Apparent mtDNA sequence heterogeneity in single human blood CD34+ cells is markedly affected by storage and transport.

Single CD34(+) cells from adult human peripheral blood show mtDNA sequence heterogeneity. In this study, we compared mtDNA sequence variation in single CD34(+) cells from peripheral blood (PB) mononuclear cells (MNCs) from the same donors but under different conditions of storage and transport: group I, MNCs from heparinized PB that inadvertently required six days to be transported to the testing laboratory; group II, MNCs which were isolated from PB within a day of phlebotomy and frozen prior to transportation and storage. We observed more cell death for MNCs of group I than group II. Concordantly, group I CD34(+) cells had a very low potential for hematopoietic colony formation in vitro compared with group II cells. CD34(+) cells of group II showed an unexpectedly higher level of mtDNA sequence heterogeneity than was present in group I cells. These observations suggest that reduced mtDNA sequence heterogeneity in single CD34(+) cells of group I was likely due to elimination of cells harboring mutations. CD34(+) cells that survive stress ex vivo may be more enriched in quiescent primitive hematopoietic stem cells, with fewer mtDNA mutations than are present in committed progenitors. Technically, attention is required to conditions of preparation of human blood samples for single cell mtDNA analysis.

Emergence of BCR-ABL-specific cytotoxic T cells in the bone marrow of patients with Ph+ acute lymphoblastic leukemia during long-term imatinib mesylate treatment.

Imatinib mesylate has been demonstrated to allow the emergence of T cells directed against chronic myeloid leukemia cells. A total of 10 Philadelphia chromosome-positive acute lymphoblastic leukemia patients receiving high-dose imatinib mesylate maintenance underwent long-term immunological monitoring (range, 2-65 months) of (p190)BCR-ABL-specific T cells in the bone marrow and peripheral blood. (p190)BCR-ABL-specific T lymphocytes were detected in all patients, more frequently in bone marrow than in peripheral blood samples (67% vs 25%, P < .01) and resulted significantly associated with lower minimal residual disease values (P < .001), whereas absent at leukemia relapse. Specific T cells were mainly effector memory CD8(+) and CD4(+) T cells, producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (median percentage of positive cells: 3.34, 3.04, and 3.58, respectively). Cytotoxic subsets able to lyse BCR-ABL-positive leukemia blasts also were detectable. Whether these autologous (p190)BCR-ABL-specific T cells may be detectable under other tyrosine-kinase inhibitors, expanded ex vivo, and exploited for immunotherapy remains to be addressed.

Angiopoietin-2 plasma dosage predicts time to first treatment and overall survival in chronic lymphocytic leukemia.

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P < .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P < .001), high beta(2)-microglobulin (P < .001), unmutated variable region of immunoglobulin heavy chain gene status (P < .001), high CD38 and zeta-chain-associated protein kinase 70 expression (P < .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

Physical contact with endothelial cells through ß1- and ß2- integrins rescues chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis and induces a peculiar gene expression profile in leukemic cells.

BACKGROUND: Chronic lymphocytic leukemia B cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interactions with endothelial cells in infiltrated tissues and during recirculation may have a pathogenic role in chronic lymphocytic leukemia. DESIGN AND METHODS: We evaluated apoptosis of leukemic cells after co-culture on a monolayer of human umbilical vein endothelial cells with addition of fludarabine and antibodies that block adhesion. Then, we compared microarray-based gene expression profiles between leukemic cells at baseline and after co-culture. RESULTS: We found that the endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days of co-culture. Moreover, the endothelial layer decreased the sensitivity of chronic lymphocytic leukemia B cells to fludarabine-induced apoptosis. Physical contact with endothelium mediated by both ß(1)- and ß(2)- integrins is essential for the survival advantage of leukemic cells. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B cells led to the almost complete abrogation of the survival advantage (>70% inhibition of viability). However, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes in chronic lymphocytic leukemia B cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, an increase of genes involved in TGFß and Wnt signaling pathways, secretion of cytokines recruiting stromal cells and macrophages and up-regulation of anti-apoptotic molecules such as Bcl2 and Survivin. CONCLUSIONS: Our study supports the notion that endothelial cells are major players in the chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly supports survival, protects from drug-induced apoptosis and extensively modifies the gene expression profile of leukemic cells.

