Comparison of multiple definitions for ventilator-associated pneumonia in patients requiring mechanical ventilation for non-pulmonary conditions: preliminary data from PULMIVAP, an Italian multi-centre cohort study.

OBJECTIVES: To compare intensivist-diagnosed ventilator-associated pneumonia (iVAP) with four established definitions, assessing their agreement in detecting new episodes. METHODS: A multi-centric prospective study on pulmonary microbiota was carried out in patients requiring mechanical ventilation (MV). Data collected were used to compare hypothetical VAP onset according to iVAP with the study consensus criteria, the European Centre for Disease Control and Prevention definition, and two versions of the latter adjusted for leukocyte count and fever. RESULTS: In our cohort of 186 adult patients, iVAPs were 36.6% (68/186, 95% confidence interval 30.0-44.0%), with an incidence rate of 4.64/100 patient-MV-days, and median MV-day at diagnosis of 6. Forty-seven percent of patients (87/186) were identified as VAP by at least one criterion, with a median MV-day at diagnosis of 5. Agreement between intensivist judgement (iVAP/no-iVAP) and the criteria was highest for the study consensus criteria (50/87, 57.4%), but still one-third of iVAP were not identified and 9% of patients were identified as VAP contrary to intensivist diagnosis. VAP proportion differed between criteria (25.2-30.1%). CONCLUSIONS: Caution is needed when evaluating studies describing VAP incidence. Pre-agreed criteria and definitions that capture VAP's evolving nature provide greater consistency, but new clinically driven definitions are needed to align surveillance and diagnostic criteria with clinical practice.

Two siblings presenting with novel ADA2 variants, lymphoproliferation, persistence of large granular lymphocytes, and T-cell perturbations.

The presence of large granular lymphocytes has been reported in patients with ADA2 deficiency and T-LGL leukemia. Here we describe two siblings with novel ADA2 variants, expanding the mutational spectrum of ADA2 deficiency. We show that lymphoproliferation, persistence of large granular lymphocytes, T-cell perturbations, and activation of PI3K pathway, measured by means of phosphorylation levels of S6, are detectable in DADA2 patients without T-LGL leukemia.

Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling.

Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients.

Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

Autologous purified peripheral blood SCT in childhood low-risk relapsed ALL.

The treatment of childhood B-cell-precursor ALL after isolated-extramedullary or late relapse is controversial. Most approaches are based on chemotherapy or allogeneic transplantation. The aim of this report is to assess the long-term outcome of children with 'low-risk' relapsed ALL treated according to a prospective purified auto-transplantation protocol. From January 1997 to March 2004, at a single pediatric Center, 30 ALL consecutive children, lacking an HLA-identical sibling, were treated according to the autologous purified peripheral blood stem cell protocol after isolated-extramedullary (7) or late medullary (24) relapse. After the 'DIAVE' mobilizing regimen a median of 11.6 × 10(6)CD34+/Kg (range 3.9-27.4) were collected. Leukaphereses were depleted by 99% of CD19+cells (range 98-100) by means of a double step immunological purification. The conditioning regimen included TBI. No early severe complications nor transplant-related deaths occurred; late effects, as expected, mostly consisted in endocrinological issues and were assessed at a median follow-up of 8.5 years. Five-year-EFS and survival were 68.5% (s.e. 7.9) and 85.7% (s.e. 5.9), respectively, for the 35 eligible patients and 70.0% (s.e. 8.4) and 86.7% (s.e. 6.2) for the 30 patients actually transplanted as per protocol. The outcome of this series favorably compares with historical data regarding both autologous transplantation and standard salvage chemotherapy.

Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction.

