Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia.

Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.

Low dose Velcade, thalidomide and dexamethasone (LD-VTD): an effective regimen for relapsed and refractory multiple myeloma patients.

From January 2004, R/R MM cases referred to the Institution received LD-VTD regimen. Patients, irrespective of age, PS and life expectancy, were enrolled in the study once they had a measurable disease. Planned therapy: Velcade 1.0 mg m(-2) i.v. twice weekly for 2 weeks of a 28-day cycle for up to 6 cycles, oral Dexamethasone 24 mg on the day of and the day following each Velcade dose and Thalidomide 100 mg each evening. DVT prophylaxis with warfarin to maintain international normalized ratio between 2.0-3.0 was planned in all patients. As of 1 June 2005, 18 were the treated patients: median age 63 years, median time from diagnosis 5.8 years, a median of 4 previous therapy lines. Seventeen were the valuable patients and 9 (53%) were the responders: 2 CR, 6 PR, 1 MR. Six were the stable disease and 2 the progressive ones. Median time to best response was 2 months. Toxicity was negligible. No case of DVT was recorded. Except for the first cycle, subsequent cycles were delivered on an outpatient basis. After a median follow-up of 11 months, 12 patients were alive and 5 died (3 disease progression, 1 heart failure, 1 intestinal bleeding). Thus, the LD-VTD regimen applied appears feasible and effective in elderly and heavily pre-treated R/R myeloma patients.

Internal tandem duplications of Flt3 gene (Flt3/ITD) predicts a poor post-remission outcome in adult patients with acute non-promyelocytic leukemia.

Despite progress in AML therapy, most patients eventually relapse, even the ones with normal or favorable karyotype. Since survival is poor once relapse occurs, new genetic tools above karyotype at diagnosis are needed to predict leukemia free survival. Recently, Flt3/ITD has been reported as an independent marker for clinical outcome in most studies concerning adult AML patients. To assess the prognostic relevance of activating mutations of Flt3, pretreatment samples of 100 not-M3 AML patients, all of them subjected to an intensive chemotherapy regimen, were analyzed for Flt3/ITD; 25/100 patients had one or more Flt3-ITD. Flt3/ITD patients had higher WBC count (P = 0.005), a lower incidence of a preceding MDS (P = 0.004) and most of them had a normal karyotype. Flt3/ITD had no impact on CR achievement while karyotype remained the most powerful prognostic factor (HR 2.8 95% CI 1.2 6.3). However, post-remission outcome was significantly worsened by the presence of Flt3/ITD. Median RFS of the Flt3/ITD patients was 5 vs. 27 months compared to the patients with wild-type Flt3 (P = 0.0002); moreover, Flt3/ITD patients had a significantly poorer post-remission survival (11 vs. 38 months, P = 0.01). On multivariate analysis, the presence of Flt3-ITD significantly affected relapse free survival and post-remission survival (HR 3.1 and 2.1, respectively). Thus, post-remission outcome highly depends on Flt3 status. Flt3 mutations identify patients at high risk of relapse, who should prospectively receive, according to age, either more aggressive or alternative therapeutic approaches.

Incidence and management of proven and probable fungal infections in patients with acute leukemia: a single center experience.

The incidence of fungal infections and the role of liposomal amphotericin B (Ambisome) in proven and probable infections were evaluated in acute leukemic patients, intolerant to conventional amphotericin B. During 1999-2002, 307 febrile episodes occurred in 231 patients. Fungi were responsible for 3% of bloodstream infections. Ambisome was employed in 5 fungal sepsis (1 Candida albicans, 1 C. famata, 1 C. tropicalis, 1 C. krusei, 1 Geotrichum capitatum) 2 Aspergillosis, 2 probable fungal pneumonia cases. A favorable response was achieved in 78% of patients (4 fungemia, 2 aspergillosis, 1 probable), an unfavorable response in 1 C. krusei fungemia and in 1 probable pneumonia. Our antimicrobial pattern documented a high resistance rate to azoles. We concluded that Ambisome is an effective and well tolerated agent and its introduction has changed the outcome for many patients, although in some refractory diseases other strategies must be considered.

The prognostic value of cytogenetics is reinforced by the kind of induction/consolidation therapy in influencing the outcome of acute myeloid leukemia--analysis of 848 patients.

