Valganciclovir suppressed Epstein Barr virus reactivation during immunosuppression with alemtuzumab.

BACKGROUND: Reactivation of latent herpes viruses occurs with immunosuppression. Alemtuzumab is an antibody targeting CD52, which is expressed on all B- and T-cells. Treatment with alemtuzumab leads to profound T-cell suppression, and reactivation of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) occurs. Valganciclovir is used as an anti-CMV prophylaxis during alemtuzumab therapy. OBJECTIVE: To determine if EBV reactivation is decreased with valganciclovir prophylaxis. STUDY DESIGN: Plasma EBV DNA was serially quantified by quantitative polymerase chain reaction with a World Health Organization EBV standard in patients receiving alemtuzumab therapy with valganciclovir as anti-CMV prophylaxis. RESULTS: Twenty-nine patients were studied. A total of 258 samples were quantified, at a median of 7 (3-25) specimens per patient. Twenty-four patients never had any quantifiable EBV DNA. Five patients (17%) developed EBV reactivation. Two patients had EBV reactivation at very low levels of about 10(3)IU/mL, 3-4 logs lower than those typically found in post-transplant lymphoproliferative diseases. Three patients had EBV reactivation at higher levels of 10(4)IU/mL, which only occurred after two courses of alemtuzumab were administered. EBV reactivation subsided spontaneously in four cases. One patient developed EBV-positive Hodgkin lymphoma, but he had also received previously another potent T-cell suppressing drug fludarabine. CONCLUSION: Valganciclovir suppressed EBV reactivation during alemtuzumab therapy. It might be a useful prophylaxis in immunocompromized patient populations at high risk of EBV reactivation.

t(8;16)(p11;p13) predisposes to a transient but potentially recurring neonatal leukemia.

A Chinese girl presented with generalized papular rash and monocytic leukemia 19 days after birth. Cytogenetic analysis showed t(8;16)(p11.2;p13.3) as the sole chromosomal abnormality. Spontaneous regression of the leukemia was observed after 2 months, although the t(8;16) translocation persisted cytogenetically. This was followed 7 months later by the development of acute myeloid leukemia with maturation and cytogenetic evolution with extra chromosomes 4 and 8. Molecular study showed that the reciprocal MYST3 and CREBBP gene fusion characteristic of t(8;16) translocation persisted throughout the clinical course, even during spontaneous regression of the neonatal leukemia, and after chemotherapy-induced remission of the subsequent acute myeloid leukemia. The genetic lesion only became undetectable at the molecular level at the age of 20 months. The possible role of MYST3 and CREBBP gene fusion in the pathogenesis of the leukemia is discussed.

Relationship of expression of aquaglyceroporin 9 with arsenic uptake and sensitivity in leukemia cells.

Arsenic trioxide (As2O3) is highly efficacious in acute promyelocytic leukemia (APL). Aquaglyceroporin 9 (AQP9) is a transmembrane protein that may be involved in arsenic uptake. In 10 of 11 myeloid and lymphoid leukemia lines, quantitative polymerase chain reaction (Q-PCR) and Western blotting showed that AQP9 expression correlated positively with As2O3-induced cytotoxicity. As a proof-of-principle, transfection of EGFP-tagged AQP9 to the hepatoma line Hep3B, not expressing AQP9 and As2O3 insensitive, led to membrane AQP9 expression and increased As2O3-induced cytotoxicity. Similarly, the chronic myeloid leukemia line K562 expressed low levels of AQP9 and was As2O3 insensitive. The K562(EGFP-AQP9) transfectant accumulated significantly higher levels of intracellular arsenic than control K562(EGFP) when incubated with As2O3, resulting in significantly increased As2O3-induced cytotoxicity. Pretreatment of the myeloid leukemia line HL-60 with all-trans retinoic acid (ATRA) up-regulated AQP9, leading to a significantly increased arsenic uptake and As2O3-induced cytotoxicity on incubation with As2O3, which might explain the synergism between ATRA and As2O3. Therefore, AQP9 controlled arsenic transport and might determine As2O3 sensitivity. Q-PCR showed that primary APL cells expressed AQP9 significantly (2-3 logs) higher than other acute myeloid leukemias (AMLs), which might explain their exquisite As2O3 sensitivity. However, APL and AML with maturation expressed comparable AQP9 levels, suggesting that AQP9 expression was related to granulocytic maturation.

Glucose-6-phosphate dehydrogenase deficiency in female octogenarians, nanogenarians, and centenarians.

