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F Pane

Publications and source records attributed to F Pane.

At least 37 records · Page 2Linked to original sources
PubMed1999

BCR/ABL mRNA and the P210(BCR/ABL) protein are downmodulated by interferon-alpha in chronic myeloid leukemia patients.

The BCR/ABL hybrid gene plays a central role in the pathogenesis of the chronic phase of chronic myeloid leukemia (CML). We used a very sensitive quantitative reverse transcriptase-polymerase chain reaction to investigate the levels of hybrid BCR/ABL mRNA in bone marrow cells of 20 patients with Philadelphia positive (Ph(+)) CML treated with interferon-alpha (IFN-alpha) as a single agent. Bone marrow samples were collected at diagnosis and at hematologic remission induced by IFN-alpha, or by hydroxyurea in case of resistance to IFN-alpha. The mean levels of BCR/ABL transcripts in bone marrow mononuclear cells of patients who showed a complete hematologic response to IFN-alpha were significantly reduced with respect to those at diagnosis (48 x 10(3) v 168 x 10(3); P <.001), whereas no difference was detected between the values at diagnosis and at hematologic remission in patients resistant to IFN-alpha. In cell culture experiments, IFN-alpha priming significantly reduced the levels of BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone marrow precursors obtained at diagnosis from patients who subsequently responded to IFN-alpha treatment (P < .005). No downmodulation was observed in bone marrow precursors from patients who subsequently proved to be IFN-resistant. These results indicate that downmodulation of BCR/ABL gene expression could be one of the mechanisms involved in the response of CML patients to IFN-alpha treatment.

Antineoplastic Agents↗
PubMed1999

Acute promyelocytic leukemia after treatment for non-Hodgkin's lymphoma with drugs targeting topoisomerase II.

We report a patient who developed acute promyelocytic leukemia (APL) concomitantly with a second relapse of non-Hodgkin's lymphoma (NHL), intermediate grade, WF type E. At diagnosis and at first NHL relapse, the patient had received the same chemotherapy regimen, which included drugs targeting DNA topoisomerase II, i.e., etoposide (total dose 5,760 mg) and idarubicin (total dose 180 mg). Thirty-eight months after initial treatment, the patient showed pancytopenia associated with lymphoma recurrence. Bone marrow examination revealed the presence of atypical promyelocytes with Auer rods; cytogenetics showed t(15;17), and molecular analysis detected promyelocytic leukemia-retinoic acid receptor alpha rearrangement. APL reached complete remission after all trans retinoic acid therapy, whereas NHL did not respond to further chemotherapy. In the literature, five other patients developed APL after treatment for lymphoma, from a total of 59 patients developing sAPL after treatment for any type of neoplasia.

Antibiotics, Antineoplastic↗
PubMed1998

Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP multicenter "AIDA" trial. GIMEMA-AIEOP Multicenter "AIDA" Trial.

Although the majority of patients with acute promyelocytic leukemia (APL) are potentially cured by treatments combining all-trans retinoic acid (ATRA) and chemotherapy (CHT), a sizable proportion (around 30%) will relapse during follow-up. Retrospective molecular monitoring studies using reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific PML/RARalpha fusion gene, have shown that a positive test usually precedes the occurrence of hematologic relapse. Prospective RT-PCR analyses were performed since 1993 at diagnosis and at preestablished time intervals during follow-up in bone marrow (BM) samples of 163 patients with PML/RARalpha+ APL enrolled in the multicenter Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial AIDA (All-trans retinoic acid plus Idarubicin). Treatment consisted of ATRA and idarubicin for induction followed by three polychemotherapy courses as consolidation. The sensitivity level of the RT-PCR assay for PML/RARalpha, as assessed by serial dilution experiments, was 10(-4). All patients were in hematologic remission and tested PCR- at the end of consolidation. Of 21 who converted to PCR-positive thereafter, 20 underwent hematologic relapse at a median time of 3 months (range, 1 to 14) from the first PCR+ result. Seventeen of these 21 (81%) PCR+ conversions were recorded within the first 6 months postconsolidation. Of 142 who tested persistently PCR- in >/=2 tests after consolidation, 8 had hematologic relapse and 134 remained in complete remission (CR) after a median follow-up of 18 months (range, 6 to 38) postconsolidation. Using a time-dependent Cox model, the relative risk of hematologic relapse of patients who converted to PCR+ was 31.8 (confidence limits 95%, 12.9 to 78.3). Our results indicate that conversion to PCR positivity for PML/RARalpha during remission is highly predictive of subsequent hematologic relapse and highlight the prognostic value of stringent molecular monitoring during the early postconsolidation phase in APL. As a result of the present study, salvage treatment in patients enrolled in the GIMEMA trial AIDA is now anticipated at the time of molecular relapse, defined as the conversion to PCR positivity in two successive BM samplings during follow-up.

Adolescent↗
PubMed1998

Fas-mediated modulation of Bcr/Abl in chronic myelogenous leukemia results in differential effects on apoptosis.

Fas-R is expressed constitutively in CD34(+) cells of patients with chronic myelogenous leukemia (CML); Fas-R triggering results in decreased proliferation rate due to apoptosis of clonogenic cells. We have already shown that alpha-interferon (IFN-alpha) enhances Fas-R expression on CML progenitor cells, thus increasing their sensitivity to Fas-R agonists. Although it appears that IFN-alpha can prime CML cells for the effects of Fas, the response to IFN-alpha in vivo is not a constant feature in CML patients. We studied the mechanisms of Fas-mediated apoptosis in 11 patients suffering from CML in chronic phase and tried to see whether there was a correlation between in vitro inducibility of apoptosis in CD34(+) CML cells after Fas-R triggering and the clinical response to IFN-alpha. After priming with IFN-alpha, Fas triggering resulted in in vitro suppression of hematopoietic cell growth in seven of eight patients who had optimal hematologic response to IFN-alpha; in the same conditions, no inhibitory response to Fas-R agonist was observed in cells from three of three patients who proved to be poor responders to IFN-alpha. In responders to IFN-alpha, Fas-R agonist induced dose-dependent apoptosis of CD34(+) cells; this effect was associated with a decrease in the bcr/abl protein level. In cells derived from patients with a poor response to IFN-alpha, the rate of apoptosis in culture remained unchanged in the presence of Fas-R agonist and no bcr/abl downmodulation was observed. Finally, we measured bcr/abl mRNA by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and found that decreased bcr/abl protein after Fas triggering was not associated with decreased amounts of specific mRNA, a finding which is consistent with a posttranscriptional regulation of the bcr/abl protein expression. It appears that Fas-mediated downmodulation of p210 bcr/abl restores susceptibility to apoptosis of CML cells; in addition, in vitro studies on CML cells may predict response to IFN-alpha treatment.

Adult↗
PubMed1998

Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis.

BACKGROUND AND OBJECTIVE: Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis). DESIGN AND METHODS: The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined). RESULTS: CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and > 1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score < 0.8 and > 1.2, respectively; p = 0.003). INTERPRETATION AND CONCLUSIONS: Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.

Adolescent↗
PubMed1997

JURL-MK1 (c-kit(high)/CD30-/CD40-) and JURL-MK2 (c-kit(low)/CD30+/CD40+) cell lines: 'two-sided' model for investigating leukemic megakaryocytopoiesis.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.

Antigens, Surface↗
PubMed1997

BCR/ABL transcripts and leukemia phenotype: an unsolved puzzle.

The Philadelphia chromosome, arising as a consequence of the t(9;22) translocation, is one of the most frequent and certainly the most known cytogenetic abnormality present in human hematological malignancies. Unlike the vast majority of the other translocations, its presence is not restricted to a specific leukemia phenotype, but is found associated with chronic myelogenous leukemia as well as with a large percentage of acute lymphoblastic leukemias, particularly in elderly patients. Although its molecular counterpart is always represented by a rearrangement between the BCR and the ABL genes, this shows a certain degree of molecular variability. The pathogenetic relationship with the different leukemia phenotypes which have been found to be associated still awaits to be fully elucidated. However, a number of old and more recent observations seem to suggest that not only qualitative differences in the type of BCR/ABL proteins expressed, but also quantitative variations in their total level within the cells may have an important role in determining the leukemia phenotype.

Animals↗
PubMed1996

Neutrophilic-chronic myeloid leukemia: a distinct disease with a specific molecular marker (BCR/ABL with C3/A2 junction)

Neutrophilic-chronic myeloid leukemia (CML-N) is a rare myeloproliferative disorder that runs a much more benign course than chronic myeloid leukemia, and for which no specific underlying molecular lesion has been described so far. We have analyzed the genomic DNA by Southern blotting and the BCR/ABL hybrid gene transcripts by reverse transcriptase-polymerase chain reaction in three patients with clinical findings of CML-N, who did have a t(9;22) chromosomal translocation. In all patients we have found a rare type of BCR/ABL rearrangement, with a breakpoint between exons c3 and c4 of the BCR gene (corresponding to BCR exons 19 and 20). This was confirmed by hybridization with an oligonucleotide probe spanning the c3/a2 region. This type of junction causes almost the entire BCR gene to fuse with ABL. The junction is in frame and it gives rise to a fusion protein of predicted 230 kD. Our data now provide a molecular diagnostic marker for CML-N, and they are consistent with the notion that the inclusion or exclusion of BCR exons in the fusion protein affects dramatically its capacity to derange myeloid proliferation and differentiation, leading to the appearance of different disease phenotypes.

Adult↗
PubMed1996

Consistent amounts of acute leukemia-associated P190BCR/ABL transcripts are expressed by chronic myelogenous leukemia patients at diagnosis.

In chronic myelogenous leukemia (CML), the Philadelphia (Ph) chromosome translocation results in the formation of BCR/ABL genes, normally transcribed in two types of hybrid transcripts with a b2a2 or b3a2 BCR/ABL junction, which give origin to 210-kD fusion proteins (P210). A third type of BCR/ABL (with e1a2 type of junction) has been identified in approximately 50% of the Ph-positive acute lymphoblastic leukemia (Ph+ALL) cases and results in the production of a BCR/ABL protein of 190 kD (P190). The presence of this transcript has been associated almost exclusively with the presence of an acute leukemia phenotype. By contrast, here we describe that in addition to transcripts with the b2a2 and b3a2 types of junction corresponding to the P210 proteins, virtually all CMLs at diagnosis bear also BCR/ABL transcripts showing the e1a2 type of junction, which correspond to the acute leukemia-associated P190 protein. With a quantitative polymerase chain reaction assay we found that the amount of the e1a2 mRNA present in CMLs in chronic phase, although in absolute amount much lower than that present in Ph+ ALLs, represents in most cases approximately 20% to 30% of the total BCR/ABL transcripts. Moreover, using a novel and very sensitive Western blot technique, we detected relevant amounts of P190 protein in addition to P210 from peripheral cells of two of the patients.

Base Sequence↗
PubMed1996

All-trans-retinoic acid (ATRA) responsive skin relapses of acute promyelocytic leukaemia followed by ATRA-induced pseudotumour cerebri.

A 30-year-old woman with acute promyelocytic leukaemia (APL) went into complete remission following idarubicin and cytarabine chemotherapy; 18 months later she developed repeated skin relapse, with no bone marrow involvement. DNA and RNA analysis of skin lesions revealed the presence of the PML/RAR alpha hybrid gene, which was not detected at the same time in bone marrow. The skin relapses were successfully treated by all-trans-retinoic acid (ATRA) as single agent over 2 years. However, prolonged administration of ATRA caused pseudotumour cerebri, which disappeared upon drug withdrawal. The absence of the hybrid gene in the bone marrow by RT-PCR analysis led to the patient being autografted.

Adult↗
PubMed1996

Pseudouridine and 1-ribosylpyridin-4-one-3-carboxamide (PCNR) serum concentrations in human immunodeficiency virus type 1-infected patients are independent predictors for AIDS progression.

A prospective study was conducted with 161 human immunodeficiency virus (HIV)-positive patients to investigate the prognostic role of 10 serum-modified nucleosides with regard to some of the most widely used parameters of AIDS progression. Serum concentrations of pseudouridine (> 3.77 nmol/mL) predicted progression to AIDS in CDC stage A2 HIV-infected patients much better than did other widely used parameters (hazard ratio, 2.7; 95% confidence interval, 1.29-6.35; P = .01; median permanence time in stage A2, 17 vs. 30.5 months; P = .03). Serum concentrations of 1-ribosylpyridin-4-one-3-carboxamide (PCNR) and beta 2-microglobulin and the CD4:CD8 cell ratio, in decreasing order and used in combination, differentiated the overall survival time probability of AIDS patients; PCNR was the best and a new independent predictor (overall survival time, > 31 months, no positive parameters; 19.3 months, one positive parameter; and 5.5 months, two positive parameters.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Simultaneous occurrence of tetrasomy 21 and trisomy 8 in a patient with early blastic metamorphosis of chronic myeloproliferative disorder.