We studied the impact of cytogenetics and kind of induction/consolidation therapy on 848 adult acute myeloid leukemia (AML) patients (age 15-83). The patients received three types of induction/consolidation regimen: standard (daunorubicin and cytosine arabinoside (3/7); two cycles); intensive (idarubicin, cytosine arabinoside and etoposide (ICE), plus mitoxantrone and intermediate-dose Ara-C (NOVIA)); and low-dose (low-dose cytosine arabinoside). CR patients under 60 years of age, if an HLA-identical donor was available received allogeneic stem cell transplantation (allo-SCT); otherwise, as part of the program, they underwent autologous (auto)-SCT. CR rates significantly associated with 'favorable' (inv(16), t(8;21)), 'intermediate' ('no abnormality', abn(11q23), +8, del(7q)) and 'unfavorable' (del (5q), -7, abn(3)(q21q26), t(6;9), 'complex' (more than three unrelated cytogenetic abnormalities)) karyotypes (88% vs 65% vs 36%, respectively; P = 0.0001). These trends were confirmed in all age groups. On therapeutic grounds, intensive induction did not determine significant increases of CR rates in any of the considered groups, with respect to standard induction. Low-dose induction was associated with significantly lower CR rates. Considering disease-free survival (DFS), multivariate analysis of the factors examined (including karyotype grouping) showed that only age > 60 years significantly affected outcome. However, in cases where intensive induction was adopted, 'favorable' karyotype was significantly related to longer DFS (P = 0.04). This was mainly due to the favorable outcome of t(8;21) patients treated with intensive induction. Patients receiving allo-SCT had significantly longer DFS (P = 0.005); in particular, allo-SCT significantly improved DFS in the 'favorable' and 'intermediate' groups (P = 0.04 and P = 0.048, respectively). In conclusion our study could provide some guidelines for AML therapy: (1) patients in the 'favorable' karyotype group seem to have a longer DFS when treated with an intensive induction/consolidation regimen, adopted before auto-SCT instead of standard induction; this underlines the importance of reinforcement of chemotherapy, not necessarily based on repeated high-dose AraC cycles. Allo-SCT, independently of induction/consolidation therapy, should be considered an alternative treatment; (2) patients in the 'intermediate' karyotype group should receive allo-SCT; (3) patients in the 'unfavorable' karyotype group should be treated using investigational chemotherapy, considering that even allo-SCT cannot provide a significantly longer DFS, but only a trend to a better prognosis.

Amifostine in the treatment of low-risk myelodysplastic syndromes.

BACKGROUND AND OBJECTIVE: The phosphorylated aminothiol agent amifostine (Ethyol) protects bone marrow and other tissues from toxicity due to ionizing radiation and antineoplastic drugs, and stimulates progenitors from normal and myelodysplastic bone marrow. Contrasting results have been published so far on the effectiveness of amifostine in correcting cytopenia in patients with myelodysplastic syndromes (MDS). DESIGN AND METHODS: In a pilot phase II study we treated 26 patients with low risk MDS (13 RA, 2 RARS, 2 CMML, 9 RAEB with blasts < 10%) with amifostine (200 mg/m(2 )x 3/week for 4 weeks). RESULTS: Hemoglobin concentration, reticulocyte, neutrophil and platelet counts increased respectively in 6 (23%), 11 (42%), 13 (50%) and 9 (34%) of patients. Red cell transfusions were reduced (> 50%) in 4/26 patients and abolished in 1/26. Unexpectedly a significant decrease in soluble transferrin receptor level at week 4 of therapy, compared to the basal level (p<0.04), was observed in the whole population of patients. INTERPRETATION AND CONCLUSIONS: Amifostine can ameliorate cytopenia in some patients with MDS, with few and mild side effects. Neutropenia is more likely to be corrected than anemia or thrombocytopenia. Mechanisms underlying this biological effect remain to be clarified.

Microdissection and FISH investigations in acute myeloid leukemia: a step forward to full identification of complex karyotypic changes.

Complex chromosomal rearrangements in malignant hemopathies frequently remain unclarified because of paucity of material for further fluorescence in situ hybridization analyses and/or lack of suitable probes. Chromosome microdissection (MD) can be an adequate approach to elucidate chromosome aberrations unrecognizable by conventional karyotyping. We applied MD in two patients with acute myeloid leukemia (AML) and unidentified chromosome changes at karyotype. Microdissection of a ring chromosome in an AML-M5 case revealed 21q polysomy. In an AML-M4 case, MD of an add(15p) disclosed a t(8;15) with over-representation of both 8q22 and 8q24 bands. YAC probes were helpful in showing duplication of the ETO gene at 8q22, and amplification of C-MYC, at 8q24.

Characterization of 12p molecular events outside ETV6 in complex karyotypes of acute myeloid malignancies.

Acute myeloid disorders with rearrangements of 12p outside the ETV6 gene were characterized by fluorescence in situ hybridization (FISH) with a panel of DNA probes. Seven patients with de novo acute myeloid leukaemia (AML), one with secondary acute myeloid leukaemia (sAML), and one in the blast phase of chronic myeloid leukaemia (CML-BP) were enrolled in the study. All AML cases showed multiple karyotypic changes. Chromosome 5 and/or 7 deletions were the most frequent accompanying changes. FISH revealed amplification, cryptic translocation, and fragmentation of chromosome 12, not discernible at karyotypic level. Different karyotypic rearrangements of 12p showed a common molecular event. Among the seven cases in which breakpoints could be determined, six were telomeric and one centromeric to ETV6. In three AML cases a new recurrent breakpoint in the telomeric region was identified distally to locus D12S158 and to pac 922B22 which is the most telomeric probe available for 12p. Accompanying cryptic deletions were also detected in five patients and the commonly deleted region, of around 700 kb, included the ETV6 gene and the D12S391 locus.