HHV-6A in syncytial giant-cell hepatitis.

Syncytial giant-cell hepatitis is a rare but severe form of hepatitis that is associated with autoimmune diseases, drug reactions, and viral infections. We used serologic, molecular, and immunohistochemical methods to search for an infectious cause in a case of syncytial giant-cell hepatitis that developed in a liver-transplant recipient who had latent infection with variant B of human herpesvirus 6 (HHV-6B) and who had received the organ from a donor with variant A latent infection (HHV-6A). At the onset of the disease, the detection of HHV-6A (but not HHV-6B) DNA in plasma, in affected liver tissue, and in single micromanipulated syncytial giant cells with the use of two different polymerase-chain-reaction (PCR) assays indicated the presence of active HHV-6A infection in the patient. Expression of the HHV-6A-specific early protein, p41/38, but not of the HHV-6B-specific late protein, p101, was demonstrated only in liver syncytial giant cells in the absence of other infectious pathogens. The same markers of HHV-6A active infection were documented in serial follow-up samples from the patient and disappeared only at the resolution of syncytial giant-cell hepatitis. Neither HHV-6B DNA nor late protein was identified in the same follow-up samples from the patient. Thus, HHV-6A may be a cause of syncytial giant-cell hepatitis.

Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors.

Kaposi sarcoma (KS) is a vascular tumor that can develop in recipients of solid tissue transplants as a result of either primary infection or reactivation of a gammaherpesvirus, the KS- associated herpesvirus, also known as human herpesvirus-8 (HHV-8). We studied whether HHV-8 and the elusive KS progenitor cells could be transmitted from the donor through the grafts. We used a variety of molecular, cytogenetic, immunohistochemical and immunofluorescence methods to show that the HHV-8-infected neoplastic cells in post-transplant KS from five of eight renal transplant patients harbored either genetic or antigenic markers of their matched donors. These data suggest the use of donor-derived HHV-8-specific T cells for the control of post-transplant KS.

Randomized trial of two schedules of low-dose gemtuzumab ozogamicin as induction monotherapy for newly diagnosed acute myeloid leukaemia in older patients not considered candidates for intensive chemotherapy. A phase II study of the EORTC and GIMEMA leukaemia groups (AML-19).

This study compared two schedules of low-dose gemtuzumab ozogamicin (GO) as induction monotherapy for untreated acute myeloid leukaemia in older patients unfit for intensive chemotherapy, to identify the more promising regimen for further study. Patients were randomized to receive either best supportive care or a course of GO according to one of two schedules: 3 mg/m(2) on days 1, 3 and 5 (arm A), or GO 6 mg/m(2) on day 1 and 3 mg/m(2) on day 8 (arm B). Primary endpoint was the rate of disease non-progression (DnP), defined as the proportion of patients either achieving a response or maintaining a stable disease following GO induction in each arm. Fifty-six patients were randomized in the two GO arms (A, n = 29; B, n = 27). The rate of DnP was 38% [90% confidence interval (CI), 23-55] in arm A, and 63% (90% CI, 45-78) in arm B. Peripheral cytopenias were the most common adverse events for both regimens. The all-cause early mortality rate was 14% in arm A and 11% in arm B. The day 1 + 8 schedule, which was associated with the highest rate of DnP, met the statistical criteria to be selected as the preferred regimen for phase III comparison with best supportive care.

Compositional properties and thermal adaptation of 18S rRNA in vertebrates.

In order to investigate the influence of temperature on the GC level of the paired sequences of ribosomal 18S RNAs in vertebrates, we have studied their base composition in cold- and warm-blooded vertebrates using a stem-by-stem comparison. We observed that a number of stems of 18S ribosomal RNAs (rRNAs) are variable among species and that the majority of such stems are GC richer in warm-blooded than in cold-blooded vertebrates. We also constructed the secondary structures of the 18S rRNAs of a polar fish, a marsupial, and a monotreme to compare them with those of temperate/tropical fishes and of eutherians, respectively. In these cases, differences similar to those already mentioned were found. We conclude that there is a correlation between stem stability and body temperature even within the relatively limited temperature range of vertebrates.

