Mario Tiribelli

Contribution of ABL Kinase Domain Mutations to Imatinib Resistance in Different Subsets of Philadelphia-Positive Patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia

Purpose: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Experimental Design: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Results: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. Conclusions: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Nilotinib for the frontline treatment of Ph(+) chronic myeloid leukemia.

Nilotinib has a higher binding affinity and selectivity for BCR-ABL with respect to imatinib and is an effective treatment of chronic myeloid leukemia (CML) after imatinib failure. In a phase 2 study, 73 early chronic-phase, untreated, Ph(+) CML patients, received nilotinib at a dose of 400 mg twice daily. The primary endpoint was the complete cytogenetic response (CCgR) rate at 1 year. With a median follow-up of 15 months, the CCgR rate at 1 year was 96%, and the major molecular response rate 85%. Responses were rapid, with 78% CCgR and 52% major molecular response at 3 months. During the first year, the treatment was interrupted at least once in 38 patients (52%). The mean daily dose ranged between 600 and 800 mg in 74% of patients, 400 and 599 mg in 18% of patients, and was less than 400 mg in 8% of patients. Dose interruptions were mainly due to nonhematologic and biochemical side effects. Myelosuppression was irrelevant. One patient progressed to blastic crisis after 6 months; one went off-treatment for lipase increase grade 4 (no pancreatitis). Nilotinib is safe and very active in early chronic-phase CML. These data support a role for nilotinib for the frontline treatment of CML. This study was registered at ClinicalTrials.gov as NCT00481052.

The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRα-positive hypereosinophilic syndrome. Results of a multicenter prospective study

Background and Objectives The hypereosinophilic syndrome (HES) may be associated with the fusion of the platelet derived growth factor receptor α (PDGFRα) gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated PDGFRα tyrosine kinase. These cases with FIP1L1-PDGFRα rearrangement have been reported to be very sensitive to the tyrosine kinase inhibitor imatinib mesylate. Design and Methods A prospective multicenter study of idiopathic or primary HES was established in 2001 (Study Protocol Registration no. NCT 0027 6929). One hundred and ninety-six patients were screened, of whom 72 where identified as having idiopathic or primary HES and 63 were treated with imatinib 100 to 400 mg daily. Results Twenty-seven male patients carried the FIP1L1-PDGFRα rearrangement. All 27 achieved a complete hematologic remission (CHR) and became negative for the fusion transcripts according to reverse transcriptase polymerase chain reaction (RT-PCR) analysis. With a median follow-up of 25 months (15–60 months) all 27 patients remain in CHR and RT-PCR negative, and continue treatment at a dose of 100 to 400 mg daily. In three patients imatinib treatment was discontinued for few months, the fusion transcript became rapidly detectable, and then again undetectable upon treatment reassumption. Thirty-six patients did not carry the rearrangement; of these, five (14%) achieved a CHR, which was lost in all cases after 1 to 15 months. Interpretation and Conclusions All patients meeting the criteria for idiopathic or primary HES should be screened for the FIP1L1-PDGFRα rearrangement. For all patients with this rearrangement, chronic imatinib treatment at doses as low as 100 mg daily ensures complete and durable responses.

Anti-CD20 therapy for chronic lymphocytic leukemia-associated autoimmune diseases.

Rituximab is active in chronic lymphocytic leukemia (CLL) and may interfere with autoantibodies production in some immune diseases. We report the results of rituximab treatment in 7 patients with CLL-associated symptomatic autoimmune diseases refractory to standard immunosuppressive therapies: warm antibody hemolytic anemia (AHA) 4 patients, cold agglutinin disease (CAD) 1, immune thrombocytopenia (IT) I , axonal degenerating neuropathy (ADN) 1. Rituximab was given at the dose of 375mg/m2 per week for 4 weeks. One patient with AHA and one with CAD achieved complete normalization of hemoglobin levels and laboratory signs of haemolysis, with response duration (RD) of 8+ and 38+ months, respectively. In the patient with IT, complete remission was reached after the first week of treatment and RD was 6 months. The patient with ADN achieved a marked neurological improvement after rituximab therapy, with RD of 12 months. Retreatment of both patients with IT and ADN was effective. Rituximab may be an alternative agent for the treatment CLL-associated autoimmune diseases.

