OBJECTIVE

The aim of this study is to describe and review the cytological features of Kaposi sarcoma-associated herpes virus (KSHV) related entities, such as multicentric Castleman's disease (MCD), plasmablastic-lymphoma (PBL) and primary effusion lymphoma (PEL), which all may present as body cavity effusions. Serous fluid cytology of MCD and PBL has not, to our knowledge, thus far been described. Although different in nature, MCD, PBL and PEL are characterized by similar morphological features.

METHODS

Body cavity effusions from four different patients with previously known or unknown KSHV-related lymphoproliferations have been examined by routine cytology, immunocytochemistry (IC) and polymerase chain reaction (PCR).

RESULTS

MCD, PBL and PEL are all characterized by increased cellularity, comprising mainly lymphoid and plasmacytoid cells with variable proportions of immunoblasts. Immunocytochemistry and PCR results show the MCD to be CD138 and KSHV positive, CD30 negative, IgM, IgH and lambda restricted but IgH polyclonal. PBL was CD138 positive, kappa restricted, weakly positive with VS38 and over 80% positive with MIB 1. PEL was CD45, EMA, CD138, KSHV, p53 and CD3 positive, CD20, EBV, CD30, CD2, CD4, ALK1, epithelial and mesothelial markers negative, and PCR monoclonal B-cell expanded (Ig-kappa bands).

CONCLUSIONS

Cytological examination of effusions in KSHV-related lymphoproliferative disorders may show similar morphological features but clonality studies and immunocytochemistry are very helpful in distinguishing between these rare benign and malignant lymphoproliferative diseases.

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-β family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.

Hepatopulmonary syndrome (HPS) is a serious pulmonary vascular disease derived from chronic liver disease, and its key pathogenesis is angiogenesis. Krüppel-like factor 6 (KLF6) mediates physiological repair and remodeling during vascular injury. However, the role of KLF6 in pulmonary microvascular endothelial cells (PMVECs) during angiogenesis of HPS and its underlying mechanism in HPS have not been investigated. Common bile duct ligation (CBDL) in rats can replicate pulmonary vascular abnormalities of human HPS. Here, we found that advanced pulmonary angiogenesis and pulmonary injury score coincided with the increase of KLF6 level in PMVECs of CBDL rat; KLF6 in PMVECs was also induced while cultured with CBDL rat serum in vitro Inhibition of KLF6 dramatically suppressed PMVEC-mediated proliferation, migration and tube formation in vivo; this may be related to the downregulation of activin receptor-like kinase-1 (ALK1) and endoglin (ENG), which are transacted by KLF6. Bone morphogenetic protein 9 (BMP9) enhanced the expression of KLF6 in PMVECs and was involved in the angiogenesis of HPS. These results suggest that KLF6 triggers PMVEC-mediated angiogenesis of HPS and is aggravated by BMP9, and the inhibition of the BMP9/KLF6 axis may be an effective strategy for HPS treatment.

Bone morphogenetic protein (BMP)-9 and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor, ALK1, and the type II receptors, the activin type-IIA and bone morphogenetic type II receptors. We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and may influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of Chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. This was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.

Keywords: BMP10; BMP9; CCL2; Endothelial; TNF.

A rechallenge is common after initial efficacy of alkylating-based chemotherapy (ALK) in pancreatic neuroendocrine tumors (PanNET). High MGMT expression seems associated with lower response to ALK. We aimed to evaluate the efficacy and toxicity of ALK rechallenge in PanNET. Secondly, to assess the evolution of MGMT expression under ALK. All consecutive patients with advanced PanNETs who received initial ALK (achieving tumor control) followed by a pause > 3 months, then an ALK rechallenge (ALK2) upon progression were retrospectively studied (Cohort A). Primary endpoint was progression-free survival under ALK2 (PFS2). The MGMT expression was retrospectively assessed by immunohistochemistry (H-score) in consecutive PanNET surgically resected following ALK (Cohort B). We foud that cohort A included 62 patients (median Ki67 8%), for which ALK1 followed by pause achieved an objective response rate of 55%, and a PFS1 of 23.7 months (95% IC, 19.8-27.6). ALK2 achieved no objective response, and stability in 62% of patients. The median PFS2 was 9.2 months (IC 95% 7.1-11.3). At multivariable analysis, a hormonal syndrome (p=0.032) and a pause longer than 12 months (p=0.041) were associated with a longer PFS2. In the cohort B (17 patients), the median MGMT H-score increased from 45 (IQR 18-105) before ALK, to 100 (IQR 56-180) after ALK (p=0.003). We conclude that after initial efficacy of ALK treatment, a pause followed by ALK rechallenge might be appropriate to prolong tumor control, improve quality of life and limit long-term adverse events. Increased MGMT expression under ALK might explain low efficacy of ALK rechallenge.

