Epstein-Barr virus-associated leukemic lymphoma after allogeneic stem cell transplantation.

Leukemic Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative diseases (PTLD) following allogeneic hematopoietic stem cell transplantation are extremely rare. We can successfully treat an EBV-associated leukemic lymphoma patient with rituximab, cidofovir, and donor lymphocyte infusion (DLI). In the present case, EBV-specific T cells that were present in the peripheral blood before rituximab administration treatment rapidly increased after DLI in association with a decrease in the EBV-DNA load.

Comprehensive profiling of Epstein-Barr virus-encoded miRNA species associated with specific latency types in tumor cells.

BACKGROUND: Epstein-Barr virus (EBV) is an etiological cause of many human lymphocytic and epithelial malignancies. EBV expresses different genes that are associated with three latency types. To date, as many as 44 EBV-encoded miRNA species have been found, but their comprehensive profiles in the three types of latent infection that are associated with various types of tumors are not well documented. METHODS: In the present study, we utilized poly (A)-tailed quantitative real-time RT-PCR in combination with microarray analysis to measure the relative abundances of viral miRNA species in a subset of representative lymphoid and epithelial tumor cells with various EBV latency types. RESULTS: Our findings showed that the miR-BHRF1 and miR-BART families were expressed differentially in a tissue- and latency type-dependent manner. Specifically, in nasopharyngeal carcinoma (NPC) tissues and the EBV-positive cell line C666-1, the miR-BART family accounted for more than 10% of all detected miRNAs, suggesting that these miRNAs have important roles in maintaining latent EBV infections and in driving NPC tumorigenesis. In addition, EBV miRNA-based clustering analysis clearly distinguished between the three distinct EBV latency types, and our results suggested that a switch from type I to type III latency might occur in the Daudi BL cell line. CONCLUSIONS: Our data provide a comprehensive profiling of the EBV miRNA transcriptome that is associated with specific tumor cells in the three types of latent EBV infection states. EBV miRNA species represent a cluster of non-encoding latency biomarkers that are differentially expressed in tumor cells and may help to distinguish between the different latency types.

Three cases of aggressive natural killer cell leukemia with a lethal hemorrhagic complication.

Aggressive natural killer cell leukemia (ANKL) is a rare malignant disease of NK cells that has a median survival of less than 2 months and a strong association with the Epstein-Barr virus. Herein, we report three Japanese cases of the disease. A 21-year male patient, a 31-year female patient, and a 76-year female patient presented with high fever, lymphadenopathy, hepatosplenomegaly, and severe liver damage. All three cases had granular lymphocytes in both peripheral blood and bone marrow. The phenotype of these cells was CD2(+)CD3(-)CD56(+)HLA-DR(+). All cases had a high copy number of serum Epstein-Barr virus DNA in the peripheral blood and were diagnosed with ANKL. Case 1 and Case 2 were treated with chemotherapy, but suffered from gross intestinal bleeding or massive bleeding in the cerebellum, resulting in death. Although not treated with chemotherapy, Case 3 also suffered gross bleeding from an atypical duodenal ulcer and died from hemorrhagic shock 15 days after admission. There have been no previous reports of such acute lethal hemorrhagic complications with ANKL. The present cases suggest that patients with ANKL need a sufficient supply of coagulation factors, and that chemotherapy for this disease should be carefully designed with promising agents. [J Clin Exp Hematopathol 52(2) : 101-106, 2012].

Human immunodeficiency virus-associated precursor T-lymphoblastic leukemia/lymphoblastic lymphoma: report of a case and review of the literature.

