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FLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD(+)ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient-specific real-time quantitative-PCR (RQ-PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild-type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient-specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1(+)ve/FLT3 ITD(-)ve at presentation, with shorter remissions being observed in four patients re-classified as FLT3 ITD(+)ve by the new assay. Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN-AML., (© 2013 John Wiley & Sons Ltd.)

The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20mg/m(2) on days 1-5 and temsirolimus 25mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3·5months, and median overall survival was 4months (9·1months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; P=0·0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen., (© 2011 Blackwell Publishing Ltd.)

Nucleophosmin gene (NPM1) mutations characterize acute myeloid leukaemia (AML) with normal karyotype and frequently co-exist with FLT3 internal tandem duplications (ITD). We evaluated bcl-2, bax, NPM1 and FLT3-ITD in 222 AML patients. Bax/bcl-2 ratio >0.35 and NPM1 without FLT3-ITD were significantly associated (P = 0.0001). NPM1-mutated (mt)/FLT3-ITD negative patients showed a higher complete remission (CR) rate (90%, P = 0.0002) and a longer overall survival (OS, P = 0.00007). NPM1-mt/FLT3-ITD negative plus bax/bcl-2 > 0.35 subset showed a very high CR rate (96%), very long OS (P = 0.00005) and disease-free survival (P = 0.004). The favourable prognosis of NPM1-mt/FLT3-ITD negative patients might be explained by a higher bax/bcl-2 ratio.

This study compared two schedules of low-dose gemtuzumab ozogamicin (GO) as induction monotherapy for untreated acute myeloid leukaemia in older patients unfit for intensive chemotherapy, to identify the more promising regimen for further study. Patients were randomized to receive either best supportive care or a course of GO according to one of two schedules: 3 mg/m(2) on days 1, 3 and 5 (arm A), or GO 6 mg/m(2) on day 1 and 3 mg/m(2) on day 8 (arm B). Primary endpoint was the rate of disease non-progression (DnP), defined as the proportion of patients either achieving a response or maintaining a stable disease following GO induction in each arm. Fifty-six patients were randomized in the two GO arms (A, n = 29; B, n = 27). The rate of DnP was 38% [90% confidence interval (CI), 23-55] in arm A, and 63% (90% CI, 45-78) in arm B. Peripheral cytopenias were the most common adverse events for both regimens. The all-cause early mortality rate was 14% in arm A and 11% in arm B. The day 1 + 8 schedule, which was associated with the highest rate of DnP, met the statistical criteria to be selected as the preferred regimen for phase III comparison with best supportive care.

This randomized phase III clinical trial explored the efficacy of DaunoXome (DNX) versus Daunorubicin (DNR) in acute myeloid leukaemia (AML) patients aged >60 years. Three hundred and one AML patients were randomized to receive DNR (45 mg/m(2) days 1-3) or DNX (80 mg/m(2) days 1-3) plus cytarabine (AraC; 100 mg/m(2) days 1-7). Patients in complete remission (CR) received a course of the same drugs as consolidation and then were randomized for maintenance with AraC+ all trans retinoic acid or no further treatment. Among 153 patients in the DNR arm, 78 (51.0%) achieved CR, 55 (35.9%) were resistant and 20 (13.1%) died during induction. Among 148 patients in the DNX arm, 73 (49.3%) achieved CR, 47 (31.8%) were resistant and 28 (18.9%) died during induction. Univariate analysis showed no difference as to induction results. After CR, DNX showed a higher incidence of early deaths (12.5% vs. 2.6% at 6 months, P = 0.053) but a lower incidence of relapse beyond 6 months (59% vs. 78% at 24 months, P = 0.064), with a cross in overall survival (OS) and disease-free survival (DFS) curves and a later advantage for DNX arm after 12 months from diagnosis. DNX seems to improve OS and DFS in the long-term follow-up, because of a reduction in late relapses.

