Inhibiting redox-mediated endothelial migration by gadofullerenes for inducing tumor vascular normalization and improving chemotherapy.

Tumor vascular normalization (TVN) reverses abnormal tumor vasculatures, which could boost anti-cancer efficiency and especially increase drug intratumoral delivery. Endothelial cells play a vital role in angiogenesis, yet continuous modulating endothelial cell migration to improve TVN is ingenious but challenging. Here we propose a potential strategy for TVN based on inhibiting endothelial migration using antioxidative fullerene nanoparticles (FNPs). We demonstrate that FNPs inhibit cell migration upon their anti-oxidation effects in vitro. The optimized alanine-modified gadofullerene (GFA) exhibits superior TVN ability and inhibits tumor growth in vivo. Mechanically, facilitated with the protein microarray, we confirm that GFA could suppress the focal adhesion pathway to restrain endothelial migration. Subsequently, remarkable anti-tumor efficacy of chemotherapy synergy was obtained, which benefited from a more normalized vascular network by GFA. Together, our study introduces the potential of FNPs as promising TVN boosters to consider in cancer nanomedicine design.

Liver mesenchymal neoplasms: something old, something new.

Histological examination of liver biopsies and resection specimens remains the gold standard to establish a definitive diagnosis of liver lesions. While hepatocellular carcinoma remains the most commonly encountered liver lesion on mass-directed biopsies, surgical pathologists must be aware of other entities that may pose diagnostic challenges, as an accurate diagnosis is key for patient management. Mesenchymal tumours of the liver are relatively uncommon, therefore many pathologists are unfamiliar with these tumours. While the clinical presentation and radiological features of these lesions often overlap, careful attention to histological clues can assist in weeding out various congeners to arrive at the most accurate diagnosis. An additional challenge when diagnosing mesenchymal tumours is the specimen type, as mass-directed core biopsies are limited and have become standard clinical practice. Besides careful attention to histological features, radiological findings and clinical history, immunohistochemical analysis and molecular studies have become of immense diagnostic value. In this review, we discuss several common and rare mesenchymal hepatic lesions as defined in the current World Health Organization (WHO) classification and most up-to-date literature. We also discuss immunohistochemistry panels and relevant molecular findings that may assist in rendering an accurate diagnosis when encountering these lesions in daily practice.

Hexokinase 2-driven glycolysis in pericytes activates their contractility leading to tumor blood vessel abnormalities.

Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role.

A unique epithelioid vascular neoplasm of bone characterized by EWSR1/FUS-NFATC1/2 fusions.

An increasing number of epithelioid vascular lesions, in particular tumors from the benign and low-grade end of the spectrum, have been characterized by recurrent gene fusions. As a result, the detection of these molecular markers have improved the classification of diagnostically challenging cases. However, despite the significant progress, there are occasional lesions that do not fit in known histologic or molecular groups. Herein, we present five such unclassified epithelioid vascular lesions, which occurred in the bone and showed a distinct morphology composed of alternating vasoformative and solid growth and mild to moderate nuclear pleomorphism. The variegated morphologic appearance resembled that of composite hemangioendothelioma, being distinct from both epithelioid hemangioma and epithelioid hemangioendothelioma, and consistently showed cytologic atypia. Due to their unusual morphologic appearance and negative molecular work-up, targeted transcriptome sequencing was performed in two cases showing the presence of NFATC2 fusions with either EWSR1 or FUS genes. Three additional bone tumors with EWSR1 gene rearrangements were identified by FISH screening of a large cohort of 45 fusion-negative epithelioid vascular neoplasms, one fused to NFATC2 while two others to NFATC1. There were three females and two males, with a wide age range at presentation, mean of 44 years. The lesions occurred in the pelvis, maxillary sinus, and humerus. Two patients presented with polyostotic disease, both located in the pelvic bones. Two patients had available follow-up, one developed two local recurrences in the humerus over a 15-year period, while the other showed no recurrence 4 years subsequent to an en-bloc resection. Tumors were positive for CD31 and ERG, while negative for EMA, CK, synaptophysin, and chromogranin. FISH confirmed this abnormality in all cases, none of them being associated with gene amplifications. Further studies are needed to establish the pathogenetic relationship of this rare molecular subset with other epithelioid vascular tumors and to determine its clinical behavior.