Nutlin-3 up-regulates the expression of Notch1 in both myeloid and lymphoid leukemic cells, as part of a negative feedback antiapoptotic mechanism

The small molecule inhibitor of the MDM2/p53 interaction Nutlin-3 significantly up-regulated the steady-state mRNA and protein levels of Notch1 in TP53(wild-type) (OCI, SKW6.4) but not in TP53(deleted) (HL-60) or TP53(mutated) (BJAB) leukemic cell lines. A direct demonstration that NOTCH1 was a transcriptional target of p53 in leukemic cells was obtained in experiments carried out with siRNA for p53. Moreover, inhibition of Notch1 expression using Notch1-specific siRNA significantly increased cytotoxicity in TP53(wild-type) leukemic cells. Of note, Nutlin-3 up-regulated Notch1 expression also in primary TP53(wild-type) B-chronic lymphocytic leukemia (B-CLL) cells and the combined use of Nutlin-3 plus pharmacological gamma-secretase inhibitors of the Notch signaling showed a synergistic cytotoxicity in both TP53(wild-type) leukemic cell lines and primary B-CLL cells. A potential drawback of gamma-secretase inhibitors was their ability to enhance osteoclastic maturation of normal circulating preosteoclasts induced by RANKL + M-CSF. Notwithstanding, Nutlin-3 completely suppressed osteoclastogenesis irrespective of the presence of gamma-secretase inhibitors. Taken together, these data indicate that the p53-dependent up-regulation of Notch1 in response to Nutlin-3 represents an antiapoptotic feedback mechanism able to restrain the potential therapeutic efficacy of Nutlin-3 in hematologic malignancies. Therefore, therapeutic combinations of Nutlin-3 + gamma-secretase inhibitors might potentiate the cytotoxicity of Nutlin-3 in p53(wild-type) leukemic cells.

Quantitative assessment of WT1 gene expression after allogeneic stem cell transplantation is a useful tool for monitoring minimal residual disease in acute myeloid leukemia.

Introduction:  WT1 overexpression is described in several oncological diseases including acute myeloid leukemia (AML). Quantification of WT1 in bone marrow samples may be useful as a marker of minimal residual disease (MRD) and may predict the relapse of AML after allogeneic hematopoietic stem cell transplant (HSCT).

Synergistic cytotoxic activity of recombinant TRAIL plus the non-genotoxic activator of the p53 pathway nutlin-3 in acute myeloid leukemia cells.

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.

Investigating factors associated with adherence behaviour in patients with chronic myeloid leukemia: an observational patient-centered outcome study

Background:Optimal adherence to imatinib therapy is of paramount importance to maximise treatment effectiveness in patients with chronic myeloid leukaemia (CML). The main objective of this study was to investigate patient-reported personal factors associated with adherence behaviour.Methods:Analysis was conducted on 413 CML patients receiving long-term therapy with imatinib. Adherence behaviour was measured with the Morisky Medication Adherence Scale and personal factors investigated included: quality of life, perceived social support, fatigue, symptom burden, psychological wellbeing and desire for additional information. Key socio-demographic and treatment-related factors were also taken into account. Univariate and multivariate logistic regression analyses were used to investigate factors associated with optimal adherence to therapy.Results:In all, 53% of patients reported an optimal adherence behaviour. The final multivariate model retained the following variables as independent predictors of optimal adherence to therapy: desire for more information (ref. no), odds ratio (OR)=0.43 (95% confidence interval (CI), 0.29–0.66; P<0.001), social support (higher score representing greater support), OR=1.29 (95% CI, 1.11–1.49; P<0.001) and concomitant drug burden (ref. no), OR=1.82 (95% CI, 1.18–2.80; P=0.006).Conclusion:This study suggests that a higher level of social support, satisfaction with information received and concomitant drug burden are the main factors associated with greater adherence to long-term imatinib therapy.

Caspofungin as first line therapy of pulmonary invasive fungal infections in 32 immunocompromised patients with hematologic malignancies

Invasive Fungal Infections (IFI) remain a severe and major complication among patients with hematologic diseases, but the recent availability of new antifungal agents (echinocandins and new azoles) have improved the chance of cure. Caspofungin (Cancidas‐Merck) is a large lipopeptