Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Combined analysis of albumin in situ hybridisation and C reactive protein immunohistochemistry for the diagnosis of intrahepatic cholangiocarcinoma: towards a molecular classification paradigm.

AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a diagnosis of exclusion that can pose a challenge to the pathologist despite thorough clinical workup. Although several immunohistochemical markers have been proposed for iCCA, none of them reached clinical practice. We here assessed the combined usage of two promising diagnostic approaches, albumin in situ hybridisation (Alb-ISH) and C reactive protein (CRP) immunohistochemistry, for distinguishing iCCA from other adenocarcinoma primaries. METHODS: We conducted Alb-ISH and CRP immunohistochemistry in a large European iCCA cohort (n=153) and compared the results with a spectrum of other glandular adenocarcinomas of different origin (n=885). In addition, we correlated expression patterns with clinicopathological information and mutation data. RESULTS: Alb-ISH was highly specific for iCCA (specificity 98.8%) with almost complete negativity in perihilar CCA and only rare positives among other adenocarcinomas (sensitivity 69.5%). CRP identified the vast majority of iCCA cases (sensitivity 84.1%) at a lower specificity of 86.4%. Strikingly, the combination of CRP and Alb-ISH boosted the diagnostic sensitivity to 88.0% while retaining a considerable specificity of 86.1%. Alb-ISH significantly correlated with CRP expression, specific tumour morphologies and small or large duct iCCA subtypes. Neither Alb-ISH nor CRP was associated with iCCA patient survival. 16 of 17 recurrent mutations in either IDH1, IDH2 and FGFR2 affected Alb-ISH positive cases, while the only KRAS mutation corresponded to an Alb-ISH negative case. CONCLUSIONS: In conclusion, we propose a sequential diagnostic approach for iCCA, integrating CRP immunohistochemistry and Alb-ISH. This may improve the accuracy of CCA classification and pave the way towards a molecular-guided CCA classification.

Differential Immunoexpression of Inhibitory Immune Checkpoint Molecules and Clinicopathological Correlates in Keratoacanthoma, Primary Cutaneous Squamous Cell Carcinoma and Metastases.

Beyond established anti-programmed cell death protein 1/programmed cell death ligand 1 immunotherapy, T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) and its ligand CD155 are promising novel inhibitory immune checkpoint targets in human malignancies. Yet, in cutaneous squamous cell carcinoma, evidence on the collective expression patterns of these inhibitory immune checkpoints is scarce. Complete tumour sections of 36 cutaneous squamous cell carcinoma, 5 cutaneous metastases and 9 keratoacanthomas, a highly-differentiated, squamoproliferative tumour, with disparately benign biologic behaviour, were evaluated by immunohistochemistry for expression of programmed cell death ligand 1 (Tumor Proportion Score, Immune Cell Score), TIGIT, CD155 and CD8+ immune infiltrates. Unlike keratoacanthomas, cutaneous squamous cell carcinoma displayed a strong positive correlation of programmed cell death ligand 1 Tumor Proportion Score and CD115 expression (p < 0.001) with significantly higher programmed cell death ligand 1 Tumor Proportion Score (p < 0.001) and CD155 expression (p < 0.01) in poorly differentiated G3-cutaneous squamous cell carcinoma compared with keratoacanthomas. TIGIT+ infiltrates were significantly increased in programmed cell death ligand 1 Immune Cell Score positive primary tumours (p = 0.05). Yet, a strong positive correlation of TIGIT expression with CD8+ infiltrates was only detected in cutaneous squamous cell carcinoma (p < 0.01), but not keratoacanthomas. Providing a comprehensive overview on the collective landscape of inhibitory immune checkpoint expression, this study reveals associations of novel inhibitory immune checkpoint with CD8+ immune infiltrates and tumour differentiation and highlights the TIGIT/CD155 axis as a potential new target for cutaneous squamous cell carcinoma immunotherapy.

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Etiology-independent activation of the LTß-LTßR-RELB axis drives aggressiveness and predicts poor prognosis in HCC.

BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

The crosstalk between glomerular endothelial cells and podocytes controls their responses to metabolic stimuli in diabetic nephropathy.

In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of "immediate early response" genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.

