LSS were observed. CONCLUSIONS: Amyloid, mostly of the ATTR subtype, was found in four out of five patients with LSS and is associated with age and ligamentum flavum thickness. Histopathological work-up of ligamentum flavum might inform future decision making.

LAG3 in gastric cancer: it's complicated.

PURPOSE: Lymphocyte activation gene 3 (LAG3) is thought to contribute to T cell exhaustion within the tumor microenvironment of solid tumors. This study aimed to analyze the spatial distribution of LAG3 + cells in relation to clinicopathological and survival data in a large set of 580 primary resected and neoadjuvantly treated gastric cancers (GC). METHODS: LAG3 expression was evaluated in tumor center and invasive margin using immunohistochemistry and whole-slide digital image analysis. Cases were divided into LAG3-low and LAG3-high expression groups based on (1) median LAG3 + cell density, (2) cut-off values adapted to cancer-specific survival using Cutoff Finder application. RESULTS: Significant differences in spatial distribution of LAG3 + cells were observed in primarily resected GC, but not in neoadjuvantly treated GC. LAG3 + cell density showed evident prognostic value at following cut-offs: in primarily resected GC, 21.45 cells/mm2 in tumor center (17.9 vs. 10.1 months, p = 0.008) and 208.50 cells/mm2 in invasive margin (33.8 vs. 14.7 months, p = 0.006); and in neoadjuvantly treated GC, 12.62 cells/mm2 (27.3 vs. 13.2 months, p = 0.003) and 123.00 cells/mm2 (28.0 vs. 22.4 months, p = 0.136), respectively. Significant associations were found between LAG3 + cell distribution patterns and various clinicopathological factors in both cohorts. In neoadjuvantly treated GC, LAG3 + immune cell density was found to be an independent prognostic factor of survival (HR = 0.312, 95% CI 0.162-0.599, p < 0.001). CONCLUSION: In this study, a higher density of LAG3 + cells was associated with favorable prognosis. Current results support the need for extended analysis of LAG3. Differences in the distribution of LAG3 + cells should be considered, as they could influence clinical outcomes and treatment responses.

Deep learning-based subtyping of gastric cancer histology predicts clinical outcome: a multi-institutional retrospective study.

INTRODUCTION: The Laurén classification is widely used for Gastric Cancer (GC) histology subtyping. However, this classification is prone to interobserver variability and its prognostic value remains controversial. Deep Learning (DL)-based assessment of hematoxylin and eosin (H&E) stained slides is a potentially useful tool to provide an additional layer of clinically relevant information, but has not been systematically assessed in GC. OBJECTIVE: We aimed to train, test and externally validate a deep learning-based classifier for GC histology subtyping using routine H&E stained tissue sections from gastric adenocarcinomas and to assess its potential prognostic utility. METHODS: We trained a binary classifier on intestinal and diffuse type GC whole slide images for a subset of the TCGA cohort (N = 166) using attention-based multiple instance learning. The ground truth of 166 GC was obtained by two expert pathologists. We deployed the model on two external GC patient cohorts, one from Europe (N = 322) and one from Japan (N = 243). We assessed classification performance using the Area Under the Receiver Operating Characteristic Curve (AUROC) and prognostic value (overall, cancer specific and disease free survival) of the DL-based classifier with uni- and multivariate Cox proportional hazard models and Kaplan-Meier curves with log-rank test statistics. RESULTS: Internal validation using the TCGA GC cohort using five-fold cross-validation achieved a mean AUROC of 0.93 ± 0.07. External validation showed that the DL-based classifier can better stratify GC patients' 5-year survival compared to pathologist-based Laurén classification for all survival endpoints, despite frequently divergent model-pathologist classifications. Univariate overall survival Hazard Ratios (HRs) of pathologist-based Laurén classification (diffuse type versus intestinal type) were 1.14 (95% Confidence Interval (CI) 0.66-1.44, p-value = 0.51) and 1.23 (95% CI 0.96-1.43, p-value = 0.09) in the Japanese and European cohorts, respectively. DL-based histology classification resulted in HR of 1.46 (95% CI 1.18-1.65, p-value < 0.005) and 1.41 (95% CI 1.20-1.57, p-value < 0.005), in the Japanese and European cohorts, respectively. In diffuse type GC (as defined by the pathologist), classifying patients using the DL diffuse and intestinal classifications provided a superior survival stratification, and demonstrated statistically significant survival stratification when combined with pathologist classification for both the Asian (overall survival log-rank test p-value < 0.005, HR 1.43 (95% CI 1.05-1.66, p-value = 0.03) and European cohorts (overall survival log-rank test p-value < 0.005, HR 1.56 (95% CI 1.16-1.76, p-value < 0.005)). CONCLUSION: Our study shows that gastric adenocarcinoma subtyping using pathologist's Laurén classification as ground truth can be performed using current state of the art DL techniques. Patient survival stratification seems to be better by DL-based histology typing compared with expert pathologist histology typing. DL-based GC histology typing has potential as an aid in subtyping. Further investigations are warranted to fully understand the underlying biological mechanisms for the improved survival stratification despite apparent imperfect classification by the DL algorithm.

