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Biomedical subjects

Daniela Diverio

Publications and source records attributed to Daniela Diverio.

At least 19 recordsLinked to original sources
PubMed2006

Podocalyxin is expressed in normal and leukemic monocytes.

We have investigated the expression of podocalyxin in primary cultures of leukemic blast cells from 73 patients with acute myeloid leukemia. Podocalyxin was expressed at moderate levels in 15 patients and at high levels in 13 patients. The analysis of membrane markers showed that Podocalyxin expression in leukemic blasts was associated with a monocytic immunophenotype. Cases of podocalyxin-positive acute myelogenous leukemia had high blast cell counts at diagnosis and elevated CD123, CD135, VLA-4 and CXCR4 expression, features associated with poor prognosis. Podocalyxin expression in leukemic blasts was coupled with the concomitant expression of VEGF-R1, -R2, -R3 and Tie-2, the capacity to release VEGF-A and angiopoietin1 and the ability to differentiate into endothelial cells under appropriate culture conditions. These findings show that podocalyxin is a marker of acute myeloid leukemia with a monocytic phenotype and suggest that podocalyxin-positive cases of acute myeloid leukemia originate from the malignant transformation of progenitors common to the myeloid and endothelial lineages. These observations suggest a possible relationship between the monocytic lineage and podocytes.

Biomarkers, Tumor↗
PubMed2006

Sudden blast crisis in patients with Philadelphia chromosome-positive chronic myeloid leukemia who achieved complete cytogenetic remission after imatinib therapy.

BACKGROUND: Most patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) in chronic phase (CP) who receive treatment with imatinib achieve complete cytogenetic remission (CCR), which is correlated tightly with long-term progression-free survival. In these patients, the occurrence of blastic crisis (BC) is rare, and its clinical biologic characteristics are not well known. METHODS: Among 164 patients who received imatinib and were followed for a median of 35 months, 11 patients (6.7%) developed a BC; this was sudden (i.e., it occurred within 3 months of a documented CCR) in 6 patients (54.5%). Those patients were analyzed with respect to their clinical and biologic features and were compared with previous reports. RESULTS: At the time of diagnosis, there were 3 low-risk patients and 3 intermediate-risk patients; 4 patients had received pretreatment with interferon, and 2 patients received only imatinib. The median CP was 18 months, and the median duration of imatinib therapy was 7 months. The median time to CCR was 3 months, and the median time from CCR to BC was 4 months. BC phenotype was lymphoid in 2 patients, myeloid in 3 patients (including 2 patients who had extramedullary localization), and biclonal in 1 patient. Karyotype evolution was detected in 4 patients, whereas a Ph-positive/Ph-negative mosaicism was evident in all 6 patients. One patient presented an M351T mutation. The overall median survival was 3 months. CONCLUSIONS: Sudden BC generally is an uncommon phenomenon that may be relatively frequent in patients with CML who receive imatinib. Clinical and biologic features also seem to characterize this peculiar type of abrupt disease evolution, which intervenes in patients' response to this drug. Close monitoring of disease markers and full disease eradication are warranted.

Adult↗
PubMed2006

Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia.

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.

Active Transport, Cell Nucleus↗
PubMed2006

Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

Active Transport, Cell Nucleus↗
PubMed2006

Denaturing high-performance liquid chromatography: a valid approach for identifying NPM1 mutations in acute myeloid leukemia.

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

Amino Acid Sequence↗
PubMed2006

Dual-color split signal fluorescence in situ hybridization assays for the detection of CALM/AF10 in t(10;11)(p13;q14-q21)-positive acute leukemia.

We developed dual-color split fluorescence in situ hybridization (FISH) assays to detect AF10 and/or CALM rearrangements. Among nine cases of acute leukemia with translocation breakpoints at 10p13 and 11q14-21, a CALM/AF10 rearrangement was found in seven and was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) in all. In 2/7 cases, FISH detected CALM/AF10 in extramedullary leukemic infiltrations in the mediastinum and breast. As expected, FISH was less sensitive than RT-PCR for disease monitoring of CALM-AF10 positive cases. This new FISH assay reliably discriminates between MLL/AF10 and CALM/AF10 genomic rearrangements, identifies variant and complex CALM/AF10 translocations and detects the CALM/AF10 rearrangement in extramedullary leukemic infiltrations.