Organising pneumonia mimicking invasive fungal disease in patients with leukaemia.

Clinical charts from 63 consecutive highly immunocompromised haematologic patients presenting with pulmonary nodular lesions on CT scan, classified as either probable or possible invasive fungal disease (IFD) according to the revised EORTC/MSG classification, were retrospectively studied. Histopathological analysis of lung tissues, available for 23 patients, demonstrated proven IFD in 17 cases (14 invasive aspergillosis and 3 invasive zygomycosis), diffuse alveolar damage in one and organising pneumonia (OP) in five cases. In the OP cases, three of which have been defined as probable IFD according to EORTC/MSG classification, extensive immunohistochemical, molecular and immunological analyses for fungi were negative. Our case descriptions extend the notion that OP may be encountered as a distinct histopathological entity in pulmonary nodular lesions in patients with leukaemia with probable/possible IFD.

Chromosome 14q32 translocations involving the immunoglobulin heavy chain locus in chronic lymphocytic leukaemia identify a disease subset with poor prognosis.

Immunophenotypic studies, fluorescence in situ hybridization (FISH) and conventional karyotyping were used to define the clinicobiological significance of 14q32 translocations involving the immunoglobulin gene locus (14q32/IGH) in 252 chronic lymphocytic leukaemia (CLL) patients. The following regions were studied: 13q14, centromere 12, 6q21; 11q22/ATM; 17p13/TP53, 14q32/IGH. Patients were classified as group 1 (favourable, i.e. 13q-single or normal), group 2 (intermediate risk, i.e. +12, 6q-, 1-2 anomalies), group 3 (unfavourable, i.e. 17p-, 11q-, complex karyotype), or group 4 (14q32/IGH translocation). Endpoints were treatment-free survival (TFS) and overall survival (OS). One hundred and ten patients were included in group 1, 99 in group 2, 25 in group 3 and 18 in group 4. 14q32/IGH translocation partners were identified in eight cases (BCL2 in five cases, BCL11A, CCND3 and CDK6 in one case each). group 4 showed shorter TFS versus groups 2 and 1 (25% patients treated at 2 months vs. 12 (P = 0.02) and 20 months (P = 0.002), respectively) and shorter OS (25% patients dead at 18 months versus 50 (P = 0.0003) and >60 months (P < 0.0001) respectively. The 14q32/IGH translocation maintained prognostic significance at multivariate analysis on TFS (P = 0.025) and OS (P < 0.001), along with advanced stage and CD38+. These findings show that the 14q32/IGH translocation predicts for an unfavourable outcome in CLL and that this cytogenetic subset might be included as a separate entity in a hierarchical cytogenetic classification of CLL.

Increased expression of angiopoietin-2 characterizes early B-cell chronic lymphocytic leukemia with poor prognosis.

We measured the angiopoietin-2 (Ang-2) expression in early chronic lymphocytic leukemia (CLL) patients, pointing our attention on the association with immunoglobulin (IgV(H)) mutational status, CD38 expression and clinical outcome. Our results indicate that Ang-2 expression is heterogeneous among Binet stage A CLL patients. CLL patients can be divided into two subgroups (Ang-2 positive and Ang-2 negative CLL) with 30% of them displaying Ang-2 RNA levels above the cut off. A shorter progression-free survival was observed in Ang-2 positive CLL subset (p=0.032). Abnormal Ang-2 expression was also associated with unmutated IgV(H) genes (p<0.0001) and increased bone marrow angiogenesis (p=0.028), suggesting a role of Ang-2 in disease-progression of early CLL patients.

Development of hypogammaglobulinemia in patients treated with imatinib for chronic myeloid leukemia or gastrointestinal stromal tumor.

Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukemia and gastrointestinal stromal tumor patients. Although several in vitro and animal studies demonstrated that imatinib affects immune response, few immune alterations are described in humans. We retrospectively studied hematologic and immunological parameters in 72 chronic myeloid leukemia and 15 gastrointestinal stromal tumor patients treated with imatinib at standard dosage and in 20 chronic myeloid leukemia patients treated before the introduction of imatinib in clinical practice. Both chronic myeloid leukemia and gastrointestinal stromal tumor patients developed a significant reduction of gammaglobulin and immunoglobulin serum levels. No significant hypogammaglobulinemia was observed in chronic myeloid leukemia patients in the pre-imatinib era. These data demonstrate that imatinib treatment induces hypogammaglobulinemia that can reach a severe entity in 10% of cases, both in chronic myeloid leukemia and in gastrointestinal stromal tumor patients. Prospective studies are needed to evaluate immune humoral alterations and to define the real incidence of infectious events, including viral reactivations.