We describe a case of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma in a 43-year-old Italian man with a history of human immunodeficiency virus infection lasting 9 years. Immunoperoxidase stains showed that neoplastic cells were positive for CD3, TdT, CD45, CD10, CD1a, CD2, CD7, CD5, and CD43 (focal). The proliferation rate was approximately 70%, assessed by Ki-67/MIB-1 staining. Flow cytometry of the marrow aspirate revealed an intermediate/cortical T-lymphoblastic phenotype: negative for surface CD3 and positive for cytoplasmic CD3, CD1a, TdT, CD2, CD7, CD5, and CD8, with partial coexpression of dimCD4. Analysis of T-cell receptor gamma polymerase chain reaction products showed clonality. T-lymphoblastic leukemia/lymphoblastic lymphoma is a very rare occurrence in the clinical setting of human immunodeficiency virus infection. It is not listed in the World Health Organization classification of lymphomas associated with human immunodeficiency virus infection. Only 4 cases of human immunodeficiency virus-associated T-lymphoblastic leukemia/lymphoblastic lymphoma are reported in the current medical literature.

Quantitative expression profiling guided by common retroviral insertion sites reveals novel and cell type specific cancer genes in leukemia.

Proviral insertional mutagenesis is a powerful tool for the discovery of cancer-associated genes. The ability of integrated proviruses to affect gene expression over long distances combined with the lack of methods to determine the expression levels of large numbers of genes in a systematic and truly quantitative manner have limited the identification of cancer genes by proviral insertional mutagenesis. Here, we have characterized a new model of proviral insertional mutagenesis-induced lymphoid tumors derived from Eed Polycomb group gene mutant mice and quantitatively determined the expression levels of all genes within 100 kb of 20 different retroviral common insertion sites (CISs) identified in these tumors. Using high-throughput quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR), we document an average of 13 CIS-associated genes deregulated per tumor, half of which are leukemia subtype-specific, while the others are coordinately deregulated in the majority of tumors analyzed. Interestingly, we find that genes located distantly from common proviral integration sites are as frequently deregulated as proximal genes, with multiple genes affected per integration. Our studies reveal an unsuspected conservation in the group of genes deregulated among phenotypically similar subtypes of lymphoid leukemias, and suggest that identification of common molecular determinants of this disease is within reach.

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma.

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFkappaB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at http://www.mouse-genome.bcm.tmc.edu/vision .

Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors.

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new beta-diketo acid analogs with the phenanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid (27f) with an IC(50) of 0.38microM against integrase strand transfer. The phenanthrene diketo acids 27d-f were more potent (IC(50)=2.7-0.38microM) than the corresponding phenanthridinone diketo acid 16 (IC(50)=65microM), suggesting that the polar amide bridge in the phenanthridinone system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible binding of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simulations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg(2+) contributes to IN inhibitory potency. Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC(50) down to 8microM for phenanthrene-3-(2,4-dioxo)butyric acid (27d), with a selectivity index of 10 against PBMCs.

Characterization of HTLV envelope seroreactivity in large granular lymphocyte leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is a rare chronic lymphoproliferative disorder of unknown etiology. We have previously reported that patients with T-LGL leukemia were seroreactive against BA21, a 34 amino acid peptide derived from HTLV-I envelope protein p21. We tested sera from 70 patients with T-LGL leukemia and found that 21/70 (30%) of them were seroreactive against fusion peptide GST-BA21. In control group of healthy blood donors 3/30 (10%) were seroreactive. We synthesized a set of overlapping peptides derived from BA21 and tested them against sera from patients. Only a single peptide (p21 env 417-430) showed reactivity. We then generated multiple fusion peptides consisting of 5-14 amino acid residues derived from this peptide and tested them against patient and control sera. Shortest peptide giving positive seroreactivity was octapeptide P8 (p21 env 418-425). Competitive Western blot assay with use of fusion peptides revealed that the minimal HTLV-I epitope responsible for seroreactivity found in patients with T-LGL leukemia is a decapeptide PP10 (p21 env 417-426). Protein Bank (NCBI) search did not reveal any significant homology between PP10 epitope and known human proteins. These results further define the epitope responsible for HTLV env seroreactivity observed in LGL leukemia.

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

Vesicular stomatitis virus: a potential therapeutic virus for the treatment of hematologic malignancy.

