Matrix Gla Protein drives stemness and tumor initiation in ovarian cancer.

Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking, and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings. Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.

CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Distinct populations of tumor-initiating cells derived from a tumor generated by rat mammary cancer stem cells.

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/(WAF1) and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased alpha-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.

Evidence of Epstein-Barr virus infection in ulcerative colitis.

BACKGROUND AND AIM: The aetiology of ulcerative colitis is still controversial, however, recent studies have emphasised the possible role of infectious agents or ingested substances and their breakdown products, which might activate immune-mediated mechanisms eventually leading to tissue damage. Aim of this investigation was to ascertain the occurrence and the potential role of Epstein-Barr virus infection in large bowel mucosa of ulcerative colitis patients. PATIENTS AND METHODS: Twenty-three biopsies and six total colectomies from 17 patients were analysed for the expression of Epstein-Barr virus proteins and RNAs. Polymerase chain reaction experiments were also carried out to detect Epstein-Barr virus DNA. For comparison, ten biopsies from patients with Crohn's disease, ten biopsies from patients with different types of colitis, seven biopsies and five surgical margins of normal colonic mucosa from the small and large bowels were studied (controls). RESULTS: Six biopsies and four colectomies from seven ulcerative colitis patients showed scattered lymphocytes expressing nuclear EBER 1-2 and harbouring polymerase chain reaction-amplifiable Epstein-Barr virus-DNA. In some cases, linear viral DNA (typical of lytic Epstein-Barr virus infection) was also found. Epithelial cells were invariably negative in all cases. All control tissues from non-ulcerative colitis patients were also invariably non-reactive. CONCLUSION: Evidence of Epstein-Barr virus infection in the mucosal inflammatory cells of ulcerative colitis patients suggests a possible role of this virus in the chronicity of ulcerative colitis.

Immunoreactivity for latent membrane protein 1 of Epstein-Barr virus in nevi and melanomas is not related to the viral infection.

Epstein-Barr virus (EBV) is a human herpes virus with oncogenic potential, associated with several malignancies. The EBV-encoded latent membrane protein 1 (LMP1) is one of nine proteins regularly expressed in virally infected and immortalised B lymphocytes. We now document the consistent immunoreactivity for LMP1 in 90% of 65 nevi and melanomas, using the monoclonal antibody cocktail CS1-4. The immunocytochemical findings, however, were not confirmed using reverse-transcription polymerase chain reaction (RT-PCR) experiments, which failed to demonstrate any actual expression of LMP1 mRNA. In situ hybridisation for EBV-encoded RNAs (EBERs 1 and 2) and PCR amplification of EBV genomic sequences also failed to document any viral infection. Several normal and neoplastic human tissues have also been immunostained for LMP1, without any positive staining, with the exception of a minor percentage of skin melanocytes and of normal blasts of the myeloid and erythroid lineages. We conclude that the vast majority of nevi and melanomas express a still uncharacterised molecule, cross-reacting with anti-LMP1 (CS1-4) antibodies, which may be considered a consistent marker of melanocytic proliferations. The immunoreactivity of normal and neoplastic human tissues for the anti-LMP1 reagent should not be taken as evidence of EBV infection.

[Lympho-epithelial carcinoma (malignant lympho-epithelial lesions) of the parotid gland. Description of a case with detection of EBV by in situ hybridization]. / Carcinoma linfoepiteliale (lesione linfoepiteliale maligna) della ghiandola parotide. Descrizione di un caso con rilevazione dell'EBV mediante ibridazione in situ.

The authors describe a case of malignant lymphoepithelial lesion (MLEL), commonly referred to as lymphoepithelial carcinoma of parotid gland, that is a very rare tumour. There is a relatively high incidence in Eskimos of Alaska and Greenland, but some cases are described in natives of south China. The immunophenotypic profile and histopathological aspect of this neoplasm are discussed, and the differential diagnosis in regard to other primitive or metastatic tumours of parotid is also considered. In our case a diffuse positivity of epithelial neoplastic cells for EBV genome was found using in situ hybridization. The possible role of EBV in the ethiopathogenesis of this rare lesion is herein discussed.

Glomus tumour: the immunohistochemical characteristics of twenty-three cases.

Twenty-three cases of glomus tumour were investigated with the antibodies keratin, vimentin, neurofilaments, desmin, actin, S100, Factor VIII-related antigen, substance P and KP1/CD68. It will be shown that the glomus cells were vimentin-positive in 11 out of 23 cases (48%), actin-positive in 16 out of 23 cases (70%) and desmin-positive in 9 out of 23 cases (39%). The KP1 monoclonal antibody demonstrated the presence of numerous mast cells in some cases. The substance P was only occasionally positive in six cases (26%). No positive staining was seen with keratin, neurofilaments, S100 and Factor VIII-related antigen. It is confirmed that glomus cells can be considered as elements of smooth muscle origin. Our study suggests a possible explanation of the mechanism which induces paroxysmal attacks in patients with glomus tumours.

Atheromatosis and double media: uncommon vascular lesions of renal allografts.

