Single-cell landscape of idiopathic multicentric Castleman disease in identical twins.

ABSTRACT: Idiopathic multicentric Castleman disease (iMCD) is a rare cytokine-driven disorder characterized by systemic inflammation, generalized lymphadenopathy, and organ dysfunction. Here, we present an unusual occurrence of iMCD in identical twins and examined the immune milieu within the affected lymphoid organs and the host circulation using multiomic high-dimensional profiling. Using spatial enhanced resolution omics sequencing (Stereo-seq) transcriptomic profiling, we performed unsupervised spatially constrained clustering to identify different anatomic structures, mapping the follicles and interfollicular regions. After a cell segmentation approach, interleukin 6 (IL-6) pathway genes significantly colocalized with endothelial cells and fibroblastic reticular cells, confirming observations using a single-cell sequencing approach (10× Chromium). Furthermore, single-cell sequencing of peripheral blood mononuclear cells revealed an "inflammatory" peripheral monocytosis enriched for the expression of S100A family genes in both twins. In summary, we provided evidence of the putative cell-of-origin of IL-6 signals in iMCD and described a distinct monocytic host immune response phenotype through a unique identical twin model.

Tumor-Infiltrating Mast Cells in Angiosarcoma Correlate With Immuno-Oncology Pathways and Adverse Clinical Outcomes.

Recent studies have described several molecular subtypes and deregulation of immuno-oncologic signaling pathways in angiosarcoma. Interestingly, mast cells were enriched in subsets of angiosarcoma, although their significance remains unknown. In this study, we aim to verify this observation using immunohistochemistry (H scores) and NanoString transcriptomic profiling and explore the association between mast cells with clinical and biological features. In the study cohort (N = 60), H scores showed a significant moderate correlation with NanoString mast cell scores (r = 0.525; P < .001). Both H score and NanoString mast cell scores showed a significant positive correlation (P < .05) with head and neck location, nonepithelioid morphology, and lower tumor grade. Mast cell enrichment significantly correlated with higher NanoString regulatory T-cell scores (H score, r = 0.32; P = .01; NanoString mast cell score, r = 0.27; P = .04). NanoString mast cell scores positively correlated with signaling pathways relating to antigen presentation (r = 0.264; P = .0414) and negatively correlated with apoptosis (r = -0.366; P = .0040), DNA damage repair (r = -0.348; P = .0064), and cell proliferation (r = -0.542; P < .001). Interestingly, in the metastatic setting, patients with mast cell-enriched angiosarcoma showed poorer progression-free survival (median, 0.2 vs 0.4 years; hazard ratio = 3.05; P = .0489) along with a trend toward worse overall survival (median, 0.2 vs 0.6 years; hazard ratio, 2.86; P = .0574) compared with patients with mast cell-poor angiosarcoma. In conclusion, we demonstrated the presence of mast cells in human angiosarcoma and provided initial evidence of their potential clinical and biological significance. Future research will be required to elucidate their specific roles and mechanisms, which may uncover novel avenues for therapeutic intervention.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Giant juvenile fibroadenomas with and without prominent pseudoangiomatous stromal hyperplasia (PASH)-like change: clinicopathological and molecular characteristics.