Fludarabine, cytarabine, and granulocyte-colony stimulating factor for the treatment of high risk myelodysplastic syndromes.

BACKGROUND: The prognosis of patients with high risk myelodysplastic syndromes (MDS) (i.e., refractory anemia with excess of blasts [RAEB] and refractory anemia with excess of blasts in transformation [RAEB-t]) usually is poor. The combination of fludarabine, cytarabine, and granulocyte-colony stimulating factor (G-CSF) (FLAG regimen) has been reported to be effective in patients with these diseases. METHODS: Forty-two patients (32 with RAEB-t and 10 with RAEB) were treated with the FLAG regimen. The median age was 61 years (range, 27-74 years). Forty patients were diagnosed with primary MDS and 2 patients had treatment-related MDS. Induction therapy was comprised of the FLAG regimen, whereas consolidation therapy included idarubicin and cytarabine. Patients with a compatible donor and who were age < 50 years were scheduled to undergo an allogeneic bone marrow transplantation (BMT), whereas for those patients without a donor and who were age < 60 years autologous BMT with peripheral blood stem cells mobilized by the consolidation regimen plus G-CSF was planned. RESULTS: Complete remission (CR) was achieved in 31 of 42 patients (74%; 95% confidence interval, 60-87%). Death during induction therapy occurred in 4 patients (9%) whereas 7 patients (17%) were resistant to the FLAG regimen. Toxicity from the consolidation regimen was negligible. All patients age < 50 years and achieving CR were eligible for allogeneic BMT procedures, with early recurrence being the only reason for exclusion. The median overall survival and disease free survival were 13 months and 18 months, respectively. Patients with favorable cytogenetics had a significantly better outcome compared with those patients with an adverse karyotype. CONCLUSIONS: The FLAG regimen is effective in patients with high risk MDS as well as in patients age > 60 years. The toxicity of the regimen is low and the majority of patients are eligible to undergo allogeneic BMT procedures after induction/consolidation therapy.

Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias.

There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT-PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1-M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for alpha-globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo-R) and thrombopoietin receptor (c-mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB-subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.

Idarubicin in induction treatment of acute myeloid leukemia in the elderly.

BACKGROUND AND OBJECTIVE: The best approach to treatment of acute myeloid leukemia (AML) in elderly patients remains controversial. Intensive chemotherapy is the treatment of choice in selected patients, but age related changes might affect the pharmacokinetics of antineoplastic agents resulting in enhanced toxicity. We report our experience in elderly patients treated with idarubicin at attenuated doses plus cytarabine and etoposide. METHODS: Sixty-six AML patients, median age 66, with progressive disease and high tumor burden received idarubicin 8 mg/sqm i.v. d 1,3,5; cytarabine 200 mg/sqm by continuous i.v. infusion d 1-7; etoposide 60 mg/sqm i.v. d 1-5. A second course with the same drugs was planned irrespective of complete remission (CR) achievement. No consolidation was given; 44% had a documented preexisting myelodysplasia, 45% had a documented preexisting myelodysplasia, 45% presented with fever. Promyelocytic leukemias were excluded. RESULTS: Thirty-five patients (53%) achieved CR and 9 PR for an overall response rate of 67%. Nine of them (13%) died early or during the aplastic phase. Preexisting myelodysplasia had no significant impact on CR achievement. Resistant disease was associated with CD7 phenotype and unfavorable karyotype. Overall survival and disease free survival were 14 and 13 months, respectively. The major toxicity consisted of infectious complications (WHO > 2 in 24% of patients). Six patients died for infection, 2 for heart failure, 1 for pulmonary embolism. INTERPRETATION AND CONCLUSIONS: This induction regimen with attenuated doses of idarubicin is feasible and effective, but long-term survival remains an unresolved problem. Alternative post remission approaches are advisable in the aim of improving the remission duration.

Low-dose cytosine arabinoside in patients with acute myeloid leukemia not eligible for standard chemotherapy.