Different transformation mechanisms have been proposed for elderly acute myeloid leukaemia (AML) and secondary AML (sAML) when compared with de novo AML or AML of younger patients. However, little is known regarding differences in the immunophenotypic profile of blast cells in these diseases. We systematically analysed, by flow cytometry, 148 patients affected by de novo (100 cases) or sAML (48 cases). By defining a cut-off level of 20% of CD34+ cells co-expressing CD90, the frequency of CD90+ cases was higher in sAML (40%) versus de novo AML (6%, P < 0.001), elderly AML (>60 years) (24%) versus AML of younger patients (10%, P = 0.010) and poor- versus good-risk karyotypes (according to the Medical Research Council classification, P < 0.001). The correlation between CD90 expression, sAML and unfavourable karyotypes was confirmed by analysing the subset of CD34+ AML cases alone (91/148). Consistently, univariate analysis showed that expression of CD90 was statistically relevant in predicting a shorter survival in CD90+ AML patients (P = 0.042). Our results, demonstrating CD90 expression in AML with unfavourable clinical and biological features, suggest an origin of these diseases from a CD90-expressing haemopoietic progenitor and indicate the use of CD90 as an additional marker of prognostic value in AML.

Concurrent resistance mechanisms, such as P-glycoprotein (PGP) and bcl-2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990 and 2000, we analysed PGP and bcl-2 by flow cytometry, using two anti-PGP (C219 and JSB-1) monoclonal antibodies (mAbs) and an anti-bcl-2 mAb in 115 de novo adult ALL patients. Both a longer overall survival (OS) and longer disease-free survival (DFS) were observed in PGP-negative patients (23%vs 0% at 3 years, P = 0.011 and 29%vs 0% at 2 years, P = 0.006 for C219 respectively; 42%vs 0% at 1.5 years, P = 0.004 and 53%vs 0% at 8.5 months, P = 0.00006 for JSB-1 respectively). Bcl-2 positivity was associated with a significantly higher complete remission rate (90%vs 66%, P = 0.01). Moreover, in 69 patients not presenting with either t(9;22) or B-mature immunophenotype, PGP negativity (JSB-1) maintained its significant favourable prognostic impact with regard to OS (41%vs 0% at 1.5 years, P = 0.009) and DFS (83%vs 0% at 6 months, P = 0.0005). Importantly, within a subset of 62 patients with normal (n = 31) or unknown (n = 31) karyotype, PGP (JSB-1)-negative patients showed both a significantly longer OS and DFS (63%vs 0% at 1.4 years, P = 0.018 and 84%vs 0% at 6 months, P = 0.001 respectively). In multivariate analysis, JSB-1 (P = 0.008) and cytogenetics (P = 0.02) were found to be independent prognostic factors with regard to DFS. Therefore, in adult ALL, PGP and bcl-2 represent sensitive indicators of clinical outcome, and potential targets of novel molecules aimed at overcoming chemoresistance and recurrent relapses.

Tumour necrosis factor alpha (TNF-alpha) is believed to play a major role in apoptotic death of bone marrow cells in myelodysplastic syndromes (MDS). We explored the efficacy and safety profile of infliximab chimaeric anti-TNF-alpha monoclonal antibody treatment in two MDS patients. They both had low-/intermediate-risk MDS, isolated anaemia and elevated circulating levels of TNF-alpha. Infliximab produced no adverse side-effects and resulted in sustained erythroid responses, one major and one minor. Laboratory studies indicated a remarkable decrease in the percentage of apoptotic stem cells in the bone marrow. This preliminary report indicates that infliximab may have an application as MDS therapy and warrants further investigation.