AIMS: Juvenile fibroadenomas (JFA) are biphasic fibroepithelial lesions (FEL) usually occurring in adolescent female patients. Giant (G) JFA, like other FEL, may exhibit prominent pseudoangiomatous stromal hyperplasia (PASH)-like change. We sought to determine clinicopathological and molecular characteristics of GJFA with and without PASH. METHODS AND RESULTS: Archives were searched for cases of GJFA (1985-2020). All were stained for androgen receptor (AR), beta-catenin, CD34 and progesterone receptor (PR). Cases were sequenced using a custom 16-gene panel - MED12 (exons 1 and 2), TERT promoter (-124C>T and -146Ctable>T), SETD2, KMT2D, RARA (exons 5-9), FLNA, NF1, PIK3CA (exons 10, 11 and 21), EGFR, RB1, BCOR, TP53, PTEN, ERBB4, IGF1R and MAP3K1. Twenty-seven GJFA from 21 female patients aged 10.1-25.2 years were identified. Size ranged from 5.2 to 21 cm. Two patients had multiple, bilateral and later recurrent GJFA. Thirteen (48%) cases showed prominent PASH-like stroma. All were positive for stromal CD34, negative for AR and beta-catenin and one case showed focal PR expression. Sequencing showed MAP3K1 and SETD2 mutations in 17 samples, with KMT2D, TP53 and BCOR aberrations in 10 (45%), 10 (45%) and seven (32%) cases, respectively. Tumours with a PASH-like pattern had higher prevalence of SETD2 (P = 0.004) and TP53 (P = 0.029) mutations, while those without PASH had more RB1 mutations (P = 0.043). MED12 mutation was identified in one case. TERT promoter mutation was observed in four (18%), including two recurrences. CONCLUSIONS: Gene mutations along more advanced phases of the proposed FEL pathogenetic pathway in GJFA are unusual, and suggest a mechanism for more aggressive growth in these tumours.

The Multi-Dimensional Biomarker Landscape in Cancer Immunotherapy.

The field of immuno-oncology is now at the forefront of cancer care and is rapidly evolving. The immune checkpoint blockade has been demonstrated to restore antitumor responses in several cancer types. However, durable responses can be observed only in a subset of patients, highlighting the importance of investigating the tumor microenvironment (TME) and cellular heterogeneity to define the phenotypes that contribute to resistance as opposed to those that confer susceptibility to immune surveillance and immunotherapy. In this review, we summarize how some of the most widely used conventional technologies and biomarkers may be useful for the purpose of predicting immunotherapy outcomes in patients, and discuss their shortcomings. We also provide an overview of how emerging single-cell spatial omics may be applied to further advance our understanding of the interactions within the TME, and how these technologies help to deliver important new insights into biomarker discovery to improve the prediction of patient response.

Bizarre giant cells in human angiosarcoma exhibit chemoresistance and contribute to poor survival outcomes.

Giant cells (GC) are a poorly understood subset of tumor cells that have been increasingly recognized as a potential contributor to tumor heterogeneity and treatment resistance. We aimed to characterize the biological and clinical significance of GC in angiosarcoma, an aggressive rare cancer of endothelial origin. Archival angiosarcoma samples were examined for the presence of GC and compared with clinicopathological as well as NanoString gene expression data. GC were examined in angiosarcoma cell lines MOLAS and ISOHAS using conventional and electron microscopy, single cell whole genome profiling, and other assays. In the cell lines, GC represented a rare population of mitotically active, non-senescent CD31+ cells, and shared similar genomic profiles with regular-sized cells, consistent with a malignant endothelial phenotype. GC remained viable and persisted in culture following exposure to paclitaxel and doxorubicin. In patient samples, GC were present in 24 of 58 (41.4%) cases. GC was correlated with poorer responses to chemotherapy (25.0% vs 73.3%, P = 0.0213) and independently contributed to worse overall survival outcomes (hazard ratio 2.20, 95% confidence interval 1.17-4.15, P = 0.0142). NanoString profiling revealed overexpression of genes, including COL11A1, STC1, and ERO1A, accompanied by upregulation of immune-related metabolic stress and metastasis/matrix remodeling pathways in GC-containing tumors. In conclusion, GC may contribute to chemoresistance and poor prognosis in angiosarcoma.

Genetic differences between benign phyllodes tumors and fibroadenomas revealed through targeted next generation sequencing.

Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.

Morphologic and genetic heterogeneity in breast fibroepithelial lesions-a comprehensive mapping study.

