Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy.

OBJECTIVES: Human leishmaniasis can be severe and fatal, yet in the Mediterranean region only a small percentage of infections progress to clinical disease. We evaluated the percentage of asymptomatic Leishmania infection in the Bologna province, northeastern Italy. METHODS: We examined the presence of specific antibodies by Western Blot (WB) and parasitic DNA by real time PCR in peripheral blood of 240 blood donors residing in the Bologna province. RESULTS: Anti-Leishmania IgG were detected by WB in 27 subjects (11.2%, 95% CI 7%-15%), while Leishmania kinetoplast DNA was detected in peripheral blood specimens of 4 out of 240 donors (1.7%, 95% CI 0.2%-3.2%). Overall, the prevalence of Leishmania infection in the blood donor cohort was 12.5%, thus indicating an elevated cumulative exposure to the Leishmania parasite in the examined municipality. CONCLUSIONS: Our results suggest that a surveillance system for monitoring Leishmania infection in blood donors and/or strategies of protozoan inactivation in whole blood should be taken into consideration in areas with circulation of the Leishmania parasite.

Cyclin D1 overexpression is a favorable prognostic variable for newly diagnosed multiple myeloma patients treated with high-dose chemotherapy and single or double autologous transplantation.

We used a sensitive real-time reverse transcription-polymerase chain reaction assay to quantify cyclin D1 mRNA levels in bone marrow samples collected at diagnosis from 74 newly diagnosed multiple myeloma (MM) patients who were randomized to undergo either single or double autologous peripheral blood stem cell transplantation as part of first-line therapy for their malignancy. In 46 cases, fluorescence in situ hybridization (FISH) analysis and/or conventional cytogenetics were performed to detect chromosome 11 abnormalities. Patients with the t(11;14) or trisomy 11 significantly overexpressed cyclin D1 (P <.0001) in comparison with patients without 11q abnormalities, who had cyclin D1 mRNA levels similar to healthy donors. Overall, 32 (43%) of 74 patients showed cyclin D1 overexpression. No difference was found between cyclin D1-positive (group A) and cyclin D1-negative (group B) patients with respect to presenting clinical and laboratory characteristics, including chromosome 13 abnormalities, as well as to response to therapy and overall survival, both of which were calculated on an intent-to-treat basis. Patients who overexpressed cyclin D1 had significantly longer duration of remission in comparison with patients who did not (41 vs 26 months, respectively; P =.02). As a result, median event-free survival (EFS) was longer in group A than in group B (33 vs 24 months, respectively; P =.055). We concluded that cyclin D1 overexpression is closely associated with 11q abnormalities and identifies a subset of MM patients who are more likely to have prolonged duration of remission and EFS following autologous transplantation.

Novel mutation and RNA splice variant of fibroblast growth factor receptor 3 in multiple myeloma patients at diagnosis.

BACKGROUND AND OBJECTIVES: The karyotypically silent t(4;14)(p16.3;q32) translocation can be found in approximately 15-20% of multiple myeloma (MM) patients and results in the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4. Point mutations in specific FGFR3 domains can be found in the translocated allele, and have been recently proven to be oncogenic. These mutations produce a constitutively activated receptor, which shows dimerization and autophosphorylation even in the absence of ligand. We investigated the presence of FGFR3 expression and activating mutations in a series of newly diagnosed MM patients. DESIGN AND METHODS: We validated a new sensitive and specific Taqman real-time reverse-transcription polymerase-chain-reaction (RT-PCR) set up to evaluate FGFR3 mRNA expression, and applied it to 78 newly diagnosed patients; in positive cases, FGFR3 mRNA transcripts were sequenced. Fluorescence in situ hybridization (FISH) was done in 32 cases with sufficient material. RESULTS: Real-time RT-PCR revealed FGFR3 mRNA expression in 10/78 (13%) patients. In two cases, sequence analysis revealed novel FGFR3 mutations. In a patient with FISH evidence of the t(4;14), a CGC to TGC transition was detected in codon 248. In a patient without the t(4;14), three additional, abnormal-sized transcripts were detected, corresponding to truncated transcripts originating from cryptic splice donor sites located within exon 7. INTERPRETATION AND CONCLUSIONS: We describe a novel FGFR3 mutation (with a demonstrated deregulatory mechanism), as well as a case of alternative splicing in the absence of t(4;14), detected in newly diagnosed MM patients overexpressing FGFR3. This implies that FGFR3 mutation can occur at an early stage of myelomagenesis and even in the absence of the t(4;14).