BACKGROUND: Age-related skewing of X-chromosome inactivation leading to glucose-6-phosphate dehydrogenase (G6PD) deficiency in elderly women in a population with prevalent G6PD gene mutations was investigated. METHODS: G6PD activity was measured biochemically. G6PD mutations were detected by polymerase chain reaction (PCR) and allele-specific extension, and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and Sequenom MassARRAY. X-chromosome inactivation was quantified by semiquantitative PCR for the HUMARA gene, before and after HpaII digestion. RESULTS: In 173 women (median age: 90 years; range, 80-107 years), 18 heterozygotes for G6PD mutations were identified. Three heterozygotes were G6PD deficient, owing to skewed X-chromosome inactivation affecting the wild-type allele. Fifteen heterozygotes, with skewing apparently affecting the mutant alleles, had normal but significantly lower G6PD levels. At 1.73%, G6PD deficiency was significantly more frequent than expected from population screening at birth. CONCLUSION: Due to skewed X-chromosome inactivation, elderly women in populations with prevalent G6PD mutations are at risk of G6PD deficiency.

Epstein-barr virus-related gastric adenocarcinoma: an early secondary cancer post hemopoietic stem cell transplantation.

BACKGROUND & AIMS: Epstein-Barr virus (EBV) infection has been associated with some cases of gastric cancer. METHODS: We studied a case of early onset gastric adenocarcinoma after nonmyeloablative hematopoietic stem cell transplantation for myeloma in a 56-year-old man. RESULTS: The development of gastric adenocarcinoma was preceded by severe graft-versus-host disease (GVHD) necessitating strong immunosuppression, which resulted in an intense reactivation of EBV infection. Three sequential gastric biopsy examinations performed at 100, 130, and 150 days after hematopoietic stem cell transplantation showed gastritis, dysplasia, and adenocarcinoma, respectively. There was no evidence of Helicobacter pylori infection. Quantitative polymerase chain reaction for circulating EBV showed a surge of EBV DNA peaking at the time of gastritis, followed by a gradual decrease afterward with adequate control of GVHD and tailing of immunosuppression. In situ hybridization for EBV-encoded early small RNA showed absence of EBV in the gastritis specimen, but the presence of EBV in the dysplastic and carcinoma specimens. Aberrant promoter methylation of E-cadherin was observed only in the carcinoma specimens, showing that infection with EBV preceded E-cadherin methylation. CONCLUSIONS: Mucosal damage caused by GVHD, immunosuppression, and EBV reactivation combined to lead to EBV infection of the gastric cells and initiation of carcinogenesis, suggesting this case to be a genuine EBV-related opportunistic malignancy post-transplantation. An interesting proposition is that this case also might reflect a compacted timeline of events in EBV-related gastric cancers developing in immunocompetent patients.

Alemtuzumab induced complete remission of therapy-resistant pure red cell aplasia.

Two patients with pure red cell aplasia (PRCA) refractory to anti-thymocyte globulin, prednisolone, cyclophosphamide, fludarabine, mitoxantrone, dexamethasone and cyclosporine, were treated with alemtuzumab (anti-CD52 antibody). Case 1, a 35-year-old man with idiopathic PRCA, remitted completely with 130 mg of alemtuzumab. Case 2, a 42-year-old man with PRCA due to T-cell large granular lymphocyte (T-LGL) leukaemia, achieved complete remission of the PRCA with 490 mg of alemtuzumab, although the T-LGL leukaemia responded only transiently. There were no significant side effects, and normalization of erythropoiesis was durable. Alemtuzumab is active in PRCA that is idiopathic or secondary to T-cell lymphoproliferative diseases.

Caveolin-1 gene is coordinately regulated with the multidrug resistance 1 gene in normal and leukemic bone marrow.

Caveolin-1 is a structural protein that may function as a scaffold for plasma membrane proteins, one of which is P-glycoprotein (P-gp), product of the multidrug resistance-1 (MDR-1) gene. We tested the hypothesis that if P-gp and caveolin-1 interacted physically, caveolin-1 and MDR-1 genes might be coordinately regulated; by quantifiying their gene expression with quantitative-polymerase chain reaction. MDR-1 and caveolin-1 gene expressions were normalized to an internal control and related to a fixed calibrator by a comparative cycle-threshold (CT) method. In four different groups of marrow samples (20 normal, 56 acute myeloid leukemias (AML) at diagnosis, 48 AMLs at relapse, and 51 regenerating marrows), caveolin-1 and MDR-1 gene expressions were positively correlated. In 65 samples with MDR-1 over-expression, caveolin-1 and MDR-1 expressions were also correlated. The coordinate expression of caveolin-1 and MDR-1 suggests that they may either interact physically, or are involved in the same aberrant pathway(s) activated during MDR-1 up-regulation.