Treatment response is a strong outcome predictor for childhood acute lymphoblastic leukemia (ALL). Here, we evaluated the predictive impact of flow cytometric blast quantification assays (absolute blast count, BC, and blast reduction rate, BRR) in peripheral blood (pB) and/or bone marrow (BM) at early time points of induction therapy (days 0, 8 and 15) on the remission status in the AIEOP-BFM-ALL 2000 protocol. At the single parameter level (905 patients), the strongest predictive parameter for the remission status as a dichotomous minimal residual disease (MRD) parameter (positive/negative) has been provided by the BC at day 15 in BM (cutoff: 17 blasts/microl; 50 vs 15%; odds ratio: 5.6; 95% confidence interval: 4.1-7.6, P<0.001), followed by the BRR at day 15 in BM and by the BC at day 8 in pB (odds ratios: 3.8 and 2.6, respectively). In the multiple regression analysis (440 patients), BC in pB (d0 and d8) and in BM (d15) as well as BRR at day 8 in pB provided significantly contributing variables with an overall correct prediction rate of 74.8%. These data show that the quantitative assessment of early response parameters, especially absolute BCs at day 15 in BM, has a predictive impact on the remission status after induction therapy.

GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease.

BACKGROUND: Periodontal disease is a degenerative illness that leads to resorption of the alveolar bone. Mesenchymal stromal cells (MSC) represent a novel tool for the production of biologic constructs for the treatment of degenerative bone diseases. The preparation of MSC differentiated into osteogenic lineage for clinical use requires the fulfillment of strict good manufacturing practice (GMP) procedures. METHODS: MSC were isolated from BM samples and then cultured under GMP conditions. MSC were characterized phenotypically and for their differentiative potential. Cells were seeded onto collagen scaffolds (Gingistat) and induced to differentiate into osteogenic lineages using clinical grade drugs compared with standard osteogenic supplements. Alizarin Red S stain was used to test the deposition of the mineral matrix. Standard microbiologic analysis was performed to verify the product sterility. RESULTS: The resulting MSC were negative for CD33, CD34 and HLA-DR but showed high expression of CD90, CD105 and HLA-ABC (average expressions of 94.3%, 75.8% and 94.2%, respectively). Chondrogenic, osteogenic and adipogenic differentiation potential was demonstrated. The MSC retained their ability to differentiate into osteogenic lineage when seeded onto collagen scaffolds after exposure to a clinical grade medium. Cell numbers and cell viability were adequate for clinical use, and microbiologic assays demonstrated the absence of any contamination. DISCUSSION: In the specific context of a degenerative bone disease with limited involvement of skeletal tissue, the combined use of MSC, exposed to an osteogenic clinical grade medium, and biomimetic biodegradable scaffolds offers the possibility of producing adequate numbers of biologic tissue-engineered cell-based constructs for use in clinical trials.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection.

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI+2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low nonspecific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection in T-ALL, bearing the advantage of surface expression in contrast to TdT.

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors.

Three different second-generation lentiviral self-inactivating vectors containing CMV, EF1alpha and PGK promoter, respectively, and all carrying the exogenous GFP gene, were compared for expression in human B-cell precursor ALL blasts. At a comparable percentage of transduction and vector DNA copy number, CMV clearly showed better efficiency of transcription. Human bone marrow stromal cells were favored compared to the MRC-5 cell line, as support for cell viability during infection. Cells were infected and analyzed after variable culture times ranging from 4 to 12 days, to reduce the possibility of pseudotransduction. In 10/14 samples, we detected more than 20% GFP-positive cells after exposure to high-titer viral supernatants. We then tested a similar vector carrying the human CD40L cDNA and, in similar infection conditions, obtained more than 20% transduction in 6/6 samples. The levels of transduction obtained were sufficient to induce the upregulation of CD83 molecule in cocultured immature dendritic cells.

Effect of Aplidin in acute lymphoblastic leukaemia cells.