Breast fibroepithelial lesions (FELs) encompass the common fibroadenoma (FA) and relatively rare phyllodes tumour (PT); the latter entity is usually classified as benign, borderline or malignant. Intratumoural heterogeneity is frequently present in these tumours, making accurate histologic evaluation challenging. Despite their rarity, PTs are an important clinical problem due to their propensity for recurrence and, in the case of malignant PT, metastasis. Surgical excision is the mainstay of management. Recent work has uncovered myriad genetic alterations in breast FELs. In this study, exome sequencing was performed on seven cases of morphologically heterogeneous breast FELs, including FAs, PTs of all grades, and a case of metaplastic spindle cell carcinoma arising in PT, in order to elucidate their intratumoural genetic repertoire. Gene mutations identified encompassed cell signalling, tumour suppressor, DNA repair and cell cycle regulating pathways. Mutations common to multiple tumour regions generally showed higher variant allele frequency. Frequent mutations included MED12, TP53, RARA and PIK3CA. Histological observations of increased cellular density and pleomorphism correlated with mutational burden. Phylogenetic analyses revealed disparate pathways of possible tumour progression. In summary, histological heterogeneity correlated with genetic changes in breast FELs.

Genomic characterisation of breast fibroepithelial lesions in an international cohort.

Fibroepithelial lesions (FELs) are a heterogeneous group of tumours comprising fibroadenomas (FAs) and phyllodes tumours (PTs). Here we used a 16-gene panel that was previously discovered to be implicated in pathogenesis and progression, to characterise a large international cohort of FELs via targeted sequencing. The study comprised 303 (38%) FAs and 493 (62%) PTs which were contributed by the International Fibroepithelial Consortium. There were 659 (83%) Asian and 109 (14%) non-Asian FELs, while the ethnicity of the rest was unknown. Genetic aberrations were significantly associated with increasing grade of PTs, and were detected more in PTs than FAs for MED12, TERT promoter, RARA, FLNA, SETD2, TP53, RB1, EGFR, and IGF1R. Most borderline and malignant PTs possessed ≥ 2 mutations, while there were more cases of FAs with ≤ 1 mutation compared to PTs. FELs with MED12 mutations had significantly higher rates of TERT promoter, RARA, SETD2, EGFR, ERBB4, MAP3K1, and IGF1R aberrations. However, FELs with wild-type MED12 were more likely to express TP53 and PIK3CA mutations. There were no significant differences observed between the mutational profiles of recurrent FAs, FAs with a history of subsequent ipsilateral recurrence or contralateral occurrence, and FAs without a history of subsequent events. We identified recurrent mutations which were more frequent in PTs than FAs, with borderline and malignant PTs harbouring cancer driver gene and multiple mutations. This study affirms the role of a set of genes in FELs, including its potential utility in classification based on mutational profiles. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Endothelin receptor blockers reduce shunting and angiogenesis in cirrhotic rats.

BACKGROUND: Angiogenesis plays a pivotal role in splanchnic hyperaemia and portosystemic collateral formation in cirrhosis. Endothelin-1 (ET-1), an endothelium-derived vasoconstrictor, has also been implicated in the pathogenesis of cirrhosis and portal hypertension. DESIGN: This study aimed to survey the influences of ET-1 in cirrhosis-related angiogenesis. Common bile duct ligation was performed on Spraque-Dawley rats to induce cirrhosis. Since the 14th day after the operation, rats randomly received distilled water (DW, control), bosentan [a nonselective ET receptor (ETR) blocker] or ambrisentan (a selective ETA R blocker) for 4 weeks. On the 43rd day, portal and systemic haemodynamics, liver biochemistry, portosystemic shunting degree, mesenteric vascular density, mRNA and/or protein expressions of relevant angiogenic factors were evaluated. RESULTS: In cirrhotic rats, bosentan significantly reduced portal pressure. Ambrisentan did not influence haemodynamics and liver biochemistry. Both of them significantly improved the severity of portosystemic collaterals and decreased the mesenteric vascular density. Compared with the DW-treated cirrhotic rats, splenorenal shunt and mesenteric inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), vascular endothelial growth factor mRNA expressions and mesenteric iNOS, COX2, VEGF, phospho-VEGF receptor 2, Akt and phospho-Akt protein expressions were down-regulated in both groups. CONCLUSIONS: In rats with liver cirrhosis, both nonselective and selective ETA R blockade ameliorate the severity of portosystemic shunting and mesenteric angiogenesis via the down-regulation of VEGF pathway and relevant angiogenic factors. ET receptors may be targeted to control the severity of portosystemic collaterals and associated complications in cirrhosis.

2'-Hydroxyflavanone ameliorates mesenteric angiogenesis and portal-systemic collaterals in rats with liver fibrosis.

BACKGROUND AND AIM: Portal-systemic collaterals lead to dreadful consequences in patients with cirrhosis. Angiogenesis participates in the development of liver fibrosis, hyperdynamic circulation, and portal-systemic collaterals. 2'-Hydroxyflavanone (2'-HF), one of the citrus fruits flavonoids, is known to have antiangiogenesis effect without adverse response. However, the relevant effects in liver fibrosis have not been surveyed. METHODS: Male Wistar rats received thioacetamide (TAA, 100 mg/kg tiw, i.p.) for 6 weeks to induce liver fibrosis. On the 29th to 42nd day, rats randomly received 2'-HF (100 mg/kg, qod, i.p.) or vehicle (corn oil). On the 43rd day, after hemodynamic measurements, the followings were surveyed: (i) severity of collaterals; (ii) mesenteric angiogenesis; (iii) mesenteric proangiogenic factors protein expressions; (iv) Mesenteric vascular endothelial cells apoptosis; and (v) Mesenteric expressions of proteins regulating apoptosis. RESULTS: Compared with the vehicle group, 2'-HF did not significantly change body weight, mean arterial pressure, heart rate, and portal pressure in TAA rats. 2'-HF significantly alleviated the severity of collaterals, but the mesenteric phospho-ERK, ERK, phospho-Akt, Akt, COX1, COX2, VEGF, and VEGFR-2 protein expressions were not altered. The apoptotic index of 2'-HF group was significantly higher and the mesenteric protein expressions of pro-apoptotic factors, NFkB 50, NFkB 65, Bax, phospho-p53, 17 kD cleaved caspase 3, and 17 kD casepase 3 were up-regulated. CONCLUSIONS: 2'-HF does not influence the hemodynamics but alleviated the severity of collaterals in rats with liver fibrosis and early portal hypertension. This is, at least partly, attributed to enhanced apoptosis of mesenteric vascular endothelial cells.

Diabetes enhances the intrahepatic vascular response to endothelin-1 in cirrhotic rats: association with the ETA receptor and pERK up-regulation.

BACKGROUND & AIMS: Cirrhosis is characterized by increased intrahepatic vascular resistance and enhanced vasocontractile responsiveness that impedes portal inflow and elevates portal pressure, in which endothelin-1 (ET-1) plays a role. Diabetes and glucose influence vasoresponsiveness but their impact on the intrahepatic vascular bed in cirrhosis is unknown. To investigate intrahepatic ET-1 vasoresponsiveness in cirrhotic rats with and without diabetes and to explore the underlying mechanisms. METHODS: Spraque-Dawley rats received common bile-duct ligation (BDL) to induce cirrhosis. Streptozotocin was injected to induce diabetes in the BDL rats (BDL/STZ). In situ liver perfusion was performed to obtain the ET-1 concentration-response curves. The basic hemodynamics and hepatic protein expressions of ET-1 receptors, pERK, ERK, pAkt, Akt, iNOS, eNOS, peNOS and calmodulin were evaluated. The circulating concentrations of N-terminal pro-brain natriuretic peptide (NT-ProBNP), blood urea nitrogen (BUN) and creatinine were also determined. RESULTS: Body weight, mean arterial pressure, heart rate and survival rate were significantly decreased in the BDL/STZ rats. The perfusion pressure changes in response to ET-1 were higher in the BDL/STZ group for all perfusates. ETA receptor and pERK expressions were enhanced in the BDL/STZ group. The circulating concentrations of NT-ProBNP, BUN and creatinine, as well as SMA flow, were not significantly different between the BDL and BDL/STZ groups. CONCLUSION: Cirrhotic rats with diabetes showed higher intrahepatic ET-1 vasoresponsiveness than normoglycemic cirrhotic rats. This effect is not affected by changes in perfused glucose concentration and may be related, at least in part, to intrahepatic ETA R receptor and pERK over-expression.

Lipopolysaccharide enhanced renal vascular response to endothelin-1 through ETA overexpression in portal hypertensive rats.

BACKGROUND AND AIM: Hypo-perfusion resulting from intense renal vasoconstriction is traditionally contributed to renal dysfunction in advanced liver disease, although cumulative studies demonstrated renal vasodilatation with impaired vascular contractility to endogenous vasoconstrictors in portal hypertension and compensated liver cirrhosis. The pathophysiology of altered renal hemodynamics remains unclear. This study, using a rat model of portal hypertension with superimposed endotoxemia, was designed to delineate the evolution of renal vascular reactivity and vaso-regulatory gene expression during liver disease progression. METHODS: Rats were randomized into sham surgery (SHAM) or partial portal vein ligation (PVL). Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS) on the seventh day following surgery. Isolated kidney perfusion was performed at 0.5 h or 5 h after LPS to evaluate renal vascular response to endothelin-1. RESULTS: In contrast to impaired vascular contractility of SHAM rats, PVL rats displayed enhanced renal vascular reactivity to endothelin-1 at 5 h following endotoxemia. There were extensive upregulations of inducible nitric oxide synthase in kidney tissues of endotoxemic rats. The changes of renal endothelin receptor type A (ETA ) level paralleled with the changes of renal vascular reactivity in LPS-treated rats. Compared with SHAM rats, PVL rats showed increased renal ETA and phosphorylated extracellular-signal-regulated kinases 1/2 (p-ERK1/2) at 5 h after LPS. CONCLUSION: LPS-induced systemic hypotension induces a paradoxical change of renal vascular response to endothelin-1 between SHAM and PVL rats. LPS-induced renal vascular hyperreactivity in PVL rats was associated with upregulation of renal ETA and subsequent activation of ERK1/2 signaling.

The ability of 17 ß-estradiol to attenuate intrahepatic vasoconstriction to endothelin-1 in female rats is lost in cirrhosis.

BACKGROUND AND RATIONALE: The control of Endothelin-1 (ET-1)-mediated intrahepatic vasoconstriction in cirrhosis is beneficial for the alleviation of relevant complications. Cirrhosis is accompanied by hypogonadism and altered sex hormone status. Besides, sex hormones have vasoactive effects, but it is unknown if they influence vascular function in cirrhosis. This study aimed to investigate the roles of sex hormones in hepatic vascular reactions to ET-1 in cirrhosis. Liver cirrhosis was induced in Spraque-Dawley male and female rats with common bile duct ligation (BDL). Sham-operated (Sham) rats were controls. On the 43rd day after operations, intrahepatic vascular concentration-response curves to ET-1 were obtained with the following preincubatioins: 1) vehicle; 2) 17ß-estradiol; 3) progesterone; 4) testosterone. Livers from sham and BDL rats were dissected for real-time polymerase chain reaction analysis of estrogen, progesterone and testosterone receptors. RESULTS: Compared with sham males perfused with vehicle, sham females presented higher perfusion pressure changes to ET-1 which was reversed only by 17 ß-estradiol. In cirrhosis, compared with males, 17 ß-estradiol no longer attenuated vascular responsiveness to ET-1 in females. In females, BDL rats had lower hepatic estrogen receptor α(ERßα) mRNA expression than that in sham rats. CONCLUSIONS: The sham females showed a stronger intrahepatic vascular constrictive effect to ET-1 than sham males, which could be reversed by 17ß-estradiol. However, the influence of 17 ß-estradiol was lost in cirrhotic females, which may be attributed, at least partly, to intrahepatic ER α down-regulation in females with cirrhosis.

Targeting antibody-mediated complement-independent mechanism in bullous pemphigoid with diacerein.

BACKGROUND: Bullous pemphigoid (BP) is an antibody-mediated blistering disease predominantly affecting the elderly. The pathogenesis involves both complement-dependent and complement-independent mechanisms. The therapeutic potential of targeting complement-independent mechanism has not yet been determined. The mainstay of treatment, corticosteroid, has many side effects, indicating the needs of better treatments. OBJECTIVE: We tempted to establish an in vitro model of BP which resembles complement-independent mechanism and to examine the therapeutic potential of a novel anti-inflammatory agent, diacerein. METHODS: Cultured HaCaT cells were treated with purified antibodies from BP patients, with or without diacerein to measure the cell interface presence of BP180, protein kinase C, and the production of proinflammatory cytokines. An open-label, randomized, phase 2 trial was conducted to compare topical diacerein and clobetasol ointments in patients with mild-to-moderate BP (NCT03286582). RESULTS: The reduced presentation of BP180 at cell interface after treating with BP autoantibodies was noticed in immunofluorescence and western blotting studies. The phenomenon was restored by diacerein. Diacerein also reduced the autoantibody-induced increase of pro-inflammatory cytokines. Reciprocal changes of BP180 and protein kinase C at the cell interface were found after treating with BP autoantibodies. This phenomenon was also reversed by diacerein in a dose-dependent manner. The phase 2 trial showed that topical diacerein reduced the clinical symptoms which were comparable to those of topical clobetasol. CONCLUSION: Diacerein inhibited BP autoantibody-induced reduction of BP180 and production of proinflammatory cytokines in vitro and showed therapeutic potential in patients with BP. It is a novel drug worthy of further investigations.

Effects of polyol and surfactant plasticisers on lyophilised rice starch wafers for buccal drug delivery.

Rice starch is a promising biopolymer for buccal formulations but typical oven drying may promote starch retrogradation that affects mechanical properties. Hence, lyophilisation was proposed here to improve starch product's stability. This study aims to investigate the effects of plasticisers (sorbitol and Tween® 80, T80) on the characteristics and drug release profiles of lyophilised rice starch wafers incorporated with propranolol hydrochloride. The wafers were prepared by lyophilising starch mixture (5%w/v) with plasticiser (0.2 and 0.3 g/g) and drug (10, 20, 30%w/w). Control wafers exhibited loose layers with rough wrinkled surface. Sorbitol resulted in a dense structure with higher puncture strength (PS) but lower water absorption capacity (WAC) while T80 loosened the flakes that reduced PS and increased WAC. Drug inclusion decreased PS and increased WAC of unplasticised wafers. T80-plasticised wafers with drug had a lower PS and higher WAC than sorbitol-plasticised wafers. Particularly, T80-plasticised wafers achieved outstandingly high PS and the lowest WAC at 30%w/w drug. Drug dissolution of wafers relied mainly on the drug crystallinity and WAC at 10 and 30%w/w drug. Plasticisers reduced and increased drug dissolution at 10 and 20%w/w drug, respectively. This study highlights the potential of lyophilisation in preparing rice starch wafers for buccal delivery.

Whole genome sequencing of HER2-positive metastatic extramammary Paget's disease: a case report.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare cancer that occurs within the epithelium of the skin, arising predominantly in areas with high apocrine gland concentration such as the vulva, scrotum, penis and perianal regions. Here, we aim to integrate clinicopathological data with genomic analysis of aggressive, rapidly-progressing de novo metastatic EMPD responding to HER2-directed treatment in combination with other agents, to attain a more comprehensive understanding of the disease landscape. METHODS: Immunohistochemical staining on the scrotal wall tumor and bone marrow metastasis demonstrated HER2 overexpression. Whole genome sequencing of the tumor and matched blood was performed. RESULTS: Notable copy number gains (log2FC > 0.9) on chromosomes 7 and 8 were detected (n = 81), with 92.6% of these unique genes specifically located on chromosome 8. Prominent cancer-associated genes include ZNF703, HOOK3, DDHD2, LSM1, NSD3, ADAM9, BRF2, KAT6A and FGFR1. Interestingly, ERBB2 gene did not exhibit high copy number gain (log2FC = 0.4) although 90% of tumor cells stained HER2-positive. Enrichment in pathways associated with transforming growth factor-beta (TGFß) (FDR = 0.0376, Enrichment Ratio = 8.12) and fibroblast growth factor receptor (FGFR1) signaling (FDR = 0.0082, Enrichment Ratio = 2.3) was detected. Amplicon structure analysis revealed that this was a simple-linear amplification event. CONCLUSION: Whole genome sequencing revealed the underlying copy number variation landscape in HER2-positive metastatic EMPD. The presence of alternative signalling pathways and genetic variants suggests potential interactions with HER2 signalling, which possibly contributed to the HER2 overexpression and observed response to HER2-directed therapy combined with other agents in a comprehensive treatment regimen.

Establishment and characterization of a patient-derived solitary fibrous tumor/hemangiopericytoma cell line model.

Solitary fibrous tumor/Hemangiopericytoma (SFT/HPC) is a rare subtype of soft tissue sarcoma harboring NAB2-STAT6 gene fusions. Mechanistic studies and therapeutic development on SFT/HPC are impeded by scarcity and lack of system models. In this study, we established and characterized a novel SFT/HPC patient-derived cell line (PDC), SFT-S1, and screened for potential drug candidates that could be repurposed for the treatment of SFT/HPC. Immunohistochemistry profiles of the PDC was consistent with the patient's tumor sample (CD99+/CD34+/desmin-). RNA sequencing, followed by Sanger sequencing confirmed the pathognomonic NAB2exon3-STAT6exon18 fusion in both the PDC and the original tumor. Transcriptomic data showed strong enrichment for oncogenic pathways (epithelial-mesenchymal transition, FGF, EGR1 and TGFß signaling pathways) in the tumor. Whole genome sequencing identified potentially pathogenic somatic variants such as MAGEA10 and ABCA2. Among a panel of 14 targeted agents screened, dasatinib was identified to be the most potent small molecule inhibitor against the PDC (IC50, 473 nM), followed by osimertinib (IC50, 730 nM) and sunitinib (IC50, 1765 nM). Methylation profiling of the tumor suggests that this specific variant of SFT/HPC could lead to genome-wide hypomethylation. In conclusion, we established a novel PDC model of SFT/HPC with comprehensive characterization of its genomic, epigenomic and transcriptomic landscape, which can facilitate future preclinical studies of SFT/HPC, such as in vitro drug screening and in vivo drug testing.

Cannabinoid receptor 2 agonist ameliorates mesenteric angiogenesis and portosystemic collaterals in cirrhotic rats.

