Total genomic alteration as measured by SNP-array-based molecular karyotyping is predictive of overall survival in a cohort of MDS or AML patients treated with azacitidine.

Metaphase cytogenetics (MC) has a major role in the risk stratification of patients with myelodysplastic syndromes (MDSs) and can affect the choice of therapies. Azacitidine (AZA) has changed the outcome of patients with MDS or acute myeloid leukemia (AML) unfit for intensive chemotherapy. Identification of patients without the benefit of AZA would allow AZA combination or other drugs in first-line treatments. New whole-genome scanning technologies such as single nucleotide polymorphism microarray (SNP-A)-based molecular karyotyping (MK) improve the risk stratification in MDS and AML. Maintenance of genomic integrity is less than three megabases (Mbs) total disruption of the genome correlated with better overall survival (OS) in patients with lower-risk MDS. In this SNP-A study, we aimed at defining a cutoff value for total genomic copy number (CN) alterations (TGA) influencing the median OS in a cohort of 51 higher-risk MDS/AML patients treated with AZA. We observed that the relative risk of worse OS increased >100 Mb of TGA, as detected by SNP-A-based MK (8 and 15 months respectively, P=0.02). Our data suggest that precise measurement of TGA could provide predictive information in poor and very poor revised International Prognostic Scoring system (IPSS-R) patients treated with AZA.

Cathepsin B release after imatinib-mediated lysosomal membrane permeabilization triggers BCR-ABL cleavage and elimination of chronic myelogenous leukemia cells.

Imatinib is the leading compound to treat patients with chronic myelogenous leukemia (CML) but the exact mechanism of its anti-leukemic effect is incompletely elucidated. Through inhibition of BCR-ABL, Imatinib blocks several downstream pathways and induces apoptosis of BCR-ABL positive cells. In this study, we analyzed further the mode of action of Imatinib in different appropriate cellular models of CML either sensitive or resistant to Imatinib and in CD34+ cells from CML patients. Pharmacological or short hairpin RNA-mediated inhibition of BCR-ABL triggers lysosomal membrane permeabilization (LMP) that culminates in activation and redistribution of Cathepsin B (CB) into the cytoplasm of CML cells, in which it triggers directly BCR-ABL degradation. Pharmacological inhibition of CB by CA-074Me or small interfering RNA-mediated knock-down of CB partly protects K562 cells from Imatinib-induced cell death and CB overexpression sensitizes these cells to Imatinib killing. Strikingly, Imatinib-triggered LMP, CB activation and BCR-ABL cleavage in CD34+ cells from CML patients and inhibition of CB confers protection against cell death in clonogenic assays of CD34+ primary cells from CML patients. Hence, we describe an original pathway by which Imatinib participates to the elimination of CML cells through LMP and CB-mediated specific degradation of BCR-ABL.

The tyrosine kinase Syk regulates the survival of chronic lymphocytic leukemia B cells through PKCdelta and proteasome-dependent regulation of Mcl-1 expression.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of mature monoclonal CD5+ B cells. The disease results mainly from a failure of cells to undergo apoptosis, a process largely influenced by the existence of constitutively activated components of B-cell receptor signaling and the deregulated expression of anti-apoptotic molecules. Recent evidence pointing to a critical role of spleen tyrosine kinase (Syk) in ligand-independent BCR signaling prompted us to examine its role in primary B-CLL cell survival. We demonstrate that pharmacological inhibition of constitutive Syk activity and silencing by siRNA led to a dramatic decrease of cell viability in CLL samples (n=44), regardless of clinical and biological status and induced typical apoptotic cell death with mitochondrial failure followed by caspase 3-dependent cell death. We also provide functional and biochemical evidence that Syk regulated B-CLL cell survival through a novel pathway involving PKCdelta and a proteasome-dependent regulation of the anti-apoptotic protein Mcl-1. Together, our observations are consistent with a model wherein PKCdelta downstream of Syk stabilizes Mcl-1 through inhibitory phosphorylation of GSK3 by Akt. We conclude that Syk constitutes a key regulator of B-CLL cell survival, emphasizing the clinical utility of Syk inhibition in hematopoietic malignancies.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells.

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Homeostatic chemokines increase survival of B-chronic lymphocytic leukemia cells through inactivation of transcription factor FOXO3a.