Study of 45 renal allograft nephrectomy specimens revealed the presence of relatively uncommon arterial vascular lesions: atheromatosis (12 cases) and a double layer of smooth muscle in the intima (Double Media) (4 cases). Histopathologic features of atheromatosis showed the presence of large lipid-laden cells localized in the intimal layer of arteries. Diagnosis of acute vascular rejection (AVR) was made in 19 cases. Diagnosis of chronic vascular rejection (CVR) was found in 4 cases. 22 cases showed lesions of both AVR and CVR. In 12 cases there was infiltration of the intima and media wall by foam cells closely resembling an atheromatous lesion. Four cases of Double Media were found in allografts with survival varying from 51 to 344 days. The presence of either atheromatous or double media does not correlate statistically with immunosuppressive treatment, blood pressure or with the presence of hypertriglyceridemia and/or hypercholesterolemia. Immunohistochemical investigation of atheromatosis revealed total negativity of the foam cells with antisera to: actin, myosin, desmin and myoglobin. Variable reactivity was observed with antisera to vimentin. Myointimal cells of Double Media expressed slight positivity for actin and vimentin. The double media lesion seems to be the result of a reparative vascular process secondary to rejection changes. Atheromatosis seems to be closely correlated to episodes of acute rejection. Vascular lesions in grafts are harbinger of poor prognosis. Double media lesion and atheromatosis do not seem to have a more unfavourable prognostic significance on the evolution of the transplants.

Ki67 immunohistochemical evaluation in colorectal cancer and normal colonic mucosa. Possible clinical applications.

Cell proliferation was evaluated by Ki67 monoclonal antibody in 33 colorectal adenocarcinomas and in the normal colonic mucosa. Immunoreactivity was assessed independently by two observers in two subsequent evaluations with a semiquantitative method, by counting at least 2000 cells in two distinct neoplastic specimens (central and peripheric section). There was an excellent intra-inter observer agreement in Ki67 score for each specimen. The tumor score range from 7 to 70% (median 48.8), without any significant correlation with sex and age of the patient and location, size, staging and grading of the neoplasm. Tumor Ki67 score was almost identical in central (46.96%) and in peripheral section (49.24%), and always higher than in normal mucosa. There was no distinction in Ki67 score in normal mucosa at various distances from the tumor. In our experience, Ki67 provides a reliable and reproducible method for assessment of proliferative activity; its clinical applications need further studies.

KP1 (CD 68) staining of malignant melanomas.

The monoclonal antibody KP1, which recognizes the CD 68 antigen on macrophages and myeloid precursors, was tested on 28 malignant (primary and metastatic) melanomas, 28 naevi, and 17 skin biopsies showing either normal (10) or hyperplastic melanocytes (7). Sixteen of 20 primary melanomas and six of eight metastatic melanomas showed variable numbers of KP1 positive tumour cells. All but five benign melanocytic proliferations (two Spitz naevi and three intradermal naevi), as well as normal and hyperplastic melanocytes were negative. These results indicate that difficulties may occur with the use of KP1 in the differential diagnosis between melanomas and neoplasms derived from histiocytes-macrophages, and that the expression of CD 68 antigen might be related to tumour progression in melanocytic cells.

[Bone marrow biopsy in HIV-positive patients with thrombocytopenia. Light and electron microscopy]. / La biopsia osteo midollare nei pazienti piastrinopenici HIV positivi. Microscopia ottica ed elettronica.

Isolated thrombocytopenia (platelets less than 100,000 mmc) may be the first clinical symptom in HIV positive patients or occur in all the evolutive phases up to overt AIDS. In this paper bone marrow lesions are evaluated at light and electron microscopy in 32 HIV positive patients with isolated thrombocytopenia (group II and III CDC 1986). At light microscopy an increase in megakaryocytes with small dysplastic changes, plasmacytosis and hypereosinophilia were the bone marrow lesions detected. Electron microscoy revealed megakaryocytes with focal nuclear alterations (hypolobation and dilatation of the perinuclear cisternae) and abnormalities in the maturation of platelets associated with cytoplasmic micro-macrovacuolation, absence of viral particles or of virus correlated structures. About 9% of HIV positive patients presented with isolated thrombocytopenia as a first clinical symptom: thrombocytopenia is not believed to have unfavourable prognostic significance in the evolution to overt AIDS.

Modulation of phagocytic activity in cultured Sertoli cells.

Phagocytic activity of rat Sertoli cells that were cultured in vitro has been evaluated as the ability to internalize polystyrene beads. Our data demonstrate that these cells are active phagocytes and that such phagocytic activity is under negative control by follicle-stimulating hormone (FSH). The hormonal control is mediated by increased intracellular levels of cAMP. Moreover Sertoli cell responds to tuftsin, an oligopeptide known to act only on macrophages and granulocytes, by increasing up to five times its phagocytic activity. Phagocytic uptake of polystyrene beads is associated with dramatic changes of the cellular shape. Such morphological modifications are significantly reduced under FSH stimulation. The physiological implications of the data are discussed.