DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p < 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Comparison of superb microvascular imaging (SMI) quantified with ImageJ to quantified contrast-enhanced ultrasound (qCEUS) in liver metastases-a pilot study.

Background: The aim of the study was to compare methods for the assessment of vascularisation of liver metastases (LM) between superb microvascular imaging (SMI), contrast-enhanced ultrasound, and microvascular density (MVD). Methods: SMI results were quantified as the vascularisation quotient (VQ), based on a grey-scale analysis with ImageJ image software. Those results were compared to contrast-enhanced ultrasonography (CEUS) values, calculated with VueBox®. MVD was measured with an anti-CD34 antibody. Results: This study included 13 patients with LM. The VQ showed a strong correlation with the quantified parameters of contrast-enhanced ultrasound. The parameters of quantified contrast-enhanced ultrasound compared with quantified SMI showed the following statistical correlations: peak enhancement (PE), in arbitrary unit (a.u.) (r=0.72104, P=0.0054), PE in Decibel (dB) (r=0.65918, P=0.00141), Wash-in- Area Under the Curve (WiAUC) in a.u. (r=0.63604, P=0.00194), Wash-in Perfusion-Index (WiPI) in a.u. (r=0.73337, P=0.0043), Wash-in Perfusion-Index (WiPI) in dB (r=0.65642, P=0.0194), Wash-in-Rate (WiR) in a.u. (r=0.7304, P=0.0036) and Wash-in-Rate (WiR) in dB (r=0.82897, P=0.0005). Conclusions: Comparison of the two methods, SMI and contrast-enhanced ultrasound (CEUS), for quantitative assessment of vascularisation of LM showed good correlation. The contrast-independent Doppler technique SMI can qualitatively assess the vascularisation of LM.

Prevalence and Prognostic Implications of PD-L1 Expression in Soft Tissue Sarcomas.

Background: PD-L1 expression differs from 19 to 92% in various cancer subtypes. Its expression carries a worse prognostic value in various malignancies and could also be used as a predictive marker for immune checkpoint inhibitor response. This study aimed to explore the prevalence of PD-L1 expression in soft tissue sarcomas and the correlation of PD-L1 expression with clinicopathological features. Patients and Methods: The tissue samples of 50 patients with STS were tested for PD-L1 expression using immunohistochemistry (IHC). We followed a 6-step proportional scoring system. The patients were treated at Ain Shams University Hospital from 2011 to 2017. We also explored the correlation of PD-L1 expression with different clinical features of the patients. The chi-square test was used to calculate the differences among variables. Results: Twelve cases (24%) showed positive PD-L1 expression with the highest prevalence in rhabdomyosarcoma and desmoid tumors (2/2 and 2/3 cases, respectively), followed by GIST in 2/4 cases and liposarcoma in 3/11 cases. Patients with positive PD-L1 expression showed a trend for worse survival, with a median overall survival of 11 months vs. 19 months for patients with negative PD-L1 expression (p-value = 0.1) and a mean PFS of 6 months vs. 11 months for patients with negative PD-L1 expression (p-value = 0.1). However, these findings did not reach statistical significance. Conclusion: Although the results did not reach statistical significance due to the small number of cases, PD-L1 expression could represent a prognostic factor for poor outcome. Larger clinical trials are recommended for the validation of PD-L1 as a poor prognostic biomarker.

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue.

BACKGROUND: Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis. METHODOLOGY/PRINCIPAL FINDINGS: Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative. CONCLUSIONS/SIGNIFICANCE: We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.

Combining Computed Tomography and Histology Leads to an Evolutionary Concept of Hepatic Alveolar Echinococcosis.

Alveolar echinococcosis (AE) is caused by the intermediate stage of Echinococcus multilocularis. We aimed to correlate computed tomography (CT) data with histology to identify distinct characteristics for different lesion types. We classified 45 samples into five types with the Echinococcus multilocularis Ulm Classification for Computed Tomography (EMUC-CT). The various CT lesions exhibited significantly different histological parameters, which led us to propose a progression model. The initial lesion fit the CT type IV classification, which comprises a single necrotic area with the central located laminated layer, a larger distance between laminated layer and border zone, a small fibrotic peripheral zone, and few small particles of Echinococcus multilocularis (spems). Lesions could progress through CT types I, II, and III, characterized by shorter distances between laminated layer and border zone, more spems inside and surrounding the lesion, and a pronounced fibrotic rim (mostly in type III). Alternatively, lesions could converge to a highly calcified, regressive state (type V). Our results suggest that the CT types mark sequential stages of the infection, which progress over time. These distinct histological patterns advance the understanding of interactions between AE and human host; moreover, they might become prognostically and therapeutically relevant.

Phase II trial evaluating the efficacy and safety of the anti-CD20 monoclonal antibody obinutuzumab in patients with marginal zone lymphoma.