B-chronic lymphocytic leukemia (B-CLL) cell is characterized by the accumulation of long-lived CD5+ B lymphocytes, whose survival in vivo is in part dependent on exogenous factors such as cytokines and/or extracellular matrix proteins. Homeostatic chemokines are critical mediators of lymphoid cell trafficking. However, how they function in cell signaling and survival remains ill-defined. In this study, we have investigated the role of the homeostatic chemokines, CXCL12, CCL21, CCL19 and CXCL13, in B-CLL cell survival. Using primary leukemic cells isolated from 26 patients, we observed that each chemokine enhances cell survival. Chemokines induced the phosphorylation of ERK1/2 and p90RSK, and of Akt and its effectors GSK3 and FOXO3a. Consistently, inhibitors against mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase inhibited chemokine-induced survival. Moreover, using a constitutively active mutated form of FOXO3a or siRNAs against FOXO3a in transfection experiments performed in primary B-CLL cells, we directly demonstrated the critical role of FOXO3a in both spontaneous and chemokine-induced B-CLL cell survival. Overall, our data support the notion that homeostatic chemokines contribute to B-CLL resistance to cell death through inactivation of the transcription factor FOXO3a, which may represent a novel therapeutic target in this hematopoietic malignancy.

Incidence and risk factors of bacterial pneumonia requiring hospitalization in HIV-infected patients started on a protease inhibitor-containing regimen.

OBJECTIVE: To describe the incidence and risk factors of bacterial pneumonia occurring in patients treated with antiretrovirals. METHODS: In the ongoing APROCO (Anti-proteases) cohort, 1281 patients at the initiation of a protease inhibitor (PI)-containing antiretroviral regimen were enrolled from 1997-1999. All events requiring hospitalization during follow up are recorded. Of these, bacterial pneumonia was defined as the occurrence of a new pulmonary infiltrate with fever and either evidence of a bacteriological cause (definite cases) or favourable outcome with antimicrobial therapy (presumptive cases). Risk factors of bacterial pneumonia were studied using survival analyses. RESULTS: During a median follow up of 43 months, 29 patients had at least one episode of bacterial pneumonia, giving an incidence of 0.8/100 patient years. The 11 definite cases were attributable to Streptococcus pneumoniae (n=9), Legionella pneumophila (n=1) and Haemophilus influenzae (n=1). In multivariate analysis, bacterial pneumonia was significantly more frequent in older patients, injecting drug users, patients having a CD4 cell count>500 cells/microL at baseline and patients who initiated PI therapy with nonboosted saquinavir. It was significantly less frequent in nonsmokers. The occurrence of bacterial pneumonia was also associated with lower self-reported adherence to antiretroviral therapy and to higher plasma HIV-1 RNA levels during follow-up. CONCLUSIONS: Bacterial pneumonia occurs rarely in patients treated with a PI-containing regimen and may be associated with virological failure.

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

In the era of highly active antiretroviral therapy, why are HIV-infected patients still admitted to hospital for an inaugural opportunistic infection?

OBJECTIVES: To identify factors related to delayed testing, and delayed or interrupted care-seeking or treatment uptake, among HIV-infected patients. DESIGN: HIV-infected patients hospitalized for an opportunistic infection (OI) cases were included in a prospective study and compared with controls matched by age and sex who had regular follow-up and treatment. Patients were asked to complete a questionnaire about their therapeutic itinerary and their socioeconomic, psychological and medical characteristics. RESULTS: Seventy patients were matched with 140 controls. According to their therapeutic itinerary prior to admission, cases were subdivided into four groups among which three will be more particularly studied: nontested patients (NT) (24%; n=17), known HIV-infected patients with no medical follow-up (NF) (30%; n=21); and noncompliant patients (NC) (36%, n=25). Characteristics of NT and NF patients included a predominantly sexual mode of contamination (P=0.01), continuing occupational activity (P=0.01) despite a low mean Karnofsky index (P=0.001) and unfavourable virological and immunological parameters. NT patients displayed a low degree of anxiety, and lacked awareness concerning risk of contamination and HIV-related symptoms. HIV-status announcement (P=0.04) and the benefits of medical follow-up (P=0.05) were less favourably perceived by NF patients than by controls, and were associated with a high degree of anxiety in NF patients. NC patients had a weaker commitment to follow-up and treatment, and more frequent treatment discontinuation associated with a higher rate of interruption of follow-up in a context of social difficulties. CONCLUSIONS: Patients ignorant of their HIV status, patients NF and NC have very specific characteristics. More appropriate approaches are needed regarding screening and access to care in order to reduce the incidence of delayed care-seeking.

[Causes of hospitalization and death in the MANIF 2000 cohort, composed of HIV-infected patients contaminated by intravenous drug use, 1995-1999]. / Motifs d'hospitalisation et mortalité dans la cohorte MANIF 2000, composée de sujets infectés par le VIH et contaminés par usage de drogues en intraveineux, 1995-1999.