The role of bariatric surgery on beta-cell function and insulin resistance in patients with nonalcoholic fatty liver disease and steatohepatitis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are strongly associated with obesity, metabolic syndrome, and insulin resistance (IR). OBJECTIVE: The aim of this study was to investigate the effects of metabolic surgery on pancreatic beta cell function and IR in patients with obesity and NAFLD. SETTING: University Hospital, Germany. METHODS: Liver biopsies were taken intraoperatively from 112 patients undergoing sleeve gastrectomy (n = 68) or Roux-en-Y gastric bypass (n = 44) and analyzed histologically for the presence of simple steatosis (NAFL) or NASH. Clinical and biochemical parameters were collected over up to 2 years. Beta cell function and IR were assessed using the homeostasis model assessment of beta-cell function (HOMA2-%B) and insulin resistance (HOMA2-IR) index. RESULTS: NASH was present in 53.6% (n = 60) of the patients and NAFL in 25.9% (n = 29). Liver enzymes, adiponectin/leptin ratio, triglycerides, and HbA1C were improved at 6 months, 1, and 2 years after surgery. HOMA2-IR was significantly lower in patients without NAFLD while HOMA2-IR did not differ between patients with NAFL and/or NASH. HOMA2-%B was highest in the NAFLD group and lowest in patients with NASH. While there was no change in HOMA2-%B and HOMA2-IR in the No-NAFLD group, HOMA2-%B decreased and IR improved in the NAFL and NASH groups. CONCLUSION: Insufficient compensatory beta-cell function may contribute to the progression from NAFL alongside with IR to NASH. Our findings suggest that bariatric surgery decreases IR while at the same time reducing compensatory insulin oversecretion. These results are associated with beneficial changes in adipose tissue function after bariatric surgery.

Deep Learning-Enabled Diagnosis of Liver Adenocarcinoma.

BACKGROUND & AIMS: Diagnosis of adenocarcinoma in the liver is a frequent scenario in routine pathology and has a critical impact on clinical decision making. However, rendering a correct diagnosis can be challenging, and often requires the integration of clinical, radiologic, and immunohistochemical information. We present a deep learning model (HEPNET) to distinguish intrahepatic cholangiocarcinoma from colorectal liver metastasis, as the most frequent primary and secondary forms of liver adenocarcinoma, with clinical grade accuracy using H&E-stained whole-slide images. METHODS: HEPNET was trained on 714,589 image tiles from 456 patients who were randomly selected in a stratified manner from a pool of 571 patients who underwent surgical resection or biopsy at Heidelberg University Hospital. Model performance was evaluated on a hold-out internal test set comprising 115 patients and externally validated on 159 patients recruited at Mainz University Hospital. RESULTS: On the hold-out internal test set, HEPNET achieved an area under the receiver operating characteristic curve of 0.994 (95% CI, 0.989-1.000) and an accuracy of 96.522% (95% CI, 94.521%-98.694%) at the patient level. Validation on the external test set yielded an area under the receiver operating characteristic curve of 0.997 (95% CI, 0.995-1.000), corresponding to an accuracy of 98.113% (95% CI, 96.907%-100.000%). HEPNET surpassed the performance of 6 pathology experts with different levels of experience in a reader study of 50 patients (P = .0005), boosted the performance of resident pathologists to the level of senior pathologists, and reduced potential downstream analyses. CONCLUSIONS: We provided a ready-to-use tool with clinical grade performance that may facilitate routine pathology by rendering a definitive diagnosis and guiding ancillary testing. The incorporation of HEPNET into pathology laboratories may optimize the diagnostic workflow, complemented by test-related labor and cost savings.

Implementation of deep learning in liver pathology optimizes diagnosis of benign lesions and adenocarcinoma metastasis.

INTRODUCTION: Differentiation of histologically similar structures in the liver, including anatomical structures, benign bile duct lesions, or common types of liver metastases, can be challenging with conventional histological tissue sections alone. Accurate histopathological classification is paramount for the diagnosis and adequate treatment of the disease. Deep learning algorithms have been proposed for objective and consistent assessment of digital histopathological images. MATERIALS AND METHODS: In the present study, we trained and evaluated deep learning algorithms based on the EfficientNetV2 and ResNetRS architectures to discriminate between different histopathological classes. For the required dataset, specialized surgical pathologists annotated seven different histological classes, including different non-neoplastic anatomical structures, benign bile duct lesions, and liver metastases from colorectal and pancreatic adenocarcinoma in a large patient cohort. Annotation resulted in a total of 204.159 image patches, followed by discrimination analysis using our deep learning models. Model performance was evaluated on validation and test data using confusion matrices. RESULTS: Evaluation of the test set based on tiles and cases revealed overall highly satisfactory prediction capability of our algorithm for the different histological classes, resulting in a tile accuracy of 89% (38 413/43 059) and case accuracy of 94% (198/211). Importantly, the separation of metastasis versus benign lesions was certainly confident on case level, confirming the classification model performed with high diagnostic accuracy. Moreover, the whole curated raw data set is made publically available. CONCLUSIONS: Deep learning is a promising approach in surgical liver pathology supporting decision making in personalized medicine.