Certain strains of vesicular stomatitis virus (VSV) have been shown to be oncolytic in a wide variety of solid tumors. In the present study, we tested the leukemolytic properties of VSV using established leukemia cell lines and primary patient material. VSV efficiently killed essentially all leukemic cell lines. In contrast, however, normal clonogenic bone marrow progenitor cells and peripheral blood cells were remarkably refractory to infection by VSV. By exploiting this large difference in susceptibility to infection we successfully purged contaminating leukemic cells from cultures of peripheral blood progenitor cells (PBPC) using VSV. VSV was also able to infect and kill leukemic cells in primary samples taken from patients with multiple myeloma (MM). This study demonstrates the potential utility of VSV in the treatment, both ex vivo and in vivo, of hematologic malignancies.

Aggressive natural killer-cell leukemia revisited: large granular lymphocyte leukemia of cytotoxic NK cells.

Aggressive natural killer-cell leukemia (ANKL) is a rare form of large granular lymphocyte leukemia, which is characterized by a systemic proliferation of NK cells. The clinical features of 22 ANKL cases were analyzed. Hepatomegaly (64%), splenomegaly (55%) and lymphadenopathy (41%) were also frequently observed. Leukemic cells were identified as CD1-, CD2+, surface CD3-, CD4-, CD5-, CD7+, CD8+/-, CD10-, CD11b+/-, CD13-, CD16+, CD19-, CD20-, CD25-, CD33(-), CD34-, CD38+, CD56+, CD122+, HLA-DR+ and TCR-. Two of the 16 cases examined for CD57 were positive and three of the seven cases examined for cytoplasmic CD3. Epstein-Barr virus was detected in the tumor cells of 11 of the 13 cases examined. No common cytogenetic abnormalities were identified and 6q anomaly was detected in only one. Three of 13 patients treated with chemotherapy containing anthracycline/anthraquinone attained complete remission, in contrast to none of the eight who were treated with regimens without anthracycline. Although the overall prognosis was poor with a median survival of 58 days, those who attained remission showed better prognosis (P=0.005). These findings suggest that ANKL is an entity of mature cytotoxic NK-cell neoplasms with distinct phenotype and disease presentations. Intensive treatment for ANKL may result in a better prognosis.

Successful treatment of natural killer (NK) cell leukemia following a long-standing chronic active Epstein-Barr virus (CAEBV) infection with allogeneic bone marrow transplantation.

The optimal treatment for natural killer (NK) cell leukemia after chronic active Epstein-Barr virus (CAEBV) infection has not been determined. A 15-year-old boy presented with NK cell leukemia following CAEBV infection for 5 years. The peripheral blood and BM had an increased number of CD3(-)CD56(+) large granular lymphocytes and a monoclonal integration of the EBV genome was detected. Chemotherapy was not sufficiently effective to control the disease. Allogeneic BMT from an HLA-identical sister was performed using a conditioning regimen consisting of total body irradiation, cyclophosphamide and thiotepa. The patient is disease-free with a perfect performance status 24 months after BMT. This is the first report to show that allogeneic BMT is potentially able to cure NK cell leukemia after CAEBV infection.

Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction.

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.

The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively.

Among the six envelope subgroups of avian leukosis virus (ALV) that infect chickens, subgroups A (ALV-A) and J (ALV-J) are the most pathogenic and widespread among commercial chicken populations. While ALV-A is predominantly associated with lymphoid leukosis (LL) and less frequently with erythroblastosis (EB), ALV-J mainly induces tumours of the myeloid lineage. In order to examine the basis for the lineage specificity of tumour induction by these two ALV subgroups, we constructed two chimeric viruses by substituting the env genes into the reciprocal proviral clones. The chimeric HPRS-103(A) virus carrying the subgroup A env gene is identical to ALV-J prototype virus HPRS-103 except for the env gene, and the chimeric RCAS(J) virus carrying the subgroup J env gene is identical to the parent replication-competent ALV-A vector RCAS except for the env gene. In experimentally inoculated chickens, HPRS-103(A) virus induced LL and EB similar to ALV-A isolates such as RAV-1, while RCAS(J) virus induced myeloid leukosis (ML) and EB, similar to ALV-J, suggesting that the env gene is the major determinant for the lineage-specific oncogenicity. There were genetic differences in susceptibility to tumour induction between line 0 and line 15(I) chickens, indicating that in addition to the env gene, other viral or host factors could also serve as determinants for oncogenicity. Induction of both LL and ML by the two chimeric viruses occurred through the activation of c-myc, while the EB tumours were induced by activation of the c-erbB oncogene.

Human leucocyte antigen-Cw-specific cytotoxic T lymphocytes generated from naive cord blood used for cord blood stem cell transplantation.

Human leucocyte antigen (HLA)-Cw-reactive cytotoxic T lymphocytes (CTL) were generated from cord blood (CB) lymphocytes of two cases used for cord blood stem cell transplantation (CBSCT). In both cases, the CTL were cytotoxic against the patient's leukaemic cells, as well as the patient's Epstein-Barr virus (EBV)-lymphoblastoid cell line (EBV-LCL) and phytohaemagglutinin blasts, and the cytotoxicity was blocked by anti-HLA-class I monoclonal antibodies. In the first case, the CTL recognized Cw 3 (Cw 9 and Cw 10)-positive EBV-LCL, while in the second case, the CTL recognized Cw1 and/or Cw7. These cases suggest that CB T cells may be competent enough for generating CTL to induce a graft-versus-leukaemia effect and/or graft-versus-host disease in patients with CBSCT and that the mismatching of Cw antigens between patient and CB may be related to the outcome of CBSCT.

Aggressive natural killer cell lymphoma/leukemia.

We report a case of aggressive natural killer (NK) cell lymphoma/leukemia in a 70-year-old woman presenting with fever, hepatosplenomegaly, retroperitoneal lymphadenopathy, and elevated serum CA19-9. The patient died 4 days after diagnosis. Neoplastic NK cells were identified in the blood and retroperitoneal lymph node biopsy. Their phenotypes were confirmed by extensive flow cytometric and immunohistochemical studies. In situ hybridization for Epstein-Barr virus (EBV)-associated RNA (EBER) was positive. Various forms of NK cell neoplasm were reviewed and discussed.

Hypersensitivity to mosquito bites as the primary clinical manifestation of a juvenile type of Epstein-Barr virus-associated natural killer cell leukemia/lymphoma.

Hypersensitivity to mosquito bites or mosquito allergy is a mysterious disorder that has been reported mainly in Japanese patients (at least 58 patients) in the first two decades of life. The skin lesion at bite sites is typically a bulla that develops into necrosis. Patients simultaneously exhibit a high temperature and general malaise and subsequently may experience lymphadenopathy and hepatosplenomegaly. Recent studies have revealed that this mosquito hypersensitivity is associated with chronic Epstein-Barr virus infection and natural killer cell leukemia/lymphoma. The natural killer cell, infected with monoclonal (or oligoclonal) Epstein-Barr virus, seems to be involved in the pathogenesis of the hypersensitivity. Half of the patients reported died of hemophagocytic syndrome (or malignant histiocytosis), granular lymphocyte proliferative disorder, or lymphomas. We propose that this disease, defined as the triad of hypersensitivity to mosquito bites, chronic Epstein-Barr virus infection, and natural killer cell leukemia/lymphoma, is a clinical entity mostly seen in Asians.

Large granular lymphocyte leukaemia occurring after renal transplantation.

Post-transplantation lymphoproliferative disorders (PTLD) are a clinicopathologically heterogeneous group of lymphoid proliferations. The majority are of B-cell origin and associated with Epstein-Barr virus (EBV) infection. In contrast, the development of T-cell PTLD is much less common and EBV does not appear to be involved in pathogenesis. In this report we describe three patients who developed large granular lymphocyte (LGL) leukaemia after renal transplantation. These patients had clonal expansion of CD3+, CD8+, CD57+, CD56- LGL. We were unable to detect CMV antigen or find evidence for EBV or human T-cell leukaemia/lymphoma virus genome in the LGL from these patients. These data show that LGL leukaemia should be included as one of the types of T-cell proliferations which can occur post transplant.