Real-time quantification of hepatitis B virus core-promoter and pre-core mutants during hepatitis E antigen seroconversion.

BACKGROUND/AIMS: Detection of hepatitis B virus (HBV) core-promoter A(1762)T-G(1764)A and pre-core G(1896)A mutants has relied on qualitative assays. We tested the hypothesis that the quantity of A(1762)T-G(1764)A and G(1896)A mutants might have clinical impact, by quantifying these mutants before and after HBe antigen (HBeAg) seroconversion in 58 patients. METHODS: A real-time quantitative-polymerase chain reaction (Q-PCR) was developed, using minor groove binder (MGB)-conjugated TaqMan probes to impart reaction specificity for wildtype/mutant HBV populations. RESULTS: Significant quantities (>20%) of core-promoter A(1762)T-G(1764)A mutant existed in 65% of patients before and after HBeAg seroconversion, and were significantly changed (>20% increase/decrease) in 13% of patients after seroconversion. Quantity of A(1762)T-G(1764)A mutants was positively correlated with alanine aminotransferase (ALT) (P<0.001) and HBV DNA (P<0.001) levels, both before and after HBeAg seroconversion. Significant quantities of pre-core G(1896)A mutant existed in about 90% of patients before and after HBeAg seroconversion, and were changed in 16% of patients after seroconversion. Quantity of G(1896)A mutant was negatively correlated with ALT (P=0.044) and HBV DNA (P=0.007) levels. CONCLUSIONS: The A(1762)T-G(1764)A and G(1896)A mutants existed in a high proportion of patients before and were unaffected after HbeAg seroconversion. The quantities of A(1762)T-G(1764)A mutant were positively and G(1896)A mutant negatively correlated with liver inflammation and viral replication.

Quantification of circulating Epstein-Barr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBV-positive lymphomas in immunocompetent patients.

In Epstein-Barr-virus (EBV)-positive lymphomas in immunocompetent patients, release of EBV DNA from tumor cells into the plasma might be useful for disease monitoring and prognostication. To test this hypothesis, we quantified serially plasma EBV DNA by quantitative polymerase chain reaction in 39 cases of EBV-positive (natural killer [NK] cell, n = 23; T cell, n = 8; B cell, n = 4; Hodgkin, n = 4) lymphomas. As control, EBV DNA was undetectable in 34 cases of EBV-negative lymphomas at diagnosis and during chemotherapy. In all cases of EBV-positive lymphomas, EBV DNA was detectable (10(5)-10(10) copies/mL) at diagnosis. It paralleled the clinical course, with EBV DNA becoming undetectable at remission and remaining elevated in refractory disease. On multivariate analysis, high-presentation EBV DNA (> 7.3 x 10(7) copies/mL) was significantly associated with an inferior overall survival (OS). Subgroup analysis of NK cell lymphomas, the largest cohort in this study, showed that presentation EBV DNA was correlated with disease stage and lactate dehydrogenase. On multivariate analysis, high-presentation EBV DNA (> 6.1 x 10(7) copies/mL) was significantly associated with an inferior disease-free survival. During treatment, patients with EBV DNA that showed further increases or failed to become undetectable had significantly inferior OS. In EBV-positive lymphomas, plasma EBV DNA is valuable as a tumor biomarker and for prognostication.

T-cell large granular lymphocyte leukemia of donor origin after allogeneic bone marrow transplantation.

A 39-year-old man with chronic myeloid leukemia in accelerated phase underwent allogeneic bone marrow transplantation (BMT). At 6 months after BMT, lymphocytosis (WBC count, 23,100/microL [23.1 x 10(9)/L]; 80% (0.80) large granular lymphocytes [LGLs]) occurred. The LGLs were CD3+CD4-CD8+, with clonally rearranged T-cell receptor gamma gene, and of donor origin, as shown by analysis of polymorphic microsatellite markers. Epstein-Barr virus was not present. The diagnosis, therefore, was consistent with T-cell large granular lymphocytic (T-LGL) leukemia. Corticosteroids controlled the LGL count, but progressive pancytopenia led to death 4 months later. Retrospective analysis showed that the T-LGL leukemia apparently had arisen as early as 3 months after BMT. The distinguishing features of this case included donor origin, neoplastic nature, and the aggressive fatal outcome.