Vascular Tumor Recapitulated in Endothelial Cells from hiPSCs Engineered to Express the SERPINE1-FOSB Translocation.

Chromosomal translocations are prevalent among soft tissue tumors, including those of the vasculature such as pseudomyogenic hemangioendothelioma (PHE). PHE shows endothelial cell (EC) features and has a tumor-specific t(7;19)(q22;q13) SERPINE1-FOSB translocation, but is difficult to study as no primary tumor cell lines have yet been derived. Here, we engineer the PHE chromosomal translocation into human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 and differentiate these into ECs (hiPSC-ECs) to address this. Comparison of parental with PHE hiPSC-ECs shows (1) elevated expression of FOSB, (2) higher proliferation and more tube formation but lower endothelial barrier function, (3) invasive growth and abnormal vessel formation in mice after transplantation, and (4) specific transcriptome alterations reflecting PHE and indicating PI3K-Akt and MAPK signaling pathways as possible therapeutic targets. The modified hiPSC-ECs thus recapitulate functional features of PHE and demonstrate how these translocation models can be used to understand tumorigenic mechanisms and identify therapeutic targets.

[Characteristics of ERG, Fli-1, CD34, CD31 and Fâ §RAg expression in hepatic malignant vascular tumors].

Objective: To investigate the expression of ERG, Fli-1, CD34, CD31 and factor Ⅷ-related antigen(FⅧRAg) in hepatic malignant vascular tumors. Methods: A retrospective analysis was conducted on 63 cases of primary hepatic malignant vascular tumors and 31 cases of hepatic other malignant spindle cell tumors collected during January 1986 to January 2014. EnVision method was used to detect the expression of ERG, Fli-1, CD34, CD31, FⅧRAg. Results: Sixty-three cases of malignant vascular tumors, including 24 cases of angiosarcoma, 38 cases of epithelioid hemangioendothelioma and 1 case of hepatic Kaposi's sarcoma. All of the cases were positive for ERG(100.0%, 63/63). Positive rate of Fli-1, CD34, CD31, FⅧRAg was 96.8% (61/63), 87.3% (55/63), 81.0% (51/63) and 41.3% (26/63), respectively. In other hepatic malignant spindle cell tumors, the positive rate of ERG, Fli-1, CD34, CD31 and FⅧRAg was 3.2% (1/31), 19.4% (6/31), 19.4% (6/31), 9.7%(3/31) and 3.2%(1/31), respectively.The sensitivity of ERG, Fli-1, CD34, CD31, FⅧRAg was 100.0%, 96.8%, 87.3%, 81.0% and 41.3%, respectively.The specificity was 96.8%, 80.6%, 80.6%, 90.3% and 96.8%, respectively. Conclusion: ERG is a more sensitive and specific diagnostic marker for hepatic malignant vascular tumors in comparison to Fli-1, CD34, CD31 and FⅧRAg.

Expression of Vascular Endothelial Growth Factor Receptors in Benign Vascular Lesions of the Orbit: A Case Series.

PURPOSE: Vascular lesions of the orbit, although not malignant, can cause morbidity because of their location near critical structures in the orbit. For the same reason, they can be challenging to remove surgically. Anti-vascular endothelial growth factor (VEGF) drugs are increasingly being used to treat diseases with prominent angiogenesis. Our study aimed to determine to what extent VEGF receptors and their subtypes are expressed on selected vascular lesions of the orbit. DESIGN: Retrospective case series of all orbital vascular lesions removed by one of the authors (JAG) at the Mayo Clinic. PARTICIPANTS: A total of 52 patients who underwent removal of vascular orbital lesions. METHODS: The pathology specimens from the patients were retrieved, their pathologic diagnosis was confirmed, demographic and clinical information were gathered, and sections from vascular tumors were stained with vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor type 1 (VEGFR1), vascular endothelial growth factor receptor type 2 (VEGFR2), and vascular endothelial growth factor receptor type 3 (VEGFR3). MAIN OUTCOME MEASURES: The existence and pattern of staining with VEGF and its subtypes on these lesions. RESULTS: There were 28 specimens of venous malformations, 4 capillary hemangiomas, 7 lymphatic malformations, and 6 lymphaticovenous malformations. All samples stained with VEGF, 55% stained with VEGFR1, 98% stained with VEGFR2, and 96% stained with VEGFR3. Most (94%) of the VEGFR2 staining was diffuse. CONCLUSIONS: Most orbital vascular lesions express VEGF receptors, which may suggest a future target for nonsurgical treatment.