In vitro studies have indicated that granulocyte-macrophage colony-stimulating factor (GM-CSF) synergizes with erythropoietin (EPO) for the production of erythroid precursors in patients with myelodysplastic syndrome (MDS). We performed a clinical trial to evaluate whether the combination of these growth factors was effective in relieving the cytopenias associated with MDS. 31 anaemic patients with low and intermediate-risk primary MDS were enrolled in a 12-week study. Therapy was initiated with GM-CSF at 1 microgram/kg/d.s.c., and then adjusted to either normalize or double the absolute neutrophil count. EPO was given subcutaneously on alternate days starting from day 2. The EPO dose was initiated at 150 U/kg and increased to 300 U/kg if after 6 weeks there was no or suboptimal erythroid response. 26 patients completed the study treatment. All evaluable cases had a neutrophil response. Clinically significant erythroid responses with increases of haemoglobin levels of at least 1 g/dl and/or reduction of transfusion needs were seen in 9/26 (34.6%), five patients improving their response after dose escalation of EPO. Treatment had no apparent effect on mean platelet counts, a single case displaying a trilineage response. An elevated bone marrow erythroid infiltration and low concentrations of circulating tumour necrosis factor-alpha were the only predictors of haemoglobin response both in univariate and in multivariate analysis. We conclude that the combination GM-CSF + EPO can abrogate neutropenia and substantially relieve transfusion requirements in a large proportion of patients with low and intermediate risk MDS. However, in vivo synergy between these growth factors for the production of erythroid precursors is not supported by our data.

Seventy-three children with acute lymphoblastic leukaemia (ALL) in first bone marrow (BM) relapse, occurring within 30 months from complete remission (CR), were enrolled in an Italian cooperative study (ALL R-87 protocol). This treatment programme consisted of an induction phase with intermediate-dose cytarabine (IDARA-C) plus idarubicin (IDA) and prednisone (PDN), followed by a multidrug consolidation therapy and bone marrow transplant (BMT). 55/73 children achieved CR (75.3%); 15 (20.5%) failed to respond and three (4.2%) died during induction. The response rate was significantly higher for children with a first CR duration > or = 12 months (P=0.0005) and for those with a white blood cell (WBC) count at relapse < 20 x 10(9)/l (P=0.004). The estimated disease-free survival (DFS +/- SE) at 82 months was 0.18 +/- 0.05 for all responders, and 0.70 +/- 0.14 for allotransplanted patients versus 0.05 +/- 0.05 for those autografted (P=0.001). The estimated probabilities of survival +/- SE and event-free survival (EFS +/- SE) at 83 months were 0.16 +/- 0.07 and 0.13 +/- 0.04, respectively. for all enrolled children. Univariate analysis showed that age < 10 years at initial diagnosis and B-lineage immunophenotype favourably influenced both DFS (P=0.001) and EFS probabilities (P=0.0014 and P=0.012, respectively), whereas a first CR duration > or = 12 months and a WBC count at relapse < 20 x 10(9)/l were associated only with a better EFS rate (P=0.026 and P=0.004, respectively). Our results show the efficacy of the IDA plus IDARA-C schedule used in the ALL R-87 protocol in high-risk relapsed ALL children. Allogeneic BMT proved effective for patients with an HLA sibling donor. In a multivariate analysis, age > or = 10 years at initial diagnosis (P=0.016) and WBC count at relapse > or = 20 x 10(9)/l (P=0.048) were independently associated with a worse disease outcome.

Sixty-one adults aged <55 years with acute lymphoblastic leukaemia (ALL) in first bone marrow relapse were enrolled in an Italian cooperative study (ALL R-87 protocol) from 12 GIMEMA Institutions. The treatment programme consisted of: (1) an induction phase with intermediate-dose cytarabine (IDARA-C 1 g/m2, 6 h daily infusion x 6 d) plus idarubicin (IDA; 5 mg/m2/d x 6 d) and prednisone (40 mg/m2/d x 21 d), (2) a consolidation phase followed by (3) bone marrow transplant (BMT). Median first complete remission (CR) duration was 8.5 months (range 1-54 months). 34/61 patients achieved CR (56%); 24 (39%) failed to respond and three (5%) died during induction. Most responders (24 patients) could not enter the BMT programme; 15 relapsed early (median time to relapse 2 months); nine were withdrawn due to toxicity and one died in CR of infection. Nine of the 34 CRs underwent BMT (five autologous and four allogeneic). Three of the four allotransplanted patients are alive in continuous CR at 22, 43 and 63 months; only one of the five who underwent an autologous BMT is alive in CR at 46 months. The estimated disease-free survival (DFS +/- SE) at 36 months was 0.16 +/- 0.08 for all responders. Univariate analysis showed that previous therapy was the only prognostic factor influencing DFS. The estimated probabilities of event-free survival (EFS +/- SE) and survival +/- SE at 37 months were 0.09 +/- 0.04 and 0.10 +/- 0.04, respectively. The EFS was significantly better in patients with a preceding CR > or = 24 months, compared to those with a shorter first remission. Our results confirm the tolerance and efficacy of IDARA-C plus IDA in inducing CR in poor-risk adult ALL. Even though the number of transplanted patients was small, allogeneic BMT seems to give a real opportunity of cure in this category of patients.