OBJECTIVE: To describe the causes of hospitalization and mortality among the MANIF 2000 cohort study, composed of HIV-infected patients contaminated through intravenous drug use. METHOD: Data collection with a standardized questionnaires with clinical, biological, therapeutic and psycho-sociologic data on inclusion and every six months. Dates and causes of hospitalization and death were collected retrospectively by nurses from the hospital clinical records. Comparison were made using the chi(2) or Mann-Whitney tests with 5% significance threshold. RESULTS: The MANIF 2000 cohort study included, between October 1995 and June 1998, 467 patients with a median age of 33 years, 30% of whom were women. On inclusion, 42.2% were still injecting drugs (half of them were on substitution therapy), 10.5% had stopped injections of drugs, 32% exhibited more than 500CD4/mm(3) and 55.7% had not taken antiretroviral treatment. As of December 31(st) 1999, 21 patients had died, i.e. a mortality rate of 19% persons/years of follow-up (10-fold greater than that expected in the general population). Less than 5 deaths were due to HIV (n=4). Suicides and liver disease each represented the same number of deaths. Out of the 335 patients not having missed more than one follow-up throughout the 24 month period, 120 had been hospitalized at least once (n=223 hospitalizations), i.e., a hospitalization rate of 2.8 per 100 persons/month of follow-up. A quarter of hospitalizations were due to benign infections or stage B or C pathologies according to the 1993 classification of HIV infections and one hospitalization out of 5 was due to psychiatric problems (in majority depressive syndromes). Other predominant causes were worsening of general state of health, trauma, problems related to alcohol consumption, drugs abuse or hepatic decompensation. CONCLUSION: HIV-infected patients contaminated by intravenous drug use represent a particular population that cumulates many risk factors and which requires careful monitoring of co-morbidities such as hepatic and psychiatric diseases in order to avoid premature death.

HIV and Leishmania coinfection: a review of 91 cases with focus on atypical locations of Leishmania.

A retrospective study was conducted in France in 1998 to determine the clinical features of visceral leishmaniasis (VL) in 91 patients infected cocomitantly with human immunodeficiency virus. Our data suggest that the clinical manifestations of VL may be influenced by the immunological status, with atypical locations of Leishmania amastigotes more frequently found in severely immunocompromised patients. In such patients, the involvement of atypical locations may lead to the discovery of VL.

Adherence to HAART in French HIV-infected injecting drug users: the contribution of buprenorphine drug maintenance treatment. The Manif 2000 study group.

OBJECTIVES: To assess adherence to highly active antiretroviral therapies (HAART) in a cohort of French patients infected by HIV through injection drug use (IDU), and the impact on adherence of buprenorphine ambulatory drug maintenance treatment (DMT) which has been widely introduced since 1996. DESIGN: Adherence assessment at first visit after initiation of HAART in the MANIF2000 cohort study. METHODS: Patient's face-to-face and self-administered questionnaires. Univariate and logistic regression adjusted odds ratios (OR) to compare characteristics of non-adherent versus adherent patients. RESULTS: Of the 164 patients, 34.8% took less than 80% of the prescribed HAART doses during the previous week. Decrease in viral load titres after initiation of HAART was significantly lower among non-adherent patients. After adjustment by logistic regression, non-adherence was associated with younger age, alcohol consumption, frequency of negative life-events during the prior 6 months and active drug use. However, IDU in buprenorphine DMT reached higher levels of adherence (78.1%) than ex-IDU (65.5%), although this difference did not reach statistical significance. CONCLUSION: Prescription of buprenorphine DMT may increase adherence to HAART among HIV-infected opiate-dependent patients. Reducing the negative impact of stressful life-events through psychosocial interventions should be considered, even for those who have stopped using drugs.

[Disseminated Penicillium marneffei infection suggesting visceral leishmaniasis in an HIV infected patient]. / Infection à Penicillium marneffei évoquant une leishmaniose viscérale chez un patient infecté par le VIH.

BACKGROUND: Penicilliosis is a fungal infection caused by Penicillium marneffei. In Southeast Asia, this infection is common in HIV-infected patients but few cases have been reported in Europe. CASE REPORT: The patient lived in southern France. He had HIV infection and was immunocompromised. The clinical signs suggested visceral leishmaniasis. Iterative travels to Southeast Asia were reported leading to the diagnosis of P. marneffei infection. DISCUSSION: In southern France, imported penicilliosis marneffei may be misdiagnosed as visceral leishmaniasis.

Clinical manifestations of visceral leishmaniasis associated with HIV infection: a retrospective study of 91 French cases.

A retrospective study was conducted in France to determine the clinical features of visceral leishmaniasis (VL) seen, between 1986 and 1997, in 91 patients co-infected with human immunodeficiency virus (HIV). Fever (87% of patients), splenomegaly (74%) and hepatomegaly (49%) were common, 43% of the patients having all of these signs and only 9% having none of them. Amastigotes were reported in atypical locations in 31 (34%) of the patients, and 15 patients had only had their VL diagnosed following accidental discovery of amastigotes in samples from their digestive tract and lungs (one), digestive tracts only (11 patients), lungs only (two), or skin (one). Some of the digestive symptoms observed are probably attributable to the intestinal infections with Leishmania. Overall, VL diagnosis was fortuitous in 27% of the subjects. Even in endemic areas, therefore, VL is not considered routinely by physicians attempting diagnoses.