We report an unusual hyperdiploid karyotype characterized by the simultaneous occurrence of tetrasomy 21 and trisomy 8 detected during early blastic evolution of a BCR-ABL-negative chronic myeloproliferative disorder. Blast cells from this patient showed a striking response to all-trans-retinoic acid (ATRA)-induced differentiation as evaluated by CD15 expression following in vitro exposure to this inducer. Our report represents the first description of such a composite karyotype in human hematologic malignancies.

Aged↗
PubMed1995

Direct detection of proviral gag segment of human immunodeficiency virus in peripheral blood lymphocytes by colorimetric PCR assay as a clinical laboratory tool applied to different at-risk populations.

We used a colorimetric polymerase chain reaction (PCR)-based assay in kit form to detect directly human immunodeficiency virus type 1 (HIV-1) proviral gag sequences in peripheral blood cells from 68 healthy blood donors, 51 subjects at risk for HIV infection, 122 patients with HIV-1 infection, 11 patients with indeterminate Western blot (immunoblot) results, 4 blood donors HIV-1 positive by enzyme immunoassay, and 13 children born to HIV-1-seropositive mothers. The results obtained in the blood donors and HIV-1-infected patients demonstrated the high degree of diagnostic specificity and sensitivity of the PCR method. HIV-1 infection was excluded in 10 of the 11 patients with indeterminate Western blot results and in all four enzyme immunoassay-positive blood donors. A diagnosis of HIV infection was ruled out by negative PCR results in 5 of 13 children from seropositive mothers, which excluded vertical transmission of the infection in these cases; these children were younger than 3 months and had positive serological results. Two at-risk patients with negative serological results had positive PCR results. All results were confirmed by conventional PCR. In conclusion, colorimetric PCR, which is commercially available in kit form, is an easy and reliable technique that can be used to detect proviral HIV-1 genomes in blood cells, and despite the limitations owing to HIV genome variability, it is useful in the clinical setting for the diagnosis of HIV infection in selected categories of patients.

Blotting, Western↗
PubMed1993

Serum pseudouridine in the diagnosis of acute leukaemias and as a novel prognostic indicator in acute lymphoblastic leukaemia.

The serum level of pseudouridine, a modified nucleoside deriving mainly from t-RNA catabolism, was evaluated in 66 acute leukaemia patients at diagnosis to investigate its diagnostic and prognostic value, and its potential as a parameter with which to classify subtypes of the disease. Serum pseudouridine, measured by high performance liquid chromatography, was increased in acute lymphoblastic leukaemia patients (90% according to the pseudouridine index, which is the serum pseudouridine/creatinine ratio), and in acute myeloblastic leukaemia patients (75% according to the pseudouridine index). The increase was higher in the L3 than in the L1 and L2 subtypes. In the acute lymphoblastic leukaemia group there was a highly significant inverse correlation between serum pseudouridine levels and the most common end-point parameters used to assess disease outcome in leukaemia (i.e., complete remission rate, disease-free survival, and overall survival). In addition, 83% of patients with serum pseudouridine values < 5.5 nmol/mL were alive and in complete remission 12 months after the initial diagnosis, while only 11% of patients with serum pseudouridine values > 5.5 nmol/mL were alive and none were disease-free after the same period. This study: 1. demonstrates that the diagnostic sensitivity of the pseudouridine index is high in adult acute lymphoblastic leukaemia and good in acute myeloblastic leukaemia; 2. suggests that the serum pseudouridine assay can contribute to the classification of adult acute lymphoblastic leukaemia; and 3. demonstrates unequivocally that both pseudouridine assay and the pseudouridine index are excellent independent prognostic markers for acute lymphoblastic leukaemia.

Adolescent↗