Elizabeth Hyjek

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration.

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF(165) induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF(165) also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF(165)-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation.

Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus

Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is invariably present in Kaposis sarcoma lesions,. KSHV contains several viral oncogenes and serological evidence suggests that KSHV infection is necessary for the development of Kaposis sarcoma, but cellular transformation by this virus has not so far been demonstrated. KSHV is found in the microvascular endothelial cells in Kaposis sarcoma lesions and in the spindle ‘tumour’ cells,, which are also thought to be of endothelial origin. Here we investigate the biological consequences of infecting human primary endothelial cells with purified KSHV particles. We find that infection causes long-term proliferation and survival of these cells, which are associated with the acquisition of telomerase activity and anchorage-independent growth. KSHV was present in only a subset of cells, and paracrine mechanisms were found to be responsible for the survival of uninfected cells. Their survival may have been mediated by upregulation of a receptor for vascular endothelial growth factor. Our results indicate that transformation of endothelial cells by KSHV, as well as paracrine mechanisms that are induced by this virus, may be critical in the pathogenesis of Kaposis sarcoma.

KSHV-positive solid lymphomas represent an extra-cavitary variant of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a unique form of non-Hodgkin lymphoma (NHL) associated with Kaposi sarcoma-associated herpesvirus (KSHV; HHV-8) that displays a distinct constellation of clinical, morphologic, immunologic, and molecular characteristics. Rare KSHV-containing immunoblastic lymphomas occurring in solid tissues have been described. Whether they represent part of the spectrum of PEL has not been determined. The morphologic, immunophenotypic, and molecular features of KSHV-positive solid lymphomas occurring in 8 HIV+/AIDS patients were systematically investigated and compared with those of 29 similarly analyzed PELs. The 8 KSHV-positive solid lymphomas were virtually indistinguishable from the 29 PELs based on morphology (immunoblastic/anaplastic), immunophenotype (CD45 positive; T cell antigen negative; CD30, EMA, CD138 positive; CD10, CD15, BCL6 negative) and genotype (100% immunoglobulin genes rearranged; no identifiable abnormalities in C-MYC, BCL6, BCL1, BCL2; and uniformly EBV positive). The only identifiable phenotypic difference was that the KSHV-positive solid lymphomas appeared to express B cell-associated antigens (25%) and immunoglobulin (25%) slightly more often than the PELs (<5% and 15%, respectively; P = 0.11 and P = 0.08, respectively). The clinical presentation and course of the patients who develop KSHV-positive solid lymphomas were also similar, except for the lack of an effusion and somewhat better survival (median 11 months vs. 3 months). However, the 3 KSHV-positive solid lymphoma patients alive without disease 11, 25, and 44 months following initial presentation were recently diagnosed patients and, unlike the other patients with KSHV-positive solid lymphomas, received anti-retroviral therapy. These findings strongly suggest that these decidedly rare KSHV-positive solid lymphomas belong to the spectrum of PEL. Therefore, we propose that the KSHV-positive solid lymphomas be designated extra-cavitary PELs.

Primary pulmonary lymphoma: a re-appraisal of its histogenesis and its relationship to pseudolymphoma and lymphoid interstitial pneumonia.

The clinical, morphological and immunohistochemical features of 15 cases of pulmonary lymphoproliferative disease are described. The diagnosis of primary pulmonary lymphoma was based in 13 cases on the demonstration of light chain restriction and in two cases on morphological characteristics. Many patients had a prolonged clinical course without significant clinical or radiographic deterioration, a feature associated with malignant lymphomas of mucosa‐associated lymphoid tissue in other sites. Lympho‐epithelial lesions were characteristic and malignant cells had the features of centrocyte‐like cells, similar to those described in gastric and salivary gland lymphomas. Germinal centres were present in three cases: some were partially overgrown by centrocyte‐like cells but residual polyclonal follicle centre cells and dendritic reticulum cells were still detectable. It is suggested that primary pulmonary lymphoma arises from centrocyte‐like cells normally present in bronchus‐associated lymphoid tissue. In addition to the malignant population, reactive follicles and polytypic plasma cells are frequently present and may prejudice interpretation of immunohistochemical features. In the light of these findings, cases previously diagnosed as pseudolymphoma or lymphoid interstitial pneumonia require careful assessment and the majority are, in reality, examples of primary pulmonary lymphomas.

BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs

BackgroundBRCA1–associated protein 1 (BAP1) is a tumor suppressor gene located on chromosome 3p21. Germline BAP1 mutations have been recently associated with an increased risk of malignant mesothelioma, atypical melanocytic tumors and other neoplasms. To answer the question if different germline BAP1 mutations may predispose to a single syndrome with a wide phenotypic range or to distinct syndromes, we investigated the presence of melanocytic tumors in two unrelated families (L and W) with germline BAP1 mutations and increased risk of malignant mesothelioma.MethodsSuspicious cutaneous lesions were clinically and pathologically characterized and compared to those present in other families carrying BAP1 mutations. We then conducted a meta-analysis of all the studies reporting BAP1-mutated families to survey cancer risk related to the germline BAP1 mutation (means were compared using t-test and proportions were compared with Pearson χ2 test or two-tailed Fisher’s exact test).ResultsMelanocytic tumors: of the five members of the L family studied, four (80%) carried a germline BAP1 mutation (p.Gln684*) and also presented one or more atypical melanocytic tumors; of the seven members of W family studied, all carried a germline BAP1 mutation (p.Pro147fs*48) and four of them (57%) presented one or more atypical melanocytic tumors, that we propose to call “melanocytic BAP1-mutated atypical intradermal tumors” (MBAITs). Meta-analysis: 118 individuals from seven unrelated families were selected and divided into a BAP1-mutated cohort and a BAP1-non-mutated cohort. Malignant mesothelioma, uveal melanoma, cutaneous melanoma, and MBAITs prevalence was significantly higher in the BAP1-mutated cohort (p ≤ 0.001).ConclusionsGermline BAP1 mutations are associated with a novel cancer syndrome characterized by malignant mesothelioma, uveal melanoma, cutaneous melanoma and MBAITs, and possibly by other cancers. MBAITs provide physicians with a marker to identify individuals who may carry germline BAP1 mutations and thus are at high risk of developing associated cancers.

Diagnostic Usefulness of HBME1, Galectin-3, CK19, and CITED1 and Evaluation of Their Expression in Encapsulated Lesions With Questionable Features of Papillary Thyroid Carcinoma

We evaluated HBME1, galectin-3 (GAL3), cytokeratin (CK)19, and a new anti-CITED1 antibody in 127 follicular adenoma (FA) and papillary thyroid carcinoma (PTC) cases. The findings were used to evaluate 11 diagnostically challenging encapsulated follicular lesions with questionable features of PTC (FL/QPTC). All 4 markers showed higher expression in PTC than FA. HBME1 was the most specific (96%), whereas CK19 was the most sensitive (96%). In addition, 100% specificity was seen with coexpression of HBME1/CK19. Negative expression of all 4 markers was 97% specific for FA. GAL3 and CITED1, less useful individually, could help in selective cases. FL/QPTC showed heterogeneous, often intermediate, staining patterns, implying that some FL/QPTCs may be biologically borderline lesions or represent a biologic spectrum of PTC. These antibodies can have a confirmatory role in distinguishing the follicular variant of PTC and FA. For FL/QPTC, these antibodies are helpful in some cases, their limitation perhaps suggesting the biologic ambiguity of these lesions.

Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms.

The distinction of hepatocellular carcinoma (HCC) from metastatic tumor in the liver often presents a diagnostic challenge that carries significant impact on prognostication and therapy. The number of diagnostically useful immunohistochemical markers of hepatocytes is limited to hepatocyte paraffin antigen (HepPar-1), polyclonal carcinoembryonic antigen, and CD10, with α-fetoprotein and glypican-3 labeling HCCs. Arginase-1 (Arg-1) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea. We used immunohistochemistry to compare the sensitivity of Arg-1 to that of HepPar-1 in 151 HCCs. We found that the overall sensitivities of Arg-1 and HepPar-1 are 96.0% and 84.1%, respectively. The sensitivities of Arg-1 in well, moderately, and poorly differentiated HCCs are 100%, 96.2%, and 85.7%, respectively, whereas, in comparison, HepPar-1 demonstrated sensitivities of 100%, 83.0%, and 46.4% for well, moderately, and poorly differentiated tumors, respectively. There were no HCCs in our study that were reactive for HepPar-1 but nonreactive for Arg-1. We also examined Arg-1 expression in nonhepatocellular tumors, including many that are potential mimics of HCC (renal cell carcinomas, neuroendocrine tumors, melanomas, gastric adenocarcinomas, and adrenocortical carcinomas) and found that only 2 non-HCC tumors were reactive for Arg-1. Arg-1 represents a sensitive and specific marker of benign and malignant hepatocytes that may ultimately prove to be a useful diagnostic tool in routine surgical pathology practice.

miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia

HOXA9 and MEIS1 have essential oncogenic roles in mixed lineage leukaemia (MLL)-rearranged leukaemia. Here we show that they are direct targets of miRNA-196b, a microRNA (miRNA) located adjacent to and co-expressed with HOXA9, in MLL-rearranged leukaemic cells. Forced expression of miR-196b significantly delays MLL-fusion-mediated leukemogenesis in primary bone marrow transplantation through suppressing Hoxa9/Meis1 expression. However, ectopic expression of miR-196b results in more aggressive leukaemic phenotypes and causes much faster leukemogenesis in secondary transplantation than MLL fusion alone, likely through the further repression of Fas expression, a proapoptotic gene downregulated in MLL-rearranged leukaemia. Overexpression of FAS significantly inhibits leukemogenesis and reverses miR-196b-mediated phenotypes. Targeting Hoxa9/Meis1 and Fas by miR-196b is probably also important for normal haematopoiesis. Thus, our results uncover a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumour suppressors, simultaneously, or, sequentially, in tumourigenesis and normal development per cell differentiation, indicating that miRNA regulation is much more complex than previously thought. HOX9AandMEIS1are key oncogenes in MLL-rearranged leukaemia. miRNA-196b is shown here to directly suppress their expression and delay MLL-fusion-mediated leukaemia, but to also cause an aggressive leukaemia phenotype when expressed ectopically, suggesting that it targets tumour suppressors as well.

Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy

Malignant cells may evade death from cytotoxic agents if they are in a dormant state. The host microenvironment plays important roles in cancer progression, but how niches might control cancer cell dormancy is little understood. Here we show that osteopontin (OPN), an extracellular matrix molecule secreted by osteoblasts, can function to anchor leukemic blasts in anatomic locations supporting tumor dormancy. We demonstrate that acute lymphoblastic leukemia (ALL) cells specifically adhere to OPN in vitro and secrete OPN when localized to the endosteal niche in vivo. Using intravital microscopy to perform imaging studies of the calvarial bone marrow (BM) of xenografted mice, we show that OPN is highly expressed adjacent to dormant tumor cells within the marrow. Inhibition of the OPN-signaling axis significantly increases the leukemic cell Ki-67 proliferative index and leads to a twofold increase in tumor burden in treated mice. Moreover, using cell-cycle-dependent Ara-C chemotherapy to produce minimal residual disease (MRD) in leukemic mice, we show that OPN neutralization synergizes with Ara-C to reduce detectable BM MRD. Taken together, these data suggest that ALL interacts with extracellular OPN within the malignant BM, and that this interaction induces cell cycle exit in leukemic blasts, protecting them from cytotoxic chemotherapy.

CXCL12 and CXCR4 in adenocarcinoma of the lung: Association with metastasis and survival

OBJECTIVES Although the chemokine CXCL12 and its receptor CXCR4 have been implicated in metastasis of non-small cell lung carcinoma, the prognostic significance of these molecules is poorly defined. This study aimed to determine whether expression of these molecules is associated with clinicopathologic features and disease-free survival in non-small cell lung carcinoma. METHODS Immunohistochemical staining for CXCL12 and CXCR4 was performed on 154 primary non-small cell lung carcinomas. Staining intensity was compared with tumor histotype, TNM stage, and disease-free survival; correlation was assessed by using the Fishers exact test, and Kaplan-Meier and Cox multivariate proportional hazards regression analysis. RESULTS Intense CXCL12 immunostaining was associated with nodal metastasis, although no difference in survival was observed. The prognostic relevance of CXCR4 was dependent on its subcellular location: in univariate analysis intense nuclear staining was significantly associated with lower T classification and improved disease-free survival in patients with adenocarcinoma, whereas cytomembranous staining was associated with distant metastasis and decreased disease-free survival. On multivariate analysis, cytomembranous CXCR4 expression conferred a significantly worse disease-free survival (relative risk, 2.8; 95% confidence interval, 1.4-5.7; P = .004). CONCLUSIONS Cytomembranous expression of the chemokine receptor CXCR4 in adenocarcinoma of the lung is an independent risk factor associated with worse disease-free survival, whereas nuclear staining confers a survival benefit. These findings are consistent with a model in which CXCR4 promotes tumor cell proliferation and metastasis when present in the cytoplasm or cell membrane, whereas localization of this molecule in the nucleus prevents it from exerting these effects.