The results of treatment with low dose cytosine arabinoside (LDARA-C) in 131 AML patients ineligible for standard regimens were analyzed retrospectively. Eighty-seven were previously untreated, 25 were refractory to conventional chemotherapy and 19 were relapsed patients. The median age was 66 years (15-84). An antecedent hematological disorder (AHD) was documented in half of the patients. Overall, 22 (17%) achieved complete remission, 14 (11%) partial remission, 77 (59%) had resistant leukemia and 18 died during induction. Median disease free survival was 57 weeks and median survival, for the 87 previously untreated patients, was 22.5 weeks. The prognostic value of initial parameters was analyzed for response. Bone marrow cellularity was the only significant factor. We observed 33% vs 81% (p < 0.01) of responses in patients with normo-hypercellular and hypocellular marrow, respectively. Accordingly, there was a trend to more responses in patients with leukocyte counts of less than 10 x 10(9)/L. M4-M5 FAB subtypes were frequently resistant to LDARA-C, resulting in a lower response rate compared to M0-M2 (18% vs 32%). Other parameters, including age, sex, hemoglobin, platelet count, AHD and fever at diagnosis, had no prognostic value. Our findings suggest that LDARA-C may be an effective treatment for some patients who are not eligible for first line conventional chemotherapy. However, this schedule is not advised in patients with monocytic leukemia or those with an hypercellular marrow.

Attenuated-dose idarubicin in acute myeloid leukaemia of the elderly: pharmacokinetic study and clinical results.

AML in the elderly is characterized by intrinsic biological features implying an enhanced chemoresistance. Intensive chemotherapy should be the treatment of choice, but the standard doses could induce unacceptable rates of aplastic deaths. We evaluated the efficacy of an induction protocol with attenuated-dose idarubicin (IDA) 8 mg/m2 for 3 d plus cytarabine and etoposide in 26 AML patients aged > 60. 18 patients (69%) achieved CR, five (19%) were non-responders and three (12%) died during induction. To compare the pharmacokinetics of IDA between elderly and young patients, we assayed daily the serum level of the drug and of its metabolite (idarubicinol, IDAol) in a group of eight elderly patients who received a dose of 8 mg/m2 (group A) and in a group of nine younger AML patients treated with 12 mg/m2 (group B). The apparent terminal half-life of IDAol was significantly longer in the elderly than in the younger patients (mean half-life 59.7 h versus 41.4 h, P < 0.05). The values of the area under the serum concentration curve of IDAol indicated that the two patient groups received a very similar exposure to the drug despite the different doses. In conclusion, this protocol, based on attenuated doses of IDA, compares well with the results obtained previously in similar age-matched patient series.

Mixed acute leukemia with genotypic lineage switch: a case report.

Morphologically well classifiable leukemias can reveal a mixed phenotype. A case of acute myeloid leukemia (CD13, CD33, CD14, CD11b) which at presentation showed a co-expression of B-lymphoid markers (CD19, CD10, CD20), at the time of the first relapse revealed a morphologic, phenotypic and genotypic switch of the blasts to a purely lymphoid form. Analysis of the immunoglobulin (Ig) H chain locus and of the T-cell receptor (TCR) genes showed at diagnosis a germline configuration of the IgH, TCR beta and tau genes, and a deletion of the TCR delta gene at the second chromosome. At relapse, monoclonal rearrangements of the IgH, TCR tau, and TCR delta were detected. At a subsequent relapse, the blasts re-expressed myeloid morphologic features and myeloid-associated antigens, while they retained the same rearranged configuration of the IgH and TCR beta and delta genes. The TCR delta gene configuration, which links each phase of the disease, may represent an early pathogenetic event and makes the emergence of a second malignancy unlikely. Each phenotypic change occurred after anti-myeloid and anti-lymphoid oriented chemotherapy. The close correlation between the progressive acquisition of different phenotypes and the switch at the genomic level represent the peculiar features of this unusual case.

CD34 expression fails to predict the outcome in adult acute myeloid leukemia.

BACKGROUND: The expression of the CD34 antigen on the blast cells of acute myeloid leukemia (AML) has been regarded as an unfavorable prognostic factor for the achievement of complete remission (CR). However, clinical reports on this issue still remain controversial. We evaluated the relationship between CD34 expression, some clinical characteristics and outcome in 80 consecutive adult AML patients. METHODS: Immunophenotyping was performed with a FACSCAN flow cytometer and CD34 was tested by HPCA-1 (My10, Becton-Dickinson). Samples were considered positive when at least 20% of cells were labeled. Promyelocytic leukemias were excluded from the study. Sixty-six patients were designed to receive intensive induction chemotherapy; 14 "low-dose" ARA-c. RESULTS AND CONCLUSIONS: Forty percent of AML were CD34 positive. In this group there was a higher incidence of less differentiated FAB subtypes (p = 0.03), but not of pre-existing myelodysplasia. No differences were found in complete remission (CR) rate, remission duration or survival. Excluding induction deaths from the analysis, the CR rate was slightly lower in CD34+ AML (55 vs 65%), without any impact on survival. We could not confirm the prognostic relevance of CD34 in adult AML, but larger studies are needed.