Acute Disease↗
PubMed2005

Diphtheria toxin fused to variant human interleukin-3 induces cytotoxicity of blasts from patients with acute myeloid leukemia according to the level of interleukin-3 receptor expression.

Leukemic blasts from patients with acute myeloid leukemia (AML) frequently express high levels of the interleukin-3 receptor alpha chain (IL-3Ralpha). In the present study, we have explored the sensitivity of primary leukemic blasts obtained from 34 patients with AML to a diphtheria toxin (DT) composed of the catalytic and translocation domains of DT (DT388) fused to IL-3 (DT388IL-3) and to DT388 fused to a variant IL-3 with increased binding affinity (DT388IL-3[K116W]). On a molar basis, DT388IL-3[K116W] was significantly more active than DT388IL-3 in mediating leukemic cell killing. The rate of cell killing induced by the 2 DT/IL-3 fusion proteins was significantly correlated with the level of IL-3Ralpha/IL-3Rbeta expressed on leukemic blasts. These observations support a potential use of DT388IL-3[K116W] in the treatment of refractory AMLs and provide a simple biochemical parameter for the selection of eligible patients.

Antineoplastic Agents↗
PubMed2005

Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance.

Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc+ AML), consequent to mutations in the NPM putative nucleolar localization signal. These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, FLT3 mutations, and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 without major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc+ from NPMc- AMLs, regardless of the presence of FLT3 mutations or non-major chromosomal rearrangements, supporting the concept that NPMc+ AML represents a distinct entity. The molecular signature of NPMc+ AML includes up-regulation of several genes putatively involved in the maintenance of a stem-cell phenotype, suggesting that NPMc+ AML may derive from a multipotent hematopoietic progenitor.

Acute Disease↗
PubMed2005

Retinoic acid targets DNA-methyltransferases and histone deacetylases during APL blast differentiation in vitro and in vivo.

The acute promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion product recruits histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities on retinoic acid (RA)-target promoters causing their silencing and differentiation block. RA treatment induces epigenetic modifications at its target loci and restores myeloid differentiation of APL blasts. Using RA-sensitive and RA-resistant APL cell lines and primary blasts, we addressed the functional relevance of the aberrant methylation status at the RA-target promoter RARbeta2 and the mechanism by which methylation is reversed by RA. RA decreased DNMT expression and activity, which correlated with demethylation at specific sites on RARbeta2 promoter/exon-1, and the ability of APL blasts to differentiate in vitro and in vivo. None of these events occurred in an RA-resistant APL cell line containing a PML-RARalpha defective for ligand binding. The specific contribution of the HDAC and DNMT pathways to the response of APL cells to RA was also tested by inhibiting these enzymatic activities with TSA and/or 5-azacytidine. In RA-responsive and RA-resistant APL blasts, TSA and 5-azacytidine induced specific changes on the chromatin state at RA-target sites, increased the RA effect on promoter activity, endogenous RA-target gene expression and differentiation. These results extend the rationale for chromatin-targeted treatment in APL and RA-resistant leukemias.

Blast Crisis↗
PubMed2005

GIMEMA-AIEOPAIDA protocol for the treatment of newly diagnosed acute promyelocytic leukemia (APL) in children.

The role of all-trans retinoic acid (ATRA) in pediatric acute promyelocytic leukemia (APL) is the topic of several ongoing studies. The results of the Italian pediatric experience with the multicentric Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA)-Italian Pediatric Hematology and Oncology Group (AIEOP) "AIDA" (ATRA and idarubicin) trial are presented. Of the 983 patients with APL enrolled in this protocol between January 1993 and June 2000, 124 (13%) had younger than 18 years. Treatment consisted of ATRA and idarubicin induction followed by 3 polychemotherapy consolidation courses. Molecular response by reverse transcriptase-polymerase chain reaction (RT-PCR) was assessed after consolidation and patients who were PCR- were randomized for different maintenances. One hundred and seven children were eligible and evaluable for induction: 103 (96%) achieved a hematologically complete remission. Overt ATRA syndrome was observed in 2 patients and pseudotumor cerebri was observed in 10 patients. Ninety-four patients were evaluable for RT-PCR analysis at the end of consolidation: 91 (97%) proved PCR+ and 3 PCR-. The overall survival and event-free survival (EFS) are 89% (95% confidence interval [c.i.]: 83%-95%) and 76% (c.i.: 65%-85%), respectively, at more than 10 years. A white blood cell (WBC) count at diagnosis of greater than 10 x 10(9)/L had a significant impact on EFS (59% vs 83% at 10 years). These results highlight the efficacy and feasibility of the AIDA protocol in the pediatric APL population.