CD30 expression in malignant vascular tumors and its diagnostic and clinical implications: a study of 146 cases.

Angiosarcoma (AS) is a rare malignant vascular tumor, whereas epithelioid hemangioendothelioma (EHE) is a vascular tumor of low-grade malignancy. CD30 is a member of the tumor necrosis factor receptor superfamily, member 8 (TNFRSF8). Although the expression of CD30 is most commonly associated with lymphoid malignancies or germ cell tumors, occasional ASs have been reported as CD30 positive. However, there are limited data to evaluate its role definitively in malignant vascular tumors. In this study, we evaluated 91 ASs, 30 EHEs from various sites, and 25 Kaposi sarcomas. Overall, CD30 was expressed in 31/91 cases (34%) of AS, in 7/30 cases (30%) of EHE, but in none of the Kaposi sarcomas. CD30 was expressed in a membranous staining pattern and positivity in tumor cells varied from focal to diffuse. The positive ASs included vasoformative more differentiated tumors and also solid, undifferentiated, lymphoma-like examples, one of which was classified as lymphoma before the era of immunohistochemistry. The CD30 expression was seen in >50% of tumor cells in a majority of ASs but only in 7% of EHEs. None of the 55 ASs studied were immunohistochemically positive for TIA-1 or Granzyme B, antigens used as more specific markers for anaplastic large-cell lymphoma. Compared with AS, normal vascular endothelia of capillaries and muscular vessels showed variable positivity. Among hemangiomas, cavernous and spindle cell hemangiomas showed most frequent endothelial CD30 positivity, whereas in most other hemangiomas, CD30 positivity was scant. In conclusion, CD30 expression occurs in a significant subset of ASs and EHEs and needs to be included in the differential diagnosis with other CD30-positive malignancies to avoid a diagnostic pitfall. It remains to be determined whether patients with strongly CD30-positive ASs could be candidates for targeted therapy using the recently introduced CD30 antibody drug conjugates.

c-myc and cutaneous vascular neoplasms.

The c-myc proto-oncogene is involved in various cellular processes including cell growth, proliferation, and apoptosis. Overexpression and deregulated expression of the gene have been previously linked to several lineage-unrelated, aggressive, and poorly differentiated tumors. The expression of c-myc has also been implicated in hematopoiesis and has been shown to play a crucial role in angiogenesis via a vascular endothelial growth factor-dependent mechanism. This gives c-myc a dual oncogenic function in that tumor growth requires both cell proliferation and angiogenesis to ensure survival and confer an effective malignancy. Amplification of c-myc has been recently reported to be a recurrent genetic alteration in angiosarcomas secondary to irradiation and/or chronic lymphedema. Of note, however, no c-myc gene abnormalities have been demonstrated in cases of primary angiosarcomas or postradiation atypical vascular lesions. More recently, our own experience indicates that c-myc amplification is not normally found in the Kaposi sarcoma and cannot be correlated with expression of the c-Myc protein. This comprehensive review outlines the structure, normal functions, and effects of the deregulated expression of c-myc with particular emphasis on its role in angiogenesis and select cutaneous vascular neoplasms.

Update on vascular neoplasms.

This article provides an update on vascular neoplasms. New immunohistochemical markers for the diagnosis of vascular neoplasms tumor 1, infantile hemangiomas, myopericytomas, perivascular epithelial cell tumors, acquired elastotic hemangiomas, vascular proliferations in radiated skin, and new histopathologic variants of AIDS-related Kaposi sarcoma are explored.