Bone morphogenetic protein 9 (BMP9) has recently been described as a crucial regulator in modulating fibroblast-type cell activation. Activin receptor-like kinase 1 (ALK1) is a high affinity receptor for BMP9 that exerts its role via Smad1/5. However, the functional roles of BMP9 in activating lung fibroblasts and the underlying signaling pathway are not completely understood. The present study aimed to explore the effect of exogenous BMP9 on human lung fibroblast HFL-1 cell proliferation and differentiation, as well as the potential role of the ALK1/Smad1/5 signaling pathway. In the present study, fibroblast proliferation was assessed using Cell Counting Kit-8 and colony formation assays, and the mRNA and protein expression of target genes was examined using reverse transcription-quantitative PCR and western blot assays, respectively. Compared with the control group, BMP9 treatment increased HFL-1 cell proliferation, mRNA and protein expression of differentiated markers, including α-smooth muscle actin, type I collagen and type III collagen, and the expression of ALK1 and phosphorylated Smad1/5 expression. Furthermore, the effects of BMP9 were partially rescued by dorsomorphin-1, an inhibitor of ALK1. The results indicated that BMP9 may serve as a key inducer of lung fibroblast activation and ALK1/Smad1/5 signaling might be associated with BMP9-mediated effects in HFL-1 cells. Therefore, the present study highlighted that the potential role of the BMP9/ALK1/Smad1/5 signaling pathway in the development of pulmonary fibrosis requires further investigation.

Keywords: activin receptor-like kinase 1; bone morphogenic protein 9; differentiation; in vitro; lung fibroblast; proliferation; signaling pathway.

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. BMP receptor ALK1 is well-known for its role in blood vessel formation; however, its role within the immune system has never been revealed. Here, we found that BMP9/BMP10-ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages displayed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

Keywords: Immunology; Macrophages.

We describe a case of ALK1 negative (-) pulmonary anaplastic large-cell lymphoma (pALCL) in an adult female with an unfavorable outcome following combination chemotherapy and present a systematic review of 39 such sporadic cases reported over the past 28 years (1990-2018). pALCL occurred in 26 males and 13 females (median age, 43 years [5-81]) and 13/39 (33.33%) were ≤18 years. The lesions were endobronchial in 21 (53.85%) and parenchymal in 18 (46.15%) cases. Twenty-six cases were ALK1-; 13 were ALK1+ (positive); and 27/34 cases had a T cell phenotype (where tested). ALK- cases were characterized by higher age (P = 0.012) at presentation, more B symptoms (P = 0.002), and more parenchymal than endobronchial lesions (P = 0.039). The median survival (N = 29/39) was 60 months; pediatric group had a better survival than adult/elderly group (log-rank, P = 0.026). pALCL is rare and may have a distinct biological behavior.

Keywords: Histopathology; outcome; pulmonary lymphoma.

Anaplastic large cell lymphoma (ALCL) is a distinct T-cell non-Hodgkin lymphoma involving both nodal and extra-nodal sites with a specific anaplastic lymphoma kinase 1 (ALK-1) gene rearrangement. The commonly involved extranodal sites include skin, bone, soft tissue, lungs, and liver. ALCL primarily involving gastrointestinal (GI) tract is rare. In this manuscript, we describe a case of primary esophageal ALK1 positive-ALCL (null phenotype) in a young female, who presented with fleshy mucosal lesion in the lower third of the esophagus and present a systematic review of 35 cases of GI-ALCL reported in the English literature over the past 28 years (1990-2018) with regard to the clinicopathological characteristics, therapy, and outcome.

Keywords: Anaplastic large cell lymphoma; CD30; anaplastic lymphoma kinase 1; gastrointestinal tract.

Epithelioid fibrous histiocytoma (EFH) is a cutaneous neoplasm driven by translocations of the Anaplastic Lymphoma Kinase (ALK) gene, which can be demonstrated by immunohistochemical (IHC) analysis. We analysed the performance of two ALK clones, D5F3 and ALK1, in a cohort of EFHs and described the range of architectural variation of these lesions. TFE3 IHC was performed in ALK-negative EFHs. We identified 21 cases of EFH, 76.2% of which showed an exophytic appearance and 19% displayed flat architecture. A well-developed epidermal collarette was present in 48% of all cases with just over a third of all the exophytic lesions presenting as dermal-based nodules. ALK D5F3 expression was identified in 76.2% (16/21) of all cases but only 68.8% were concordantly positive with the ALK1 clone, indicative of a false negative stain with ALK1 in 31.2% of the cases. For the subset of cases showing positivity for the ALK1 clone, a marked decrease in the percentage of immunolabelled cells was identified when compared with D5F3 (5-50% vs. 100%, respectively). Five cases (23.8%) did not demonstrate ALK expression for either clone, with 3 of those cases showing nuclear positivity for TFE3 IHC, and the remaining 2 cases being double negative (ALK-/TFE3-). In summary, we identified that the prototypically described exophytic appearance with epidermal collarette is present in only less than half of the cases. We also demonstrated that the ALK1 antibody is suboptimal in EFH and should not be utilised in this setting. A subset of ALK-negative cases express TFE3, but double-negative cases occur.

Keywords: ALK; ALK1; D5F3; clone; epithelioid fibrous histiocytoma.

Activin A, a member of transforming growth factor-β superfamily, is involved in the regulation of cellular differentiation and promotes tissue healing. Previously, we reported that expression of activin A was upregulated around the damaged periodontal tissue including periodontal ligament (PDL) tissue and alveolar bone, and activin A promoted PDL-related gene expression of human PDL cells (HPDLCs). However, little is known about the biological function of activin A in alveolar bone. Thus, this study analyzed activin A-induced biological functions in preosteoblasts (Saos2 cells). Activin A promoted osteoblastic differentiation of Saos2 cells. Activin receptor-like kinase (ALK) 1, an activin type I receptor, was more strongly expressed in Saos2 cells than in HPDLCs, and knockdown of ALK1 inhibited activin A-induced osteoblastic differentiation of Saos2 cells. Expression of ALK1 was upregulated in alveolar bone around damaged periodontal tissue when compared with a nondamaged site. Furthermore, activin A promoted phosphorylation of Smad1/5/9 during osteoblastic differentiation of Saos2 cells and knockdown of ALK1 inhibited activin A-induced phosphorylation of Smad1/5/9 in Saos2 cells. Collectively, these findings suggest that activin A promotes osteoblastic differentiation of preosteoblasts through the ALK1-Smad1/5/9 pathway and could be used as a therapeutic product for the healing of alveolar bone as well as PDL tissue.

Keywords: ALK1; activin A; osteoblastic differentiation; periodontal ligament; preosteoblast.

Editing linear polyubiquitination of protein substrates by LUBAC and OTULIN is known to play a critical role in immune responses. A recent study by Fu et al. reveals how reversible linear polyubiquitination of the activin receptor-like kinase (ALK1) controls developmental angiogenesis and how its dysfunction leads to vascular malformations in humans.

Keywords: ALK1; BMP9; LUBAC; OTULIN; angiogenesis; arteriovenous malformation.

Objective: To analyze the molecular regulation network of circular RNA (circRNA) in colon cancer (CC) by bioinformatics method.

Methods: hsa_circ_0007843 and hsa_circ_0007331 proved to be associated with CC in previous studies were chosen as the research object. ConSite database was used to predict the transcription factors associated with circRNA, and the CC-associated transcription factors were screened out after intersection. The CircInteractome database was used to predict the RNA-binding proteins (RBPs) interacting with circRNAs and screen out the CC-associated RBPs after an intersection. Furthermore, the CircInteractome database was used to predict the miRNAs interrelated with circRNAs, and the HMDD v3.2 database was used to search for miRNAs associated with CC. The target mRNAs of miRNA were predicted by the miRWalk v3.0 database. CC-associated target genes were screened out from the GeneCards database, and the upregulated genes were enriched and analyzed by the FunRich 3.1.3 software. Finally, the molecular regulatory network diagram of circRNA in CC was plotted.

Results: The ConSite database predicted a total of 14 common transcription factors of hsa_circ_0007843 and hsa_circ_0007331, among which Snail, SOX17, HNF3, C-FOS, and RORα-1 were related to CC. The CircInteractome database predicted that the RBPs interacting with these two circRNAs were AGO2 and EIF4A3, and both of them were related to CC. A total of 17 miRNAs interacting with hsa_circ_0007843 and hsa_circ_0007331 were predicted by CircInteractome database. miR-145-5p, miR-21, miR-330-5p, miR-326, and miR-766 were associated with CC according to the HMDDv3.2 database. miR-145-5p and miR-330-5p, lowly expressed in CC, were analyzed in the follow-up study. A total of 676 common target genes of these two miRNAs were predicted by the miRWalk3.0 database. And 57 target genes were involved in the occurrence and development of CC from the GeneCards database, with 23 genes downregulated and 34 genes upregulated. Additionally, GO analysis showed that the 34 upregulated genes were mainly enriched in biological processes such as signal transduction and cell communication. KEGG pathway analysis showed that the upregulated genes were closely related to integrin, ErbB receptor, and ALK1 signal pathways. Finally, a complete regulatory network of hsa_circ_0007843 and hsa_circ_0007331 in CC was proposed, whereby each one of the participants was either directly or indirectly associated and whose deregulation may result in CC progression.

Conclusion: Predicting the molecular regulatory network of circRNAs by bioinformatics provides a new theoretical basis for further occurrence and development pathogenesis of CC and good guidance for future experimental research.

Objective: Hereditary hemorrhagic telangiectasia is the only condition associated with multiple inherited brain arteriovenous malformations (AVMs). Therefore, a mouse model was developed with a genetics-based approach that conditionally deleted the causative activin receptor-like kinase 1 (Acvrl1 or Alk1) gene. Radiographic and histopathological findings were correlated, and AVM stability and hemorrhagic behavior over time were examined.

Methods: Alk1-floxed mice were crossed with deleter mice to generate offspring in which both copies of the Alk1 gene were deleted by Tagln-Cre to form brain AVMs in the mice. AVMs were characterized using MRI, MRA, and DSA. Brain AVMs were characterized histopathologically with latex dye perfusion, immunofluorescence, and Prussian blue staining.

<strong class="sub-title"> Results: </strong> Brains of 55 Tagln-Cre+;<em>Alk1</em>2f/2f mutant mice were categorized into three groups: no detectable vascular lesions (group 1; 23 of 55, 42%), arteriovenous fistulas (AVFs) with no nidus (group 2; 10 of 55, 18%), and nidal AVMs (group 3; 22 of 55, 40%). Microhemorrhage was observed on MRI or MRA in 11 AVMs (50%). AVMs had the angiographic hallmarks of early nidus opacification, a tangle of arteries and dilated draining veins, and rapid shunting of blood flow. Latex dye perfusion confirmed arteriovenous shunting in all AVMs and AVFs. Microhemorrhages were detected adjacent to AVFs and AVMs, visualized by iron deposition, Prussian blue staining, and macrophage infiltration using CD68 immunostaining. Brain AVMs were stable on serial MRI and MRA in group 3 mice (mean age at initial imaging 2.9 months; mean age at last imaging 9.5 months).

Conclusions: Approximately 40% of transgenic mice satisfied the requirements of a stable experimental AVM model by replicating nidal anatomy, arteriovenous hemodynamics, and microhemorrhagic behavior. Transgenic mice with AVFs had a recognizable phenotype of hereditary hemorrhagic telangiectasia but were less suitable for experimental modeling. AVM pathogenesis can be understood as the combination of conditional Alk1 gene deletion during embryogenesis and angiogenesis that is hyperactive in developing and newborn mice, which translates to a congenital origin in most patients but an acquired condition in patients with a confluence of genetic and angiogenic events later in life. This study offers a novel experimental brain AVM model for future studies of AVM pathophysiology, growth, rupture, and therapeutic regression.

Keywords: Alk1; arteriovenous malformation; hereditary hemorrhagic telangiectasia; magnetic resonance imaging; transgenic mice; vascular disorders.

Chondrocytes in mice developing Osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine Transforming Growth Factor (TGF)-β, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-β receptor ALK5 mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-β signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-β co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-β-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure nor validated biomarker. This article is protected by copyright. All rights reserved.

Keywords: ALK; BMP; TGF-β; Tdgf1; ageing; bone; calcification; cartilage; joint.

Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a rare variant of the inflammatory myofibroblastic tumor. It has an aggressive clinical course and a high rate of recurrence. EIMS primarily affects children and young adults. Hereby, we report this entity in a 4-month-old infant who presented with an abdominal mass. Imaging studies revealed a large hypodense mesentery-based lesion involving the right half and mid-region of the abdomen. The mass with an attached segment of the small bowel was excised in toto. Grossly, a large encapsulated tumor was identified arising from the mesentery of the small bowel. The histological examination showed a tumor consisting of epithelioid to spindle cells loosely arranged in a myxoid background with numerous blood vessels and lymphoplasmacytic inflammatory infiltrate. On immunohistochemistry, the tumor cells showed positivity for ALK1 (nuclear), desmin, SMA, CD68, and focal positivity for CD30. A final diagnosis of EIMS of the small intestine was rendered. To the best of our knowledge, this case is the youngest reported case in literature.

Keywords: Anaplastic Lymphoma Kinase; Epithelioid Cells; Intestine, Small; Mesentery; Sarcoma.

The diagnosis of high-grade endometrial stromal sarcoma has become more refined following molecular characterization of these tumors. Recently BCOR internal tandem duplications (ITD) have been identified in a small number of high-grade endometrial stromal sarcoma. Here we present an additional case of this rare entity in a young woman in her late teens. She presented with menorrhagia and underwent resection of 2 uterine lesions. The tumor was a spindle cell neoplasm composed of long fascicles with low to moderate cellularity, mild to moderate cytologic atypia, and up to 2 mitotic figures per 10 high power fields. Necrosis was not identified. Immunohistochemical stains showed the tumor to be positive for cyclin D1 in >50% of tumor cells, focally positive for CD10, and negative for SMA, desmin, h-caldesmon, and ALK1. BCOR ITD was confirmed by polymerase chain reaction with subsequent Sanger sequencing. Clues to the diagnosis of BCOR ITD uterine sarcoma include young patient age, uniform nuclear features, and diffuse positivity for cyclin D1. These features should prompt further molecular interrogation for definitive diagnosis, which is important for prognostication.

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

Keywords: activin receptor-like kinase; angiogenesis; bone morphogenetic protein; endothelial cell; macrocyclic kinase inhibitor.

Background: The etiology of pulmonary arterial hypertension (PAH) in the Han Chinese population is poorly understood.

Objectives: The aim of this study was to assess gene variants and associated functional annotations for PAH in Han Chinese patients.

Methods: This is an ethnicity-based multi-centre study. Blood samples were collected from 20 PAH patients who volunteered for the study, and genetic tests were performed. The DAVID database was used to functionally annotate the genes BMPR2, ALK1, KCNK3, CAV1, and ENG. Associated diseases, functional categories, gene ontology, and protein interactions were analysed using the Functional Annotation Tool in the DAVID database. GEO and ClinVar databases were also used for further comparison with gene mutations in our study.

Results: PAH patient with gene mutations were female predominant except for a single male with a BMPR2 mutation. Locus variants in our study included 'G410DfsX1' in BMPR2, 'ex7 L300P,' 'ex4 S110PfsX40,' and 'ex7 E295Afs96X' in ALK1, 'c.-2C>A (IVS1-2 C>A)' in CAV1, and 'ex8 D366Q' in ENG were not found in the ClinVar database associated with PAH. In addition to BMP and TGF-β pathways, gene ontology of input genes in the DAVID database also included pathways associated with nitric oxide signaling and regulation.

Conclusions: This Multi-centre study indicated that 'G410DfsX1' in BMPR2, 'ex7 L300P,' 'ex4 S110PfsX40,' 'ex7 E295Afs96X' in ALK1, 'c.-2C>A (IVS1-2 C>A)' in CAV1, and 'ex8 D366Q' in ENG were identified in Han Chinese patients with PAH. Females were more susceptible to PAH, and a relatively young age distribution was observed for patients with BMPR2 mutations.

Keywords: ClinVar database; DAVID database; gene annotation; gene mutation; heritable pulmonary arterial hypertension; pulmonary arterial hypertension.

Objective: Intraoral, primary, CD30-positive (CD30⁺) T-cell lymphoproliferative disorders (TLPDs) are uncommon, and their clinicopathologic presentation and management can vary and may be challenging. Herein, we present a retrospective study of 4 examples of self-regressing primary CD30⁺ TLPD affecting the gingiva.

Study design: Archived files were retrospectively reviewed for oral CD30⁺ TLPDs featuring (1) proper immunohistochemical documentation, (2) Epstein-Barr virus negativity, (3) adequate follow-up information corroborating regression, and (4) no history of hematopoietic malignancy or related-mucocutaneous disease.

Results: Three women and 1 man (age range, 55-82 years; mean, 68.3 years) presented with rapidly growing gingival ulcers. Microscopic evaluation revealed diffuse infiltration by sheets of large, atypical cells admixed with lymphocytes and eosinophils, showing angiocentric distribution, focal neurotropism, and muscle infiltration. The lesional cells consistently stained for CD3 and CD30 and were variably immunoreactive against CD2, CD4, CD5, CD7, and CD8, but were negative for ALK1 and EBV-encoded small RNA. TCR-γ gene rearrangement studies revealed a monoclonal T-cell population in 1 case. All lesions showed complete regression 2 to 8 weeks postoperatively (mean follow-up, 4.5 weeks).

Conclusions: Notwithstanding their alarming clinicopathologic appearance, there are CD30⁺ TLPDs confined to the oral cavity that have an indolent course. However, clinical staging is essential to exclude aggressive systemic malignancy.

<b>0bjective</b> To investigate the clinicopathological features,immunohistochemical phenotypes, molecular genetic alterations,diagnosis and differential diagnosis of inflammatory myofibroblastic tumor (IMT) of the urinary bladder. <b>Methods:</b> Ten cases of IMT of the urinary bladder (three cases at Ningbo Diagnostic Pathology Center from September 2011 to December 2020, five in-house diagnosed cases and two consultation cases at Shanghai Rui Jin Hospital from June 2011 to December 2020) were collected retrospectively. The clinicopathologic features and immunophenotypic profiles were studied by light microscopy and immunohistochemistry (EnVision method). The translocation of ALK gene was detected by fluorescence in situ hybridization (FISH). <b>Results:</b> Of the 10 patients, eight were male and two were female. The patients' age range was 16 to 62 years (median 36 years).The main clinical presentation was hematuria and urinary irritation. Three cases were located at the dome of the urinary bladder, four cases were in the left lateral wall and the remaining three cases were in the right lateral wall. The tumor size ranged from 1.5 cm to 8.5 cm. In eight cases, the tumors were mainly submucosal, and in some cases extending to the muscular layer of the urinary bladder. In two cases, the tumors were mainly located in the muscular layer and focally extended to the submucosa and adventitia. Histologically, four cases had the nodular fasciitis-like pattern, three cases had fibrohistiocytoma-like pattern, two cases had mixed histologic patterns and the remaining case showed leiomyosarcoma-like histologic features. Immunohistochemically, the tumor cells expressed SMA (10/10),calponin (9/10),desmin (6/10) and CKpan (9/10). Cytoplasmic staining for <em>ALK1</em> and ALK (5A4) was detected in 7 of 10 cases and 8 of 10 cases, respectively. Nuclear and cytoplasmic staining for ALK (D5F3) was detected in 7 of 10 cases. Among eight cases with material available for FISH analysis, ALK rearrangement was present in five cases. Follow-up data were available in eight patients and none had local recurrence nor distant metastasis. <b>Conclusion:</b> IMT of the urinary bladder is an uncommon mesenchymal neoplasm with intermediate malignant potential.It has special clinicopathologic features, and a minority of cases have local tumor recurrence.

目的： 探讨膀胱炎性肌纤维母细胞肿瘤（IMT）临床病理学特点、免疫表型、分子遗传学改变、诊断与鉴别诊断。 方法： 收集宁波市临床病理诊断中心2011年9月至2020年12月诊断的3例和上海交通大学医学院附属瑞金医院2011年6月至2020年12月诊断的5例及2例会诊病例共10例IMT患者的临床和病理学资料，采用EnVision法进行免疫组织化学标记，应用荧光原位杂交（FISH）检测ALK基因的重排情况。 结果： 患者男性8例，女性2例。年龄范围16~62岁，中位年龄36岁。主要临床症状为血尿和尿路刺激症状，3例位于膀胱顶部，左侧壁4例，右侧壁3例。肿瘤最大径1.5~8.5 cm。8例病变主要位于黏膜下，部分区域累及肌层；2例病变主要位于肌层内，膨胀性浸润性生长，局灶侵及黏膜固有层及外膜。镜下特点，4例为结节性筋膜炎样型，3例为纤维组织细胞瘤样型，2例为混合型，1例呈平滑肌肉瘤样。免疫组织化学示10例病例平滑肌肌动蛋白（SMA）均阳性，9例Calponin阳性，6例结蛋白阳性，9例广谱细胞角蛋白（CKpan）阳性，9例间变性淋巴瘤激酶（ALK）阳性［其中7例ALK1胞质阳性，8例ALK（5A4）胞质阳性，7例ALK（D5F3）核质阳性］。8例行FISH检测，其中5例显示ALK基因重排阳性。随访8例，无复发及转移。 结论： 膀胱IMT是一种少见的中间型的具有恶性潜能的间叶性肿瘤，具有特殊的临床病理特征，较少发生复发。.