How entanglements determine the morphology of semicrystalline polymers.

Crystallization of polymers from entangled melts generally leads to the formation of semicrystalline materials with a nanoscopic morphology consisting of stacks of alternating crystalline and amorphous layers. The factors controlling the thickness of the crystalline layers are well studied; however, there is no quantitative understanding of the thickness of the amorphous layers. We elucidate the effect of entanglements on the semicrystalline morphology by the use of a series of model blends of high-molecular-weight polymers with unentangled oligomers leading to a reduced entanglement density in the melt as characterized by rheological measurements. Small-angle X-ray scattering experiments after isothermal crystallization reveal a reduced thickness of the amorphous layers, while the crystal thickness remains largely unaffected. We introduce a simple, yet quantitative model without adjustable parameters, according to which the measured thickness of the amorphous layers adjusts itself in such a way that the entanglement concentration reaches a specific maximum value. Furthermore, our model suggests an explanation for the large supercooling that is typically required for crystallization of polymers if entanglements cannot be dissolved during crystallization.

DNA methylation-based classifier differentiates intrahepatic pancreato-biliary tumours.

BACKGROUND: Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is challenging. Both tumours have similar morphological and immunohistochemical pattern and share multiple driver mutations. We hypothesised that DNA methylation-based machine-learning algorithms may help perform this task. METHODS: We assembled genome-wide DNA methylation data for iCCA (n = 259), PAAD (n = 431), and normal bile duct (n = 70) from publicly available sources. We split this cohort into a reference (n = 399) and a validation set (n = 361). Using the reference cohort, we trained three machine learning models to differentiate between these entities. Furthermore, we validated the classifiers on the technical validation set and used an internal cohort (n = 72) to test our classifier. FINDINGS: On the validation cohort, the neural network, support vector machine, and the random forest classifiers reached accuracies of 97.68%, 95.62%, and 96.5%, respectively. Filtering by anomaly detection and thresholds improved the accuracy to 99.07% (37 samples excluded by filtering), 96.22% (17 samples excluded), and 100% (44 samples excluded) for the neural network, support vector machine and random forest, respectively. Because of best balance between accuracy and number of predictable cases we tested the neural network with applied filters on the in-house cohort, obtaining an accuracy of 95.45%. INTERPRETATION: We developed a classifier that can differentiate between iCCAs, intrahepatic metastases of a PAAD, and normal bile duct tissue with high accuracy. This tool can be used for improving the diagnosis of pancreato-biliary cancers of the liver. FUNDING: This work was supported by Berlin Institute of Health (JCS Program), DKTK Berlin (Young Investigator Grant 2022), German Research Foundation (493697503 and 314905040 - SFB/TRR 209 Liver Cancer B01), and German Cancer Aid (70113922).

Cortical Desmoid of the Distal Femur-Incidentaloma or Insertional Tendinopathy?