Sixty-nine anaplastic large cell lymphomas (ALCLs) were selected from an Italian comparative trial on MACOP-B and F-MACHOP. As no significant difference in effectiveness of the protocols emerged, they were considered homogeneously treated. The ALCLs were divided into two groups according to previously defined criteria: 41 were common type (ALCLs-CT) and 28 Hodgkin-related (ALCLs-HR). T-cell phenotype was most common (58%), while B-cell, null and hybrid forms accounted for 27%, 13% and 2%. Clinically, ALCLs CT and HR differed as to mean age (27 v 34.3 years) and presentation; all ALCLs-HR showed mediastinal involvement, with bulky disease in 57%, and more frequent occurrence in stage II. In contrast, ALCLs-CT showed mediastinal masses in 58.5%, infrequently revealed bulky disease (24%), and were not specifically associated to stage. Among the ALCLs-CT, 68.4% achieved complete remission (CR), 24.4% partial remission (PR), one (2.4%) was resistant to therapy, and two (4.8%) had fatal drug toxicity. Of the ALCLs-HR, 67.8% reached CR, 14.3% PR, and 17.9% did not respond. In CR, ALCLs-CT showed a greater tendency to relapse (32.1% v 14.2%). At present, 65.8% of ALCLs-CT and 67.8% of ALCLs-HR are alive with overall survival/disease-free survival averages of 31/27 and 29/24 months respectively. Our data emphasize that, independently of subtype, ALCLs benefit from the application of third-generation protocols for high-grade non-Hodgkin's lymphomas.

One hundred and forty-seven patients aged < 55 years with advanced acute lymphoblastic leukaemia (ALL) were enrolled in an Italian cooperative study (ALL R-87). This protocol consists of an induction phase with idarubicin (IDA) plus intermediate-dose cytarabine (IDARA-C), followed by a consolidation phase and bone marrow transplant (BMT). Complete remission (CR) was achieved in 97/147 patients (66%) with a CR rate of 77% in children versus 51% in adults (P < 0.01). 48 responders (50%) underwent BMT. Probability of event-free survival (EFS +/- SE) was 10.2 +/- 3.1% at 56 months. EFS was 14.3 +/- 4.51% at 56 months for children versus 3.8 +/- 3.41% at 37 months for adults (P < 0.0001). Among patients treated in first relapse, EFS was 14.2 +/-7.79% for patients with CR > 18 months verus 6.6 +/- 3.17% for those with CR < 18 months (P < 0.0001). Projected disease-free survival (DFS +/- SE) was 15.4 +/- 4.61% at 55 months for all responders and 43.3 +/- 14.34% at 52 months for allografted patients. Projected overall probability of survival +/- SE for all patients was 18.8 +/- 4.13% at 56 months. This study confirms the efficacy of IDA plus IDARA-C in poor-risk. ALL patients. A more intensive post-remission therapy or alternative approach must be designed to improve long-term results.