Complex variant Philadelphia translocations involving the short arm of chromosome 6 in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES: Around 5% of chronic myeloid leukemias (CML) are characterized by complex variant Philadelphia (Ph) translocations involving one or more chromosomal regions in addition to 9 and 22. The BCR/ABL1 fusion gene is usually found on der(22). The additional gene(s) involved in complex variant Ph rearrangements have not been characterized. DESIGN AND METHODS: We performed fluorescent in situ hybridization (FISH) in three complex variant Ph translocations involving the short arm of chromosome 6 in addition to 9 and 22. The BCR/ABL1 D-FISH probe was applied to localize the BCR/ABL1 fusion gene as well as the 5'ABL1 and the 3'BCR. Locus-specific probes were used to narrow the 6p breakpoint. RESULTS: In all cases the BCR/ABL1 fusion gene was located on the Ph chromosome whereas the reciprocal ABL1/BCR gene was detected only in patient #2. On 6p, breakpoints were narrowed to three different regions: centromeric to the human major histocompatibility complex (MHC), between PAC 524E15 and PAC162J16, in the first patient, and telomeric to the MHC, between PAC 329A5 and PAC 145H9, and between PAC 136B1 and PAC 206F19, in the second and third patients, respectively. In patients #2 and 3 a chromosomal rearrangement different from a true complex variant was discovered. In both cases, a classical t(9;22) was associated with an additional translocation involving the der(9)t(9;22). INTERPRETATION AND CONCLUSIONS: Rearrangements at 6p in complex Ph aberrations involve more than one gene/locus. Classical t(9;22), masked by additional chromosomal rearrangements, can resemble complex variant Ph translocations, and can be detected only using appropriate FISH probes.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage.

OBJECTIVE: The aim of this study was to determine whether expression of the CD86 costimulatory molecule in acute myeloid leukemia (AML) can identify blast cells committed to the monocytic/dendritic lineage. MATERIAL AND METHODS: One hundred ten consecutive AML patients observed at diagnosis were studied by flow cytometry. In selected experiments, in vitro cultures with CD34(+)CD86(+) or CD34(-)CD86(+) blasts were performed in the presence of granulocyte-macrophage colony-stimulating actor (GM-CSF) with or without tumor necrosis factor-alpha (TNF-alpha) or GM-CSF + interleukin-4 (IL-4), respectively, to induce a dendritic differentiation, documented by morphologic and immunophenotypic assays. T-cell alloreactivity to CD86(+) AML cells and leukemic dendritic cells (AML-DC) was tested in mixed leukocyte cultures and anti-leukemic cytotoxic assays. RESULTS: CD86 was expressed in 54% AML, whereas CD80 and CD1a were only occasionally positive. CD86(+) AML samples included M5 and M4, but also 47% M0-M1 FAB types, and were more frequently CD14(+) (p < 0.00001) and CD34(-) (p = 0.00005) than CD86(-)AML. Six different patterns of CD86(+) AML were identified, according to CD34 or CD14 total or partial coexpression. Four samples enriched in CD34(+)CD86(+) AML cells differentiated into AML-DC CD86(+), CD80(+), CD40(+), CD11c(+), HLA-DR(++), CD14(+/-) that also were CD1a(+) or CD83(+), after 6 days of in vitro culture with GM-CSF +/- TNF-alpha. CD34(-)CD86(+) AML cells differentiated into AML-DC after 3 to 5 days (n = 5 experiments), and trisomy 8 was found in two AML and AML-DC samples by fluorescence in situ hybridization analysis. Finally, AML-DC induced more potent allo-T-cell proliferation, cytokine release, and anti-leukemic cytotoxicity than CD86(+) blasts. CONCLUSIONS: In AML, CD86 is a marker of monocytic/dendritic lineage. Because CD86(+) blasts may differentiate into DC rapidly, CD86(+)AML patients could be optimal candidates for immunotherapy studies, both in autologous and allogeneic settings.

Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state.

The inv(16) cytogenetic subtype of acute myeloid leukemia (AML) has a relatively good prognosis. Many patients achieve complete remission (CR). The prognostic uncertainty of negative qualitative reverse transcription-polymerase chain reaction (RT-PCR) assays suggests the need to identify prognostically significant critical thresholds by real-time RT-PCR. A reliable and sensitive (10(-5)) real-time RT-PCR assay was set up for the evaluation of relevant CBFbeta-MYH11/ABL transcript ratios and was applied to the 21 patients with inv(16) AML routinely referred for cytogenetic and molecular monitoring in Seràgnoli Institute (Bologna, Italy) since 1990. Among the 18 patients who underwent ablative chemotherapy, all achieved CR with a 3-year disease-free survival probability of 63% (95% CI, 40%-87%) and no recorded events after 26 months. Five patients had relapses; 2 died of disease and 3 entered second CR. Analysis of the 125 bone marrow (or peripheral blood) samples studied by real-time RT-PCR showed that transcript ratios of samples taken during CR at any time before a relapse were always greater than 0.12%, whereas those of samples taken during first or second CR from patients who did not subsequently have relapses were always less than 0.25%. This suggests that transcript ratios greater than 0.25% may correspond to high risk for relapse, whereas ratios below 0.12% might indicate the patient is in a curable state. If confirmed, such thresholds could open the way to a new phase in post-CR therapeutic decision making for patients with inv(16) AML.