Reverse translation of phase I biomarker findings links the activity of angiotensin-(1-7) to repression of hypoxia inducible factor-1α in vascular sarcomas.

BACKGROUND: In a phase I study of angiotensin-(1-7) [Ang-(1-7)], clinical benefit was associated with reduction in plasma placental growth factor (PlGF) concentrations. The current study examines Ang-(1-7) induced changes in biomarkers according to cancer type and investigates mechanisms of action engaged in vitro. METHODS: Plasma biomarkers were measured prior to Ang-(1-7) administration as well as 1, 2, 3, 4, and 6 hours after treatment. Tests for interaction were performed to determine the impact of cancer type on angiogenic hormone levels. If a positive interaction was detected, treatment-induced biomarker changes for individual cancer types were assessed. To investigate mechanisms of action, in vitro growth assays were performed using a murine endothelioma cell line (EOMA). PCR arrays were performed to identify and statistically validate genes that were altered by Ang-(1-7) treatment in these cells. RESULTS: Tests for interaction controlled for dose cohort and clinical response indicated a significant impact of cancer type on post-treatment VEGF and PlGF levels. Following treatment, PlGF levels decreased over time in patients with sarcoma (P = .007). Treatment of EOMA cells with increasing doses of Ang-(1-7) led to significant growth suppression at doses as low as 100 nM. PCR arrays identified 18 genes that appeared to have altered expression after Ang-(1-7) treatment. Replicate analyses confirmed significant changes in 8 genes including reduction in PlGF (P = .04) and hypoxia inducible factor 1α (HIF-1α) expression (P < .001). CONCLUSIONS: Ang-(1-7) has clinical and pre-clinical activity for vascular sarcomas that is linked to reduced HIF-1α and PlGF expression.

RUNX-1 and CD44 as markers of resident stem cell derivation in undifferentiated intimal sarcoma of pulmonary artery.

AIMS: To report a rare case of undifferentiated intimal sarcoma (UIS) of the pulmonary artery in a 44-year-old woman, and to study it by electron microscopy and a novel immunohistochemical (IHC) panel that recognizes markers of endothelial and haematopoietic stemness, in order to extend current knowledge about the histogenesis of this rare neoplasm. METHODS AND RESULTS: Immunohistochemical reactions for CD31, CD34, α-smooth muscle actin (α-SMA), caldesmon, calponin, actin, desmin, epithelial membrane antigen, WT1, CD117, Ki67, nestin, RUNX-1, platelet-derived growth factor, NG2, CD44, CD90 and CD105 were performed manually or automatically. Neoplastic cells were negative for CD31 and CD34, but positive for calponin, nestin, WT1, CD44, and RUNX-1. Electron microscopy was performed after osmium tetroxide fixation and staining with lead citrate and uranyl acetate. Ultrastructurally, tumour cells had slightly irregular nuclei, cisternae of rough endoplasmic reticulum, and punctate intercellular junctions. CONCLUSION: We report a case of pulmonary artery UIS expressing previously unreported markers, i.e. RUNX-1, nestin, WT1, and CD44, that are commonly seen in different stages of the vascular differentiation hierarchy. These findings, together with the negativity for mature endothelial and smooth muscle markers, raise the question of whether this neoplasm may derive from a vessel wall-resident stem cell, such as the haemangioblast or an embryonic-like stem cell.

Ultrastructure of myopericytoma: a continuum of transitional phenotypes of myopericytes.