B cells and herpesviruses: a model of lymphoproliferation.

Unlike alpha- and beta-herpesviruses, human gamma-herpesviruses, including the Epstein-Barr virus (EBV) and the human herpesvirus-8 (HHV-8), are B lymphotropic viruses. Primary infection with EBV, in otherwise healthy subjects, causes a benign lymphoproliferative syndrome, the mononucleosis syndrome. However, several epidemiologic and biologic studies have shown a pathogenetic role of EBV in the development of human B cell lymphomas, both in immunocompetent and in immunosuppressed individuals. HHV-8 is the necessary etiologic agent of a lymph vascular tumor, the Kaposi sarcoma, but it is also implicated in the pathogenesis of rare B cell lymphoproliferative disorders, mainly occurring in the setting of immunosuppression. The aim of this review is to provide an updated description of the different strategies used by these two herpesviruses to influence B cell fate decisions. Both EBV and HHV-8 have evolved specific mechanisms in order to: (1) interact with the B cell developmental machinery; (2) allow infected B cells to escape from the control of the immune system; (3) affect the B cell cycle checkpoints; (4) mimic and influence B cellular proliferation and differentiation pathways. Understanding the mechanisms of herpesvirus induced B cell lymphoproliferation will provide the basis for novel treatment approaches in patients with EBV and HHV-8 related lymphomas.

Immunoglobulin mutational status detected through single-round amplification of partial V(H) region represents a good prognostic marker for clinical outcome in chronic lymphocytic leukemia.

The immunoglobulin (Ig) mutational status in B-cell chronic lymphocytic leukemia (CLL) distinguishes two subsets of patients with different prognosis. Ig status detection is commonly performed with a panel of V(H) family-specific primers. Although this method detects clonal VDJ rearrangement in virtually all cases, it is technically cumbersome and therefore not widely used clinically. Here, we describe a simple and rapid method to establish the mutational status of IgV(H) in CLL. The method is based on a consensus V(H) FR2 primer, used in both polymerase chain reaction (PCR) and sequencing reactions. Overall, monoclonal B-cell populations were detected in 163 of 189 CLL patients (86%). The prognostic value of IgV(H) mutational status was then evaluated by analyzing survival in 146 CLL cases using different V(H) homology cutoffs. CLL prognostic groups were best separated by the classical 98% cutoff: median survival was 127 and 206 months in unmutated and mutated CLL cases, respectively (P = 0.0023). V(H) FR2 consensus and V(H) family PCR were compared in 41 cases, correctly assigning all cases by both methods. Therefore, we suggest a sequential strategy to detect immunoglobulin mutational status in CLL patients by first using the approach described in this study followed by alternative V(H) family-specific PCRs for negative cases.

Efficacy of imatinib mesylate as maintenance therapy in adults with acute lymphoblastic leukemia in first complete remission.

Seven Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) patients in first complete remission received maintenance therapy with imatinib alone. Two-year progression-free survival was 75%. Quantitative polymerase-chain-reaction (qPCR) monitoring of BCR-ABL showed that: (i) persisting molecular complete response (CR) was associated with long-lasting CR; (ii) molecular relapse did not invariably mean hematologic relapse; (iii) only the wide and rapid increment of BCR-ABL values was predictive of leukemia relapse.

Cytomegalovirus and Clostridium Difficile co-infection in severe ulcero-hemorrhagic colitis during induction chemotherapy for acute lymphoblastic leukemia.

Here we describe the first case of a biopsy-proven Cytomegalovirus ulcero-hemorrhagic colitis, associated with Clostridium Difficile co-infection, occurring during standard induction chemotherapy for common B cell acute lymphoblastic leukemia. We discuss the case and focalize clinical management and diagnostic issues arising from it.