UNLABELLED: Angiogenesis in liver cirrhosis leads to splanchnic hyperemia, increased portal inflow, and portosystemic collaterals formation, which may induce lethal complications, such as gastroesophageal variceal hemorrhage and hepatic encephalopathy. Cannabinoids (CBs) inhibit angiogenesis, but the relevant influences in cirrhosis are unknown. In this study, Spraque-Dawley rats received common bile duct ligation (BDL) to induce cirrhosis. BDL rats received vehicle, arachidonyl-2-chloroethylamide (cannabinoid receptor type 1 [CB(1) ] agonist), JWH-015 (cannabinoid receptor type 2 [CB(2) ] agonist), and AM630 (CB(2) antagonist) from days 35 to 42 days after BDL. On the 43rd day, hemodynamics, presence of CB receptors, severity of portosystemic shunting, mesenteric vascular density, vascular endothelial growth factor (VEGF), VEGFR-1, VEGFR-2, phospho-VEGFR-2, cyclooxygenase (COX)-1, COX-2, and endothelial nitric oxide synthase (eNOS) expressions as well as plasma VEGF levels were evaluated. Results showed that CB(1) and CB(2) receptors were present in left adrenal veins of sham rats, splenorenal shunts (the most prominent intra-abdominal shunts) of BDL rats, and mesentery of sham and BDL rats. CB(2) receptor was up-regulated in splenorenal shunts of BDL rats. Both acute and chronic JWH-015 treatment reduced portal pressure and superior mesenteric arterial blood flow. Compared with vehicle, JWH-015 significantly alleviated portosystemic shunting and mesenteric vascular density in BDL rats, but not in sham rats. The concomitant use of JWH-015 and AM630 abolished JWH-015 effects. JWH-133, another CB(2) agonist, mimicked the JWH-015 effects. JWH-015 decreased mesenteric COX-1, COX-2 messenger RNA expressions, and COX-1, COX-2, eNOS protein expressions. Furthermore, JWH-015 decreased intrahepatic angiogenesis and fibrosis. CONCLUSIONS: CB(2) agonist alleviates portal hypertension (PH), severity of portosystemic collaterals and mesenteric angiogenesis, intrahepatic angiogenesis, and fibrosis in cirrhotic rats. The mechanism is, at least partly, through COX and NOS down-regulation. CBs may be targeted in the control of PH and portosystemic collaterals.

Effects of simvastatin on the portal-systemic collateral vascular response to endothelin-1 and shunting degree in portal hypertensive rats.

OBJECTIVE: Endothelin-1 (ET-1) exerts vasoconstrictive effect on portal-systemic collateral vascular bed of portal hypertensive rats. Statins are lipid-lowering agents with nitric oxide (NO)-related vasodilatory effects. Considering NO-associated vascular hyporesponsiveness to vasoconstrictors and shunting formation in portal hypertension, this study investigated the effects of simvastatin on 1) the portal-systemic collateral vascular responsiveness to ET-1 and 2) the portal-systemic shunting degree. MATERIALS/METHODS: Portal hypertension was induced by partial portal vein ligation (PVL) in Sprague-Dawley rats. Simvastatin (20 mg/kg/day) or distilled water (control) was randomly administered by oral gavage since 2 days prior to until 7 days after PVL. Systemic and portal hemodynamics were measured on the 8th day. In another series, collateral perfusion with Krebs solution at different flow rates was performed to get flow-pressure curves which serve as an index of shunting degree. To survey the direct vascular effect, PVL rats randomly underwent preincubation with 1) Krebs solution, that is, the control group; or Krebs solution plus 2) simvastatin; 3) simvastatin + N (ω)-nitro-L-arginine (NNA, a NO synthase inhibitor); 4) simvastatin + indomethacin (a cyclooxygenase inhibitor), followed by ET-1 to evaluate the collateral vascular responsiveness. RESULTS: Chronic simvastatin treatment significantly reduced portal pressure. The flow-pressure curves were similar between two groups. Simvastatin preincubation reduced collateral perfusion pressure changes to ET-1 (p < 0.05), which were partially reversed by NNA (p < 0.05), but not by indomethacin. Conclusions. Chronic simvastatin treatment significantly improved portal hypertension. The effect was at least partially exerted by decreased portal-systemic collateral vascular resistance through NO-mediated vascular hyporesponsiveness. The severity of portal-systemic collaterals was not influenced by simvastatin.