The authors report the ultrastructural characteristics of myopericytoma, a recently described variant of perivascular (pericytic) tumors, mainly with regard to their myopericytic cells and vessels. Myopericytes range between pericytes and vascular smooth muscle cells (SMCs) in a morphologic continuum. The principal findings of the intermediate phenotypes are (1) elongated or annular morphology with processes of varying length and thickness (usually long and thin); (2) a continuous, irregularly thickened and zonally duplicated basement membrane; (3) heterocellular "peg and socket" junctions with neighboring endothelial cells, and scarce specialized junctions between myopericytes; (4) numerous micropinocytotic vesicles, whether continuous or forming focal rows; (5) abundant thin microfilaments, grouped in bundles with dense bodies and adhesion plaques; (6) poorly developed synthetic system (RER and Golgi); (7) pseudointracellular bodies formed by invagination of basement and plasma membranes, with numerous endocytic vesicles; and (8) zones of cytoplasmic rarefaction near micropinocytotic vesicles and intracellular organelles. The ultrastructure of myopericytes therefore makes it possible to distinguish them from pericytes, SMCs, and fibroblast/myofibroblasts, which is useful for myopericytoma diagnosis. The main pattern of the vessels, with perivascular concentric and multilayered growth of myopericytes (a thick wall in contrast to a small lumen) and lack of elastic material, also supports an intermediate form between pericytic and muscular microvasculature. The presence of myopericytes more similar to SMCs and of hemangiopericytoma-like vessels concurs with transitional forms with angioleyomyoma and true hemangiopericytoma, histogenetically representing a morphologic continuum for the perivascular tumors.

Occasional staining for p63 in malignant vascular tumors: a potential diagnostic pitfall.

Expression of p63, a putative marker for epithelial or myoepithelial differentiation, has been used to distinguish spindle cell carcinoma from sarcoma. The specificity of p63 for epithelial differentiation has not been thoroughly evaluated however, since p63 expression has been explored in only a handful of mesenchymal tumors. After observing unexpected immunohistochemical staining for p63 in an angiosarcoma of the breast, we evaluated a series of benign and malignant vascular tumors to determine the frequency of such a finding. Nuclear immunoreactivity to p63 was detected, at least focally, in 24% of malignant vascular tumors other than Kaposi sarcoma, which was uniformly negative. Benign vascular tumors were also negative for p63. Although p63 expression in tumors of vascular differentiation is unusual, it may be seen occasionally in some malignant vascular tumors. Thus, p63 is not entirely specific for epithelial differentiation. Since soft tissue angiosarcomas and hemangioendotheliomas sometimes express cytokeratins, the finding of nuclear p63 represents another potential pitfall in the differential diagnosis between poorly-differentiated carcinomas and vascular neoplasms.

Biological distinctions between juvenile nasopharyngeal angiofibroma and vascular malformation: an immunohistochemical study.

The exact nature of juvenile nasopharyngeal angiofibroma (JNA) is still in dispute. In recent years, the main controversy of its nature has focused on hemangioma and vascular malformation. In this study, the immunolocalization of vascular endothelial growth factor (VEGF), VEGF receptor-1/fms-like tyrosine kinase-1 (VEGFR-1/Flt-1), VEGF receptor-2/fetal liver kinase-1 (VEGFR-2/Flk-1), proliferating cell nuclear antigen (PCNA), and CD34 was investigated in 28 cases of JNA and 20 cases of orbital cavernous hemangiomas (OCH). The immunostaining levels of VEGF, Flt-1, and Flk-1 were higher and more frequent in vascular endothelial cells of JNA than those of OCH (p<0.05). The average microvessel density (MVD) marked by CD34 in JNA was (49.3 ± 9.1)/HPF (high power field), which was higher than OCH (29.1 ± 6.7)/HPF (p<0.05). Immunoreactivity of PCNA was localized in both endothelial and stromal cell components of JNA, but was predominantly seen in the stromal cells. However, no PCNA immunoreactivity was identified in any of the stromal and endothelial cells in cases of OCH. The immunostaining levels of CD34, VEGF, Flt-1, Flk-1, and PCNA in JNA were higher than those in OCH. These data support the view that JNA has biological characteristics of an angiogenic histogenetic tumor. In the future, anti-angiogenic therapy may represent a novel treatment strategy for JNA.

Unclassified epithelioid vascular tumor with hemangioendotheliomatous features and lymphatic differentiation.