Marginal zone lymphoma (MZL) belongs to the group of indolent B-cell non-Hodgkin's lymphomas, which is characterized by an indolent course. In this mostly elderly patient population, the development of chemotherapy-free approaches is of particular interest. In this situation, single-agent treatment with the next-generation anti-CD20 antibody obinutuzumab is an attractive approach, which promises high efficacy without major toxicity. We describe here an open-label, multicentric Phase II trial evaluating the efficacy and safety of obinutuzumab in de novo MZL patients, who are treatment naive for systemic therapy and not eligible for or failed local treatment. ClinicalTrials.gov identifier NCT03322865.

Pancreatic cancer-derived organoids - a disease modeling tool to predict drug response.

BACKGROUND: Organotypic cultures derived from pancreatic ductal adenocarcinoma (PDAC) termed pancreatic ductal cancer organoids (PDOs) recapitulate the primary cancer and can be derived from primary or metastatic biopsies. Although isolation and culture of patient-derived pancreatic organoids were established several years ago, pros and cons for individualized medicine have not been comprehensively investigated to date. METHODS: We conducted a feasibility study, systematically comparing head-to-head patient-derived xenograft tumor (PDX) and PDX-derived organoids by rigorous immunohistochemical and molecular characterization. Subsequently, a drug testing platform was set up and validated in vivo. Patient-derived organoids were investigated as well. RESULTS: First, PDOs faithfully recapitulated the morphology and marker protein expression patterns of the PDXs. Second, quantitative proteomes from the PDX as well as from corresponding organoid cultures showed high concordance. Third, genomic alterations, as assessed by array-based comparative genomic hybridization, revealed similar results in both groups. Fourth, we established a small-scale pharmacotyping platform adjusted to operate in parallel considering potential obstacles such as culture conditions, timing, drug dosing, and interpretation of the results. In vitro predictions were successfully validated in an in vivo xenograft trial. Translational proof-of-concept is exemplified in a patient with PDAC receiving palliative chemotherapy. CONCLUSION: Small-scale drug screening in organoids appears to be a feasible, robust and easy-to-handle disease modeling method to allow response predictions in parallel to daily clinical routine. Therefore, our fast and cost-efficient assay is a reasonable approach in a predictive clinical setting.

Drug-induced liver injury associated with the biosimilar glatiramer acetate (Clift®).

A 23-year old female was diagnosed with relapsing-remitting multiple sclerosis with two symptomatic attacks. Immunomodulatory treatment with Clift® (Glatiramer Acetate biosimilar) was initiated. Shortly after administration, an asymptomatic increase in liver enzymes was noticed, and therapy was paused. However, we observed an enormous increase in liver enzymes within a few days. Histological work up of a liver biopsy showed microfocal liver necrosis accompanied with increased numbers of CD38-positive lymphocytes as shown by immunohistology, indicating a drug-induced liver injury. Subsequently, under oral prednisolone treatment, liver enzymes normalized. This case highlights the importance of tight monitoring of liver function in the initial phase of a new immunotherapy to unravel asymptomatic hepatotoxicity in time and prevent further damage.

Clinical utility of a protein-based oncopanel in patients with end-stage head and neck cancer.

Aim: In a prospective clinical initiative, we selected heavily pretreated head and neck carcinoma patients and assessed the clinical utility of a protein-based oncopanel for identification of potential targetable markers. Patients & methods: Tumor samples of 45 patients were evaluated using a 12-marker immunohistochemistry panel. The primary end point was the prevalence of potentially actionable markers. Results: At least one expressed marker in each case could be identified. We noted a high prevalence of EGFR (80%, 39/45) and MET (57.4%, 28/45). Three patients received oncopanel-based therapy with variable results. Conclusion: Despite the limited number of treated subjects, oncopanel analysis in end-stage head and neck cancer is operationally and technically feasible. Combination with targeted next generation sequencing might provide additional therapy options.

Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Cyclin H expression is increased in GIST with very-high risk of malignancy.

BACKGROUND: Risk estimation of gastrointestinal stromal tumours (GIST) is based on tumour size and mitotic rate according to the National Institutes of Health consensus classification. The indication for adjuvant treatment of patients with high risk GIST after R0 resection with small molecule inhibitors is still a controversial issue, since these patients represent a highly heterogeneous population. Therefore, additional prognostic indicators are needed. Here, we evaluated the prognostic value of cyclin H expression in GIST. METHODS: In order to identify prognostic factors of GIST we evaluated a single centre cohort of ninety-five GIST patients. First, GISTs were classified with regard to tumour size, mitotic rate and localisation according to the NIH consensus and to three additional suggested risk classifications. Second, Cyclin H expression was analysed. RESULTS: Of ninety-five patients with GIST (53 female/42 male; median age: 66.78a; range 17-94a) risk classification revealed: 42% high risk, 20% intermediate risk, 23% low risk and 15% very low risk GIST. In patients with high risk GIST, the expression of cyclin H was highly predictive for reduced disease-specific survival (p = 0.038). A combination of cyclin H expression level and high risk classification yielded the strongest prognostic indicator for disease-specific and disease-free survival (p < or = 0.001). Moreover, in patients with tumour recurrence and/or metastases, cyclin H positivity was significantly associated with reduced disease-specific survival (p = 0.016) regardless of risk-classification. CONCLUSION: Our data suggest that, in addition to high risk classification, cyclin H expression might be an indicator for "very-high risk" GIST.