Adolescent↗
PubMed2005

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

BACKGROUND: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein. METHODS: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease. RESULTS: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells. CONCLUSIONS: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.

Adolescent↗
PubMed2005

FLT3 inhibition in t(4;11)+ adult acute lymphoid leukaemia.

The present study was designed to investigate, in t(4;11)+ adult lymphoid leukaemia (ALL) blast cells, the pathogenetic role of the FLT3 protein, its level of mRNA and protein expression, the degree of constitutive phosphorylation, the possible presence of mutations of the sequence, the capacity of signal transduction and the potential therapeutic role of specific inhibitors. We evaluated nine adult ALL patients carrying this translocation. The increased FLT3 mRNA levels, determined by oligonucleotide microarray analysis, was in agreement with the increased protein expression evaluated by Western blot. The protein was constitutively phosphorylated in all cases analysed. Polymerase chain reaction detected no internal tandem duplication or point mutations. The signal transduction apparatus, after stimulation with the specific ligand, was preserved. We then investigated the effect of specific FLT3 inhibition on signal transduction and survival. The PKC412 inhibitor specifically inhibited ligand-induced phosphorylation; the same inhibitor reduced the survival of leukaemic cells when compared with untreated cells. These data indicate that the FLT3 protein might play a role in this subgroup of ALL with a particularly poor prognosis. Specific inhibition of the kinase receptor must be hypothesised as an innovative therapeutic tool for t(4;11)+ ALL patients.

Adult↗
PubMed2005

TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.

BACKGROUND AND OBJECTIVES: The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, many cancer cells are resistant to apoptosis induction by TRAIL. The present study was designed to evaluate the sensitivity to TRAIL-induced apoptosis in acute myeloblastic leukemias (AML). DESIGN AND METHODS: TRAIL/TRAIL receptor (TRAIL-R) expression and sensitivity to TRAIL-mediated apoptosis were explored in 79 AML patients, including 17 patients with acute promyelocytic leukemia (APL). RESULTS: In non-APL AML we observed frequent expression of TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4), while TRAIL-R1 and TRAIL-R2 expression was restricted to AML exhibiting monocytic features. Total leukemic blasts, as well as AML colony-forming units (AML-CFU), were invariably resistant to TRAIL-mediated apoptosis. APL express membrane-bound TRAIL on their surface and exhibit a pattern of TRAIL-R expression similar to that observed in the other types of AML. Before, during and after retinoic acid treatment APL cells are TRAIL-resistant. The induction of granulocytic maturation of APL cells by retinoic acid was associated with a marked decline of TRAIL expression. INTERPRETATION AND CONCLUSIONS: The analysis of experimental APL models (i.e., U937 cells engineered to express PML/RAR-Eo and NB4 cells) provided evidence that PML/RAR-Eo expression was associated with downmodulation of TRAIL-R1 and with resistance to TRAIL-mediated apoptosis. We suggest that AML blasts, including APL blasts, are resistant to TRAIL-mediated apoptosis, a phenomenon seemingly related to the expression of TRAIL decoy receptors on these cells. Finally, APL blasts express membrane-bound TRAIL that could confer an immunologic privilege to these cells.

Acute Disease↗
PubMed2004

PAX5 expression in acute leukemias: higher B-lineage specificity than CD79a and selective association with t(8;21)-acute myelogenous leukemia.

The transcription factor PAX5 plays a key role in the commitment of hematopoietic precursors to the B-cell lineage, but its expression in acute leukemias has not been thoroughly investigated. Hereby, we analyzed routine biopsies from 360 acute leukemias of lymphoid (ALLs) and myeloid (AMLs) origin with a specific anti-PAX5 monoclonal antibody. Blasts from 150 B-cell ALLs showed strong PAX5 nuclear expression, paralleling that of CD79a in the cytoplasm. Conversely, PAX5 was not detected in 50 T-cell ALLs, including 20 cases aberrantly coexpressing CD79a. Among 160 cytogenetically/molecularly characterized AMLs, PAX5 was selectively detected in 15 of 42 cases bearing the t(8;21)/AML1-ETO rearrangement. Real-time reverse transcription-PCR studies in t(8;21)-AML showed a similar up-regulation of PAX5 transcript in all of the 8 tested samples (including 4 cases that were negative at anti-PAX5 immunostaining), suggesting that PAX5 is expressed in t(8;21)-AML more widely than shown by immunohistochemistry. Interestingly, PAX5(+) t(8;21)-AML also expressed CD79a and/or CD19 (major transcriptional targets of PAX5 in B-cells) in 10 of 12 evaluable cases. Our results indicate that PAX5 is a more specific marker than CD79a for B-cell ALL diagnosis. Moreover, among AMLs, PAX5 expression selectively clusters with t(8;21), allowing its immunohistochemical recognition in a proportion of cases, and likely explaining a peculiar biological feature of this subset of myeloid leukemias, i.e. the aberrant expression of B-cell genes.

Antibodies, Monoclonal↗
PubMed2004

Gemtuzumab ozogamicin (Mylotarg) as a single agent for molecularly relapsed acute promyelocytic leukemia.

The anti-CD33 antibody calicheamicinconjugate gemtuzumab ozogamicin (GO) was used to treat 16 patients with acute promyelocytic leukemia (APL) who had relapsed at the molecular level. Of these patients, 8 were experiencing a first, 5 a second, 2 a third, and 1 a fourth relapse. GO was administered at 6 mg/m2 for 2 doses, and patients achieving a new molecular remission (MR) (ie, negativity of the reverse transcriptase-polymerase chain reaction [RT-PCR] test for PML/RARalpha) received a third dose. MR was obtained in 9 (91%) of 11 patients tested after 2 doses and in 13 (100%) of 13 patients tested after the third dose. Of the 3 remaining patients, 1 achieved MR after one GO administration and received no further therapy owing to hepatic toxicity, and 2 showed disease progression during treatment. Quantitative RT-PCR studies showed that responding patients experienced a dramatic decline (at least 2 logs) of the PML/RARalpha transcript after the first GO dose. Of 14 responders, 7 remained in sustained MR for a median of 15 months (range, 7-31 months) while 7 experienced relapse at 3 to 15 months. GO was administered again in 2 patients with relapse, and both obtained a new MR. These data indicate that GO is highly effective as a single treatment for patients with molecularly relapsed APL including those with very advanced disease.

Adolescent↗
PubMed2004

Immunophenotypic features of acute myeloid leukemias overexpressing the interleukin 3 receptor alpha chain.

In a previous study we have shown that the interleukin-3 receptor alpha chain (IL-3Ralpha) is over expressed in about 45% of acute myeloid leukemias (AMLs) and this phenomenon was associated with high blast cell counts at diagnosis, high rate of cycling of leukemic blasts and with a worse prognosis. Here we have investigated the immunophenotypic features of 125 AML patients subdivided into three groups (IL-3R(high), IL-3R(middle) and IL-3R(low)) according to the level of IL-3Ralpha expression. AMLs over expressing the IL-3Ralpha represent a subgroup of AMLs with a peculiar immunophenotype mainly consisting in the elevated expression of CD34 and several receptor membrane tyrosine kinases, such as c-kit and flt3, and in a usually low expression of myeloid-associated antigens such as CD11b, CD14 and CD15. These findings suggest that IL-3Ralpha + + + AMLs are blocked at an early stage of differentiation and express at elevated levels several growth factor receptors. It is proposed that these findings may further help to understand the mechanisms involved in the development of high-risk acute leukemias.

Acute Disease↗