Reported herein is an unusual vascular tumor that arose in the little finger of a 10-year-old boy. The tumor consisted of a vaguely demarcated nodular mass, which was located in the subcutaneous layer and measured 1 cm in diameter. Microscopic characteristic findings of this tumor were epithelioid nests, admixed with focal components with retiform features, intraluminal papillary tufts, and spindle cells. These features resemble those of subtypes of hemangioendotheliomas. On immunohistochemistry CD31, CD34 and D2-40 were diffusely expressed in the tumor cells, representing prominent lymphatic differentiation. These findings are not characteristic of any established types of vascular tumors. The differential diagnosis for the current peculiar tumor are discussed.

Immunohistochemical evaluation of vascular urinary bladder tumors from cows with enzootic hematuria.

Fifty-six endothelial-derived urinary bladder tumor samples collected from 26 animals with bovine enzootic hematuria were selected for immunohistochemical studies. Expression of factor VIII-related antigen (FVIIIra), CD31, muscle-specific actin, uroplakin III (UPIII), and the cell cycle-related proteins cyclin D1 and p53 was evaluated in hemangiomas, "hemangioendotheliomas" (a vascular tumor that histologically is intermediate in appearance between a hemangioma and a conventional hemangiosarcoma), and hemangiosarcomas. Although CD31 expression was seen in all endothelial tumors tested, FVIIIra was not expressed in poorly differentiated endothelial tumor cells from solid areas or in 7 muscle-invasive hemangiosarcomas. Cyclin D1 overexpression was seen in 53% of hemangiomas, 82% of hemangioendotheliomas, and 95% of hemangiosarcomas. P53 immunoreactivity was only seen in muscle-invasive hemangiosarcomas. The UPIII staining pattern, normally very intense on the apical aspect and cytoplasm of superficial urothelial cells, was altered in the urothelium in an estimated 25% of hemangiomas, most hemangioendotheliomas, and most hemangiosarcomas. In conclusion, CD31 is a better marker than FVIIIra in the characterization of bovine endothelial tumors. The cell cycle regulatory pathways involving cyclin D1 and p53 seem to be impaired in endothelial urinary bladder tumors, p53 immunoreactivity positively correlating with enhanced invasion.

The transcription factor LMO2 is a robust marker of vascular endothelium and vascular neoplasms and selected other entities.

The transcription factor LMO2 is involved in vascular and hematopoietic development and hematolymphoid neoplasia. We have demonstrated that LMO2 is expressed nearly ubiquitously in native and neoplastic vasculature, including lymphatics. LMO2 reactivity is otherwise virtually absent in nonhematolymphoid tissues except in breast myoepithelium, prostatic basal cells, and secretory phase endometrial glands. Vasculature is LMO2- in adult and fetal heart, brain of older adults, hepatic sinusoids, and hepatocellular carcinoma. LMO2 is uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms with the exception of epithelioid vascular neoplasms of pleura and bone. Among nonvascular neoplasms, LMO2 reactivity is present in giant cell tumor of tendon sheath, juvenile xanthogranuloma, a subset of gastrointestinal stromal tumors, small round blue cell tumors, and myoepithelial-derived neoplasms. The restricted expression pattern, nuclear localization, and crisp staining of LMO2 in paraffin blocks make it an attractive candidate for the diagnostic immunohistochemistry laboratory.

Malignant myopericytoma-like tumor in a Fischer rat.

Myopericytoma is a perivascular tumor that has been recently described in humans, but not in laboratory rodents. The authors encountered an intra-abdominal tumor resembling human malignant myopericytoma in a Fischer rat. Grossly, the tumor was found as two brown-colored masses located in the mesentery of rectum. Microscopically, the tumor was composed of oval to spindle-shaped cells, which were arranged in sheets around numerous thin-walled branching vessels and partly showed a concentric perivascular growth pattern. Mitoses were frequently seen, and the tumor cells showed a local invasion. Immunohistochemically, the tumor cells were strongly positive for alpha-smooth muscle actin and weakly positive for vimentin and desmin. Ultrastructurally, the tumor cells had dendritic processes, actin-like thin filaments with dense bodies, basement membranes, hemidesmosomes, and micropinocytotic vesicles. These findings suggest that the most appropriate term for diagnosis of the present case could be a malignant myopericytoma.