Atherosclerosis (AS) is a chronic inflammatory disease that can be caused by the proliferation and migration of human vascular smooth muscle cells (HVSMCs). Here, we found that lncRNA XIST was related to the abnormal proliferation and migration of HVSMCs, and thus, the mechanism by which XIST regulated HVSMCs was further investigated. HVSMCs were treated with oxidized low-density lipoprotein (ox-LDL, 100 μg/ml) as AS models. CCK8 assays, flow cytometry, Transwell assays and wound healing assays were applied to evaluate cell viability, cell cycle analysis, and cell migration, respectively. A dual-luciferase reporter assay was employed to verify the binding relationships between XIST and miR-761, miR-761, and BMP9. Ox-LDL induced the proliferation and migration of HVSMCs, upregulated the expression of XIST, downregulated miR-761 expression, and activated the BMP9/ALK1/endoglin pathway. Luciferase assays revealed that XIST sponged miR-761. XIST knockdown ameliorated ox-LDL-mediated effects in HVSMCs, which were largely abolished by miR-761 silencing. BMP9 was targeted-inhibited by miR-761. MiR-761 overexpression alleviated ox-LDL-mediated effects in HVSMCs. However, BMP9 overexpression abolished miR-761-mediated effects in HVSMCs treated with ox-LDL. Our findings suggested that XIST knockdown suppressed the proliferation and migration of HVSMCs by promoting miR-761, which targeted-inhibited the BMP9/ALK1/endoglin pathway.

Keywords: HVSMCs; atherosclerosis; migration.

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1^- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1^- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk1^2f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk1^2f/2f;confetti^+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk1^2f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1^- ECs at early stage of bAVM development, suggesting that Alk1^- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1^- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

Keywords: Alk1; Arteriovenous malformation; Bone marrow derived endothelial cells; Clonal expansion; Endothelial cells.

Studies have confirmed that circular RNA (circRNA) has a stable closed structure, which plays an important role in the progression of tumors. Cancers with positive fusion genes can produce associated fusion circRNA (F-cirRNA). However, there are no reports concerning a role for F-circRNA of the echinoderm microtubule associated-protein like 4-anaplastic lymphoma kinase variant 1 (EML4-ALK1) in non-small cell lung cancer (NSCLC). Our study confirmed the existence of fusion circEA1 (F-circEA1) in NCI-H3122 cells (carrying the EML4-ALK1 gene), F-circEA1 was expressed both in the cytoplasm and nucleus as determined by fluorescence in situ hybridization (FISH) and Sanger sequencing. CCK8 and transwell assays showed that F-circEA1 was beneficial to cell proliferation, metastasis, and invasion. Overexpression of F-circEA1 can also promote cell proliferation, migration and invasion in A549 and SPCA1 cells (non-small cell lung cancer cell line not carrying the EML4-ALK1 gene). Interference with F-circEA1, induced cell cycle arrest and promoted apoptosis as determined by flow cytometry, and increased drug sensitivity to crizotinib in H3122 cells. F-circEA1 directly affected the expression of parental gene EML4-ALK1. Further research found that F-circEA1 can affect the downstream signaling pathway of ALK. In vivo, the growth rate of xenogeneic tumors was reduced and the protein expression level of EML4-ALK1 was significantly decreased in transplanted tumors measured by immunohistochemistry (IHC) after interference with F-circEA1. In conclusion, F-circEA1 can be considered as a proto-oncogene that regulates cell proliferation and apoptosis by affecting the expression of the parental gene EML4-ALK1 and its ALK downstream signaling pathway in non-small cell lung cancer.

Keywords: Apoptosis; Circular RNA; EML4-ALK; F-circEA1; NSCLC.