A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

An integrated cellular and molecular model of gastric neuroendocrine cancer evolution highlights therapeutic targets.

Gastric neuroendocrine carcinomas (G-NEC) are aggressive malignancies with poorly understood biology and a lack of disease models. Here, we use genome sequencing to characterize the genomic landscapes of human G-NEC and its histologic variants. We identify global and subtype-specific alterations and expose hitherto unappreciated gains of MYC family members in a large part of cases. Genetic engineering and lineage tracing in mice delineate a model of G-NEC evolution, which defines MYC as a critical driver and positions the cancer cell of origin to the neuroendocrine compartment. MYC-driven tumors have pronounced metastatic competence and display defined signaling addictions, as revealed by large-scale genetic and pharmacologic screening of cell lines and organoid resources. We create global maps of G-NEC dependencies, highlight critical vulnerabilities, and validate therapeutic targets, including candidates for clinical drug repurposing. Our study gives comprehensive insights into G-NEC biology.

ATTR- and AFib amyloid - two different types of amyloid in the annular ligament of trigger finger.

INTRODUCTION: Histological examination of tissue specimens obtained during surgical treatment of trigger finger frequently encountered unclassifiable amyloid deposits in the annular ligament. We systematically explored this unknown type by a comprehensive analysis using histology, immunohistochemistry, and quantitative mass spectrometry-based proteomics. METHODS: 205 tissue specimens of annular ligaments were obtained from 172 patients. Each specimen was studied by histology and immunohistochemistry. Tissue specimens obtained from ten patients with histology proven amyloid in annular ligament were analysed by label-free quantitative proteomics. Histological and immunohistochemical findings were correlated with patient demographics. RESULTS: Amyloid was present as band like deposits along the surface of annular ligament, dot like or patchy deposits within the matrix. Immunohistochemistry identified ATTR amyloid in 92 specimens (mostly patchy in the matrix), while the band like deposits of 100 specimens remained unclassifiable. Proteomic profiles identified the unknown amyloid as most likely of fibrinogen origin. The complete cohort was re-examined by immunohistochemistry using a custom-made antibody and confirmed the presence of fibrinogen alpha-chain (FGA) in a hitherto unclassifiable type of amyloid in annular ligament. CONCLUSION: Our study shows that two different types of amyloid affect the annular ligament, ATTR amyloid and AFib amyloid, with distinct demographic patient characteristics and histomorphological deposition patterns.

Frequency of deep-seated cerebral microbleeds in patients with lobar hemorrhages and histopathological evidence for cerebral amyloid angiopathy.

