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.

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.

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.

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.

Continuous-infusion cyclophosphamide in combination with teniposide and dexamethasone in refractory myeloma.

Forty-three consecutive patients with refractory myeloma, median age 60, received monthly courses of teniposide 30 mg/m2 i.v. on days 1-2, dexamethasone 40 mg i.v. on days 1-7 and cyclophosphamide 200 mg/m2 by continuous i.v. infusion for seven days. Major response (decrease > 50% of M-protein) was achieved in 18 of 37 evaluable patients and minor response in 9, with an overall response rate of 73%. Response was irrespective of disease status, time from diagnosis and previous treatments, while beta 2 microglobulin > 6 mg/l was a powerful prognostic factor. All patients experienced transient granulocytopenia but extramedullary toxicity was negligible. Median survival of the whole group is 20 months, with 74% of responding patients projected to be alive at 30 months. In refractory myeloma cyclophosphamide appears to be more active when given by continuous infusion.

Immunophenotype of acute lymphoblastic leukemia cells: the experience of the Italian Cooperative Group (Gimema).

The immunophenotype of 304 adult lymphoblastic leukemias (> 18 years) diagnosed on the basis of the FAB criteria was determined at the time of diagnosis using a panel of monoclonal antibodies. The series comprised cases diagnosed and immunophenotyped in 43 Italian centers (GIMEMA Cooperative Group) between April 1988 and June 1991. The immunophenotypic characterization consisted of two consecutive steps. The initial screening was based on the reactivity for TdT, HLA-Dr, CD7, CD10, CD13, CD19, CD24, CD33 and CD41. According to the results obtained, the second level of investigation assessed the positivity for intra cytoplasmic (Cy) Ig, CD1a, CD2, CD3, CD4, CD5, CD8 and CD20. Based on the hierarchical expression of the different B- and T-cell related antigens, each case was assigned to a given differentiation stage. B-lineage ALL were classified in five subgroups (B0-B4) and T-lineage ALL in four subgroups (T0-T3). Cases in which the blasts were lymphoid according to the FAB criteria, but expressed myeloid antigens in association with B- and T-lymphoid markers were defined as hybrid leukemias. As expected, CD10+ cases (B2-B3) were the most frequent within the B-lineage ALL (83.2% of cases). CyIg+ (B3) accounted for about 20% of CD10+ ALL. Twenty eight cases (13.4%) were at a pre-cALL stage (B0-B1) and of these, 8 (3.8% of the total series) were positive only for TdT and HLA-Dr (B0). Intermediate and mature thymic phenotypes (T2-T3) were predominant within the T-ALL (67.2%) groups. Five cases, were positive only for TdT and CD7 (CD5+), and classified as T0. 9.2% of cases fulfilled the definition of hybrid leukemia, largely in view of the co-expression of B-lymphoid and myeloid markers.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Chemoprophylaxis of bacterial infections in granulocytopenic patients with ciprofloxacin vs ciprofloxacin plus amoxicillin.

Ciprofloxacin was compared to ciprofloxacin plus amoxicillin as antibacterial prophylaxis in 53 evaluable patients with neutropenic episodes, because an oral penicillin may help to decrease the incidence of gram-positive infections. The two groups were randomized and evaluated in a number of febrile episodes, in days at fever/at risk, in mean interval of first febrile episode, in duration of antibiotic therapy and in causative organisms in febrile episodes. In conclusion, no significant difference was observed between the two groups in prevention of gram-positive bacteremias.

Ceftriaxone plus conventional or single-daily dose amikacin versus ceftazidime/amikacin as empiric therapy in febrile neutropenic patients.

Results of antibiotic treatment in 144 febrile episodes during drug-induced granulocytopenia are reported. 63 episodes were treated with ceftazidime plus amikacin and 81 with ceftriaxone (CFX) using single daily doses combined with amikacin either divided in three doses (CFX-1 group) or administered as single doses (CFX-2 group). The response rates were 51, 80, and 57% respectively. In this study CFX was more effective than ceftazidime and amikacin provided better results when administered using the standard 8-hour scheduling.

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.

Teniposide, dexamethasone and continuous-infusion cyclophosphamide in advanced refractory myeloma.

Twenty-one patients with advanced refractory myeloma (nine primary unresponsive to melphalan, 12 relapsed and resistant to first-line salvage therapy) received monthly 7 d courses of cyclophosphamide by continuous infusion, associated with teniposide and dexamethasone. Six patients died within 2 months from start of treatment, before receiving an adequate trial. Among the 15 evaluable patients, there were eight objective responses and three improvements (decrease of monoclonal protein greater than 50% or greater than 30%, respectively). After a median follow-up of 10 months, all responding patients are alive and none of them is still relapsed. In heavily pretreated patients, ineligible for more aggressive approaches, cyclophosphamide by continuous-infusion exerted a good antineoplastic activity without increasing toxicity.

Continuous-infusion cyclophosphamide plus vincristine, cytosine-arabinoside and prednisone for refractory acute lymphoblastic leukemia in adults.

BACKGROUND: Despite more aggressive salvage regimens, the prognosis of refractory acute lymphoblastic leukemia (ALL) is still poor. Hence, alternative approaches are warranted in heavily pretreated patients. Some studies suggest the enhancement of cyclophosphamide (CY) activity when given by continuous infusion. METHODS: To evaluate the effectiveness of this scheduling in refractory ALL, we treated 15 adult patients (4 primary resistant, 11 relapsed and refractory to salvage regimens) with a seven-day course of CY 350 mg/m2/day by continuous i.v. infusion, associated with vincristine, cytosine-arabinoside and prednisone. RESULTS: The median time for hematologic recovery was 18 days, with negligible extramedullary toxicity. Two patients died while aplastic, 3 were non responders, 10 (66%) achieved complete remission after the first cycle. The response duration ranged from 5 to 32 (median 14) weeks, and the median survival of responders was 23 weeks. It is noteworthy that half of the responding patients had been resistant to prior CY in different schedules and other drug combinations. CONCLUSIONS: These data seem to confirm that CY exerts its best antineoplastic activity by continuous infusion.

13-cis-Retinoic acid treatment in patients with myelodysplastic syndrome.

Twenty patients with primary myelodysplastic syndromes (16 refractory anemia without or with ringed sideroblasts, 2 refractory anemia with excess blasts, 2 refractory anemia with excess blasts in transformation) received 13-cis-retinoic acid at a dosage of 50-100 mg/m2/day for a minimum of 4 weeks. Twelve patients obtained an increase of hemoglobin levels greater than 1 g/dl and 7 showed an associated increase of granulocyte count greater than 50% of baseline values. No significant biochemical signs of dyslipidemia or liver damage were noted. A sustained response was noted only in refractory anemia without or with ringed sideroblasts and normal or hypercellular bone marrow. Five patients are still on therapy from 23 to 82 weeks without transfusion requirement and all have shown an improvement in performance status. We conclude that 13-retinoic acid may only be clinically useful in selected patients since in myelodysplastic syndromes with blast excess the drug does not seem to improve the course of the disease.