The increasing incidence of bacteraemia caused by Gram-positive bacteria in neutropenic patients prompted the authors to evaluate, in a prospective trial, the role of teicoplanin in the treatment of this infection. Over a 15-month period, 76 cases of bacteraemia (out of 265 evaluable episodes of fever) were observed at the Division of Haematology, University La Sapienza, Rome. Of the 76 cases studied, 46 (60%) were caused by Gram-positive bacteria and 28 (37%) were caused by Gram-negative bacilli. All febrile episodes were treated randomly and empirically with piperacillin plus amikacin with or without teicoplanin. Overall, 41 (54%) of the 76 cases of bacteraemia responded to the initial antibiotic regimen; with subsequent modifications the response rate rose to 96%. In the treatment of Gram-positive bacteraemia, first-line administration of teicoplanin was found to be associated with early defervescence and with a significantly higher rate of success without modification of treatment (P less than 0.01). Addition of teicoplanin as second-line therapy produced a favourable outcome in 12 (70%) out of 17 cases of bacteraemia unresponsive to the initial piperacillin + amikacin regimen. No cases of Gram-positive bacteraemia associated with septic shock or adult respiratory distress syndrome were observed in either treatment group. Only two late deaths were observed, and these occurred in patients with streptococcal septicaemia who were not receiving early teicoplanin. The above data do not endorse the use of glycopeptide antibiotics in the early treatment of fever in neutropenic patients: rather, these compounds should be reserved for proven or presumed Gram-positive infections which do not respond to initial beta-lactam/aminoglycoside treatment.

In this study we analysed serum IL-2 levels in 61 patients with multiple myeloma (MM). Patients serum IL-2 levels were significantly higher than normal controls. Moreover, higher serum IL-2 levels were associated with a prolonged actuarial survival. In particular, 87% of the MM patients with IL-2 greater the or equal to 10 U/ml are still alive at 5 years while only 13% of the remaining patients with IL-2 less than 10 U/ml are alive. The multivariate analysis confirmed these data indicating that high serum IL-2 levels are the most useful predictor index of longer survival in MM patients. Furthermore, among the 50 patients in whom serum beta-2-microglobulin (SB2M) determination was available we observed that all patients with serum IL-2 levels greater than or equal to 10 U/ml had SB2M less than 6 micrograms/ml, whereas in patients with serum IL-2 less than 10 U/ml SB2M ranged from 1.3 to 15 micrograms/ml. Using these two parameters we were able to identify three groups of patients with different survival duration. Group A (9 patients) defined by serum IL-2 greater than or equal to 10 U/ml and SB2M less than 6 micrograms/ml in which all patients are alive: group B (26 patients) characterized by serum IL-2 less than 10 U/ml and SB2M less than 6 micrograms/ml in which 24% of patients are alive and group C (15 patients) characterized by serum IL-2 levels less than 10 U/ml and SB2M greater than or equal to 6 micrograms/ml in which the actuarial survival curve drops to 0 at 2.5 years. A statistically significant difference was observed between groups A and B (P less than 0.05), groups A and C (P less than 0.01) and groups B and C (P less than 0.01). These data could reflect the existence of an active T cell control on B cell neoplasia and may suggest the opportunity of a more extensive use of recombinant biological modifiers such as IL-2 in the therapeutic strategy of MM.

E-rosetting of leukaemic blast cells is one of the markers of T-cell acute lymphoblastic leukaemia (ALL). In children, E+ ALL has a bad prognosis. In adults, data are scarce. This report provides information on 25 E+ ALL adult patients who have a minimum follow-up time of 36 months. Twenty-two of 25 patients (88%) achieved complete remission (CR) (median duration 16 months), and six of them were alive, relapse-free, and off therapy after 36-81 months, with a 26% projected 6-year relapse-free survival. In 97 patients with E-SmIg- ALL, who were treated at the same Institutions, over the same period of time, and by the same modalities, the outcome of therapy was almost identical: CR 80%, median duration of first CR 15 months, projected 6-year relapse-free survival 15%. The white blood cell (WBC) count at presentation influenced significantly and to the same degree first CR length in both E+ and E- cases. In this adult series, WBC count was not as high as in children. Moreover, a high Hb concentration, a very high WBC count, lymphadenomegaly, and mediastinal involvement, were found more frequently in adolescents and young adults than in adults. Based on these data, it is suggested that in adults E-rosetting as such is not a marker of a poorer prognosis, that some of the typical features of children E+ ALL weaken with age, and that in adults the disease can have a less aggressive character.