Background: Cerebral amyloid angiopathy (CAA) is a common disease and the most common cause of lobar hemorrhages in the elderly. Usually, deep-seated microhemorrhages preclude the diagnosis of CAA. In this study, we sought to estimate the frequency of deep-seated microbleeds on MRI in patients with lobar hemorrhages and histopathological evidence for cerebral amyloid angiopathy. In addition, we describe a cohort of patients with cortical and deep-seated microbleeds on MRI and a histopathological specimen available from lobar hematoma evacuation. Methods: Retrospective database search for histopathological specimens from lobar hematoma evacuation and review of imaging findings (CT and MRI) and patient charts was performed. Results: Between 1 January 2012 and 31 December 2020, 88 specimens from 88 patients were available. A total of 56 specimens were excluded (no brain tissue in the specimen n = 4, other diagnosis n = 8, no MRI n = 43, and no BOLD-based sequence n = 1). Of the remaining 32 patients, 25 patients (78%) did not harbor deep-seated lesions on MRI, of which 17 patients had histopathological features of CAA. A total of seven patients harbored deep-seated CMB. Of these seven patients, three (3/20, 15%) had histopathological features of CAA. Conclusion: Approximately 15% of patients with histopathologically diagnosed CAA harbor deep-seated microbleeds. This finding may add to the discussion on how to identify patients with CAA and deep-seated CMB.

Hepatic myofibroblasts exert immunosuppressive effects independent of the immune checkpoint regulator PD-L1 in liver metastasis of pancreatic ductal adenocarcinoma.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) represents the 4th most common cause of cancer-related deaths in Western countries. Most patients are diagnosed at advanced stages, often already with metastases. The main site of metastasis is the liver and hepatic myofibroblasts (HMF) play a pivotal role in metastatic outgrowth. Immune checkpoint inhibitors (ICI) targeting programmed death ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) improved treatment of several cancers but not of PDAC. Therefore, this study aimed to better understand the impact of HMF on PD-L1 expression and immune evasion of PDAC cells during liver metastasis. Methods: Formalin-fixed and paraffin embedded biopsy samples or diagnostic resection specimens from liver metastases of 15 PDAC patients were used for immunohistochemical analyses. Serial sections were stained with antibodies directed against Pan-Cytokeratin, αSMA, CD8, and PD-L1. To investigate whether the PD-1/PD-L1 axis and HMF contribute to immune escape of PDAC liver metastases, a stroma enriched 3D spheroid coculture model was established in vitro, using two different PDAC cell lines, HMF, and CD8+ T cells. Here, functional and flow cytometry analyses were conducted. Results: Immunohistochemical analysis of liver tissue sections of PDAC patients revealed that HMF represent an abundant stroma population in liver metastases, with clear differences in the spatial distribution in small (1500 µm) and large (> 1500 µm) metastases. In the latter, PD-L1 expression was mainly located at the invasion front or evenly distributed, while small metastases either lacked PD-L1 expression or showed mostly weak expression in the center. Double stainings revealed that PD-L1 is predominantly expressed by stromal cells, especially HMF. Small liver metastases with no or low PD-L1 expression comprised more CD8+ T cells in the tumor center, while large metastases exhibiting stronger PD-L1 expression comprised less CD8+ T cells being mostly located at the invasion front. HMF-enriched spheroid cocultures with different ratios of PDAC cells and HMF well mimicking conditions of hepatic metastases in situ. Here, HMF impaired the release of effector molecules by CD8+ T cells and the induction of PDAC cell death, an effect that was dependent on the amount of HMF but also of PDAC cells. ICI treatment led to elevated secretion of distinct CD8+ T cell effector molecules but did not increase PDAC cell death under either spheroid condition. Conclusion: Our findings indicate a spatial reorganization of HMF, CD8+ T cells, and PD-L1 expression during progression of PDAC liver metastases. Furthermore, HMF potently impair the effector phenotype of CD8+ T cells but the PD-L1/PD-1 axis apparently plays a minor role in this scenario suggesting that immune evasion of PDAC liver metastases relies on other immunosuppressive mechanisms.