The cytotoxic effect of Aplidin was investigated on fresh leukaemia cells derived from children with B-cell-precursor (BCP) acute lymphoblastic leukaemia (ALL) by using stromal-layer culture system and on four cell lines, ALL-PO, Reh, ALL/MIK and TOM-1, derived from patients with ALL with different molecular genetic abnormalities. In ALL cell lines Aplidin was cytotoxic at nanomolar concentrations. In the ALL cell lines the drug-induced cell death was clearly related to the induction of apoptosis and appeared to be p53-independent. Only in ALL-PO 20 nM Aplidin treatment caused a block of vascular endothelial growth factor (VEGF) secretion and downregulation of VEGF-mRNA, but Aplidin cytotoxicity does not seem to be related to VEGF inhibition since the sensitivity of ALL-PO cells to Aplidin is comparable to that observed for the other cells used. Aplidin induced a G(1) and a G(2) M block in ALL cell lines. In patient-derived leukaemia cells, Aplidin induced a strong cytotoxicity evidenced in a stroma-supported immunocytometric assay. Cells from children with genetic abnormalities such as t(9;22) and t(4;11) translocations, associated with an inferior treatment outcome, were sensitive to Aplidin to the same extent as that observed in other BCP-ALL cases. Aplidin exerted a strong cell killing effect (>88%) against primary culture cells from five relapsed ALL cases, at concentrations much lower than those reported to be achieved in plasma of patients receiving Aplidin at recommended doses. Taken together these data suggest that Aplidin could be a new anticancer drug to be investigated in ALL patients resistant to available therapy.

Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases.

T-cell depletion is an essential step in reducing the risk of graft-versus-host disease (GVHD) in patients with inherited metabolic storage diseases (IMSD) undergoing hematopoietic stem cell transplantation. This goal can be achieved either by selective removal of T cells or by positive selection of CD34+ cells. Large-scale preparations of purified CD34+ cells from bone marrow products have not been extensively described. We report our results with bone marrow CD34+ cell enrichment using the CliniMACS system in eight children with IMSD. The median recovery of positively selected CD34+ cells was 46.2% with a purity of 97.5%, and a residual T cell content of 0.04 x 10(6). A median of 5.5 x 10(6)/kg of CD34+ cells was infused. All patients engrafted at a median time of 12 days and none of the patients developed GVHD. This method is technically feasible and can be successfully used to transplant children with IMSD.

Rituximab for immune hemolytic anemia following T- and B-Cell-depleted hematopoietic stem cell transplantation.

The treatment of immune-mediated hemolytic anemia (IHA) complicating hematopoietic stem cell transplantation (HSCT) is often unsatisfactory. We report a case of IHA which occurred after T- and B-cell depleted unrelated donor HSCT carried out for mucopolysaccharidosis type I-H (Hurler syndrome) which was successfully treated with anti-CD20+ monoclonal antibody

Peripheral blood stem cell collection in children with acute leukemia: effectiveness of the 'DIAVE' mobilizing regimen.

Few experiences of peripheral blood (PB) hematopoietic stem cell mobilization for autologous transplantation have been reported to date in children with acute leukemia (AL). The five-drug-chemotherapy 'DIAVE' (dexamethazone, idarubicine, cytosine-arabinoside, vincristine, etoposide), followed by G-CSF, previously reported as consolidation, was adopted as a mobilization regimen in 29 children (median age: 8 years, range: 3-21; median weight: 34 kg, range: 15-73) with ALL in second remission (CR2: 21), in CR3 (2) or ANLL in CR1 (6). A median peak of 94 x 10(6) CD34(+)cells/l (range: 10-604) was reached at a median time of 12 days (range: 10-18) after the beginning of the mobilizing regimen, which was well tolerated. A median of 10.9 x 10(6) CD34(+)cells/kg (range: 2.4-56.6) were collected in 25 patients (86%), approaching 40 x 10(6)/l CD34(+) cells in the PB (ALL in CR2: 20/21, in CR3: 0/2; ANLL: 5/6) by means of one (20) or two (5) leukaphereses; a median of 2.5 blood volumes was processed. Patients with ANLL mobilized more cells than patients with ALL; moreover, the shorter the interval between remission and mobilizing therapy, the higher was the yield. The products collected underwent purification, aiming at achieving complete removal of possibly contaminating leukemic cells, in 21 cases; also, unmanipulated aliquots were stored as rescues for all but one patient. All the 23 patients undergoing transplantation engrafted (ANC >0.5 x 10(9)/l) at a median of 12 days. In conclusion, the DIAVE regimen compares favorably with conventional mobilizing regimens, usually containing cyclophosphamide, in terms of low toxicity, collection time predictability, and efficacy, as shown by the high proportion of patients mobilizing, the large amounts of stem cell collected by means of one or two leukaphereses only, and the prompt engraftment after infusion.