BACKGROUND: The cortical desmoid (DFCI) of the posteromedial femoral condyle is considered an asymptomatic incidental finding in adolescents without clinical relevance. The aim of this study was to evaluate the clinical relevance of DFCI from both a tumor orthopedic and sports medicine point of view. METHODS: n = 23 patients (13.74 ± 2.74 years; nineteen female, four male) with DFCI of the posteromedial femoral condyle were included. A localized posteromedial knee pain on exertion was differentiated from non-specific knee pain. Symptom duration, additional pathologies, number of MRIs, sports activity and training intensity, downtime, therapeutic modalities, and relief/remission of symptoms were documented. The Tegner activity scale (TAS) and Lysholm score (LS) were collected. The influence of specific posteromedial pain, MRI presence of paratendinous cysts, sports level as well as physiotherapy on downtime and LS/TAS was statistically analyzed. RESULTS: 100% reported knee symptoms at initial presentation. A localized posteromedial pain was documented in 52%. In 16/23 (70%) additional functional pathologies were diagnosed. Patients were physically highly active with high training intensities (6.52 ± 5.87 h/week) and performance level (65% competitive vs. 35% recreational). Patients underwent 1.91 ± 0.97 MRIs (max four). Symptom duration was 10.48 ± 11.02 weeks. A follow-up examination was performed after 12.62 ± 10.41 months (n = two lost to follow-up). 17/21 had physiotherapy, on average 17.06 ± 13.33 units. Overall downtime was 13.39 ± 12.50 weeks, the return-to-sports (RTS) rate 81%. 100%/38% reported a relief/remission of complaints. LS was 93.29 ± 7.95, median TAS before onset of knee complaints/at follow-up 7 (6-7)/7 (5-7). Specific posteromedial pain, presence of paratendinous cysts, sports level and physiotherapy had no significant influence on downtime and outcome parameters (n.s.). CONCLUSIONS: DFCI as a pathognomonic finding is recurrently encountered in the MRIs of children and adolescents. This knowledge is essential to spare patients from overtreatment. Contrary to the literature, the present results implicate a clinical relevance of DFCI particularly in those who are physically highly active with localized pain on exertion. Structured physiotherapy as basic treatment is recommended.

Predictive Factors for Adverse Event Outcomes After Transarterial Radioembolization with Yttrium-90 Resin Microspheres in Europe: Results from the Prospective Observational CIRT Study.

BACKGROUND: Using data collected in the prospective observational study CIRSE Registry for SIR-Spheres Therapy, the present study aimed at identifying predictors of adverse events (AEs) following transarterial radioembolization (TARE) with Yttrium-90 resin microspheres for liver tumours. METHODS: We analysed 1027 patients enrolled between January 2015 and December 2017 and followed up for 24 months. Four hundred and twenty-two patients with hepatocellular carcinoma (HCC), 120 with intrahepatic carcinoma (ICC), 237 with colorectal liver metastases and 248 with liver metastases from other primaries were included. Prognostic factors were calculated with a univariable analysis by using the overall AEs burden score (AEBS). RESULTS: All-cause AEs were reported in 401/1027 (39.1%) patients, with AEs associated with TARE, such as abdominal pain (16.6%), fatigue (17%), and nausea (11.7%) reported most frequently. Grade 3 or higher AEs were reported in 92/1027 (9%) patients. Reports on grade ≥ 3 gastrointestinal ulcerations (0.4%), gastritis (0.3%), radiation cholecystitis (0.2%) or radioembolization-induced liver disease (0.5%) were uncommon. Univariable analysis showed that in HCC, AEBS increased for Eastern Cooperative Oncology Group (ECOG) 0 (p = 0.0045), 1 tumour nodule (0.0081), > 1 TARE treatment (p = 0.0224), no prophylactic embolization (p = 0.0211), partition model dosimetry (p = 0.0007) and unilobar treatment target (0.0032). For ICC, > 1 TARE treatment was associated with an increase in AEBS (p = 0.0224), and for colorectal liver metastases, ECOG 0 (p = 0.0188), > 2 prior systemic treatments (p = 0.0127), and 1 tumour nodule (p = 0.0155) were associated with an increased AEBS. CONCLUSION: Our study confirms that TARE is a safe treatment with low toxicity and a minimal impact on quality of life.

First proficiency testing for NGS-based and combined NGS- and FISH-based detection of FGFR2 fusions in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma harbours druggable genetic lesions including FGFR2 gene fusions. Reliable and accurate detection of these fusions is becoming a critical component of the molecular work-up, but real-world data on the performance of fluorescence in situ hybridisation (FISH) and targeted RNA-based next-generation sequencing (NGS) are very limited. Bridging this gap, we report results of the first round robin test for FGFR2 fusions in cholangiocarcinoma and contextualise test data with genomic architecture. A cohort of 10 cholangiocarcinoma (4 fusion positive and 6 fusion negative) was tested by the Institute of Pathology, University Hospital Heidelberg, Germany. Data were validated by four academic pathology departments in Germany. Fusion-positive cases comprised FGFR2::BICC1, FGFR2::DBP, FGFR2::TRIM8, and FGFR2::ATE1 fusions. In a second step, a round robin test involving 21 academic and non-academic centres testing with RNA-based NGS approaches was carried out; five participants performed FISH testing in addition. Thirteen of 16 (81%) centres successfully passed the NGS only and 3 of 5 (60%) centres passed the combined NGS + FISH round robin test. Identified obstacles were bioinformatic pipelines not optimised for the detection of FGFR2 fusions and assays not capable of detecting unknown fusion partners. This study shows the benefit of targeted RNA-NGS for the detection of FGFR2 gene fusions. Due to the marked heterogeneity of the genomic architecture of these fusions, fusion partner agnostic (i.e. open) methodological approaches that are capable of identifying yet unknown fusion partners are superior. Furthermore, we highlight pitfalls in subsequent bioinformatic analysis and limitations of FISH-based tests.

Young's modulus of the different crystalline phases of poly (l-lactic acid).

Young's modulus of α'- and α-crystals of poly (l-lactic acid) (PLLA), more precisely, of aggregates of isotropically arranged lamellae, has been estimated based on dynamic-mechanical analysis of sets of isotropic film samples containing largely different though well-defined amounts of crystals. Evaluation of the modulus of elasticity of these film samples yielded the dependence of Young's modulus as a function of the enthalpy-based crystallinity, increasing with the crystal fraction in the assessed range, from zero to about 75% crystallinity. Extrapolation towards 100% crystallinity suggests values of Young's modulus of around 3.7 and 4.6 GPa for isotropic aggregates of α'- and α-crystals, respectively, being only slightly higher than the modulus of the unaged glassy amorphous phase of 3.0 GPa. Noting the inherent anisotropy of the crystal modulus, suggested in the literature, the average modulus determined in this work seems to be controlled by weaker interchain secondary bonding but not the modulus in chain direction. Great effort has been undertaken to minimize errors by keeping the lamellar thickness in samples of different crystallinity constant, and by providing evidence for independence of the moduli on the spherulitic superstructure.

Completion Pancreatectomy After Pancreatoduodenectomy: Who Needs It?

OBJECTIVE: The objective of this study was to identify the indications for and report the outcomes of completion pancreatectomy (CPLP) in the postoperative course after pancreatoduodenectomy (PD). BACKGROUND: CPLP may be considered or even inevitable for damage control after PD. METHODS: A prospectively maintained database of all patients undergoing PD between 2001 and 2019 was searched for patients who underwent CPLP in the postoperative course after PD. Baseline characteristics, perioperative details, and outcomes of CPLP patients were analyzed and specific indications for CPLP were identified. RESULTS: A total of 3953 consecutive patients underwent PD during the observation period. CPLP was performed in 120 patients (3%) after a median of 10 days following PD. The main indications for CPLP included postpancreatectomy acute necrotizing pancreatitis [n=47 (39%)] and postoperative pancreatic fistula complicated by hemorrhage [n=41 (34%)] or associated with uncontrollable leakage of the pancreatoenteric anastomosis [n=23 (19%)]. The overall 90-day mortality rate of all 3953 patients was 3.5% and 37% for patients undergoing CPLP. CONCLUSIONS: Our finding that only very few patients (3%) need CPLP suggests that conservative, interventional, and organ-preserving surgical measures are the mainstay of complication management after PD. Postpancreatectomy acute necrotizing pancreatitis, uncontrollable postoperative pancreatic fistula, and fistula-associated hemorrhage are highly dangerous and represent the main indications for CPLP after PD.

Spatiotemporal analysis of tumour-infiltrating immune cells in biliary carcinogenesis.

BACKGROUND: Intraductal papillary neoplasms (IPN) and biliary epithelial neoplasia (BilIN) are well-defined precursor lesions of biliary tract carcinoma (BTC). The aim of this study was to provide a comprehensive characterisation of the inflammatory microenvironment in BTC precursor lesions. METHODS: Immunohistochemistry was employed to assess tumour-infiltrating immune cells in tissue samples from patients, for whom precursor lesions were identified alongside invasive BTC. The spatiotemporal evolution of the immune microenvironment during IPN-associated carcinogenesis was comprehensively analysed using triplet sample sets of non-neoplastic epithelium, precursor lesion and invasive BTC. Immune-cell dynamics during IPN- and BilIN-associated carcinogenesis were subsequently compared. RESULTS: Stromal CD3+ (P = 0.002), CD4+ (P = 0.007) and CD8+ (P < 0.001) T cells, CD20+ B cells (P = 0.008), MUM1+ plasma cells (P = 0.012) and CD163+ M2-like macrophages (P = 0.008) significantly decreased in IPN compared to non-tumorous biliary epithelium. Upon transition from IPN to invasive BTC, stromal CD68+ (P = 0.001) and CD163+ (P < 0.001) macrophages significantly increased. In contrast, BilIN-driven carcinogenesis was characterised by significant reduction of intraepithelial CD8+ T-lymphocytic infiltration from non-tumorous epithelium via BilIN (P = 0.008) to BTC (P = 0.004). CONCLUSION: IPN and BilIN are immunologically distinct entities that undergo different immune-cell variations during biliary carcinogenesis. Intraepithelial CD8+ T-lymphocytic infiltration of biliary tissue decreased already at the IPN-precursor stage, whereas BilIN-associated carcinogenesis showed a slowly progressing reduction towards invasive carcinoma.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Genomic architecture of FGFR2 fusions in cholangiocarcinoma and its implication for molecular testing.

BACKGROUND: Cholangiocarcinoma (CCA) is a primary malignancy of the biliary tract with a dismal prognosis. Recently, several actionable genetic aberrations were identified with significant enrichment in intrahepatic CCA, including FGFR2 gene fusions with a prevalence of 10-15%. Recent clinical data demonstrate that these fusions are druggable in a second-line setting in advanced/metastatic disease and the efficacy in earlier lines of therapy is being evaluated in ongoing clinical trials. This scenario warrants standardised molecular profiling of these tumours. METHODS: A detailed analysis of the original genetic data from the FIGHT-202 trial, on which the approval of Pemigatinib was based, was conducted. RESULTS: Comparing different detection approaches and displaying representative cases, we described the genetic landscape and architecture of FGFR2 fusions in iCCA and show biological and technical aspects to be considered for their detection. We elaborated parameters, including a suggestion for annotation, that should be stated in a molecular diagnostic FGFR2 report to allow a complete understanding of the analysis performed and the information provided. CONCLUSION: This study provides a detailed presentation and dissection of the technical and biological aspects regarding FGFR2 fusion detection, which aims to support molecular pathologists, pathologists and clinicians in diagnostics, reporting of the results and decision-making.

Influence of carnosine and carnosinase-1 on diabetes-induced afferent arteriole vasodilation: implications for glomerular hemodynamics.

Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Two cohorts of mice including appropriate controls were studied: i.e., diabetic mice that received oral carnosine supplementation (cohort 1) and human (h)CN1 transgenic (TG) diabetic mice (cohort 2). The lumen area ratio (LAR) of the afferent arterioles and glomerular parameters were measured by conventional histology. Three-dimensional analysis using a tissue clearing strategy was also used. In both cohorts, LAR was significantly larger in diabetic BTBRob/ob versus nondiabetic BTBRwt/ob mice (0.41 ± 0.05 vs. 0.26 ± 0.07, P < 0.0001 and 0.42 ± 0.06 vs. 0.29 ± 0.04, P < 0.0001) and associated with glomerular size (cohort 1: r = 0.55, P = 0.001 and cohort 2: r = 0.89, P < 0.0001). LAR was partially normalized by oral carnosine supplementation (0.34 ± 0.05 vs. 0.41 ± 0.05, P = 0.004) but did not differ between hCN1 TG and wild-type BTBRob/ob mice. In hCN1 TG mice, serum CN1 concentrations correlated with LAR (r = 0.90, P = 0.006). Diabetic mice displayed decreased nephrin expression and increased glomerular hypertrophy. This was not significantly different in hCN1 TG BTBRob/ob mice (P = 0.06 and P = 0.08, respectively). In conclusion, carnosine and CN1 may affect intraglomerular pressure in an opposing manner through the regulation of afferent arteriolar tone. This study corroborates previous findings on the role of carnosine in the progression of DKD.NEW & NOTEWORTHY Dysregulation in glomerular hemodynamics favors hyperfiltration in diabetic kidney disease (DKD). Although carnosine supplementation ameliorates features of DKD, its effect on glomerular vasoregulation is not known. We assessed the influence of carnosine and carnosinase-1 (CN1) on afferent glomerular arteriole vasodilation and its association with glomerular size, hypertrophy, and nephrin expression in diabetic BTBRob/ob mice. Our results provide evidence that carnosine feeding and CN1 overexpression likely affect intraglomerular pressure through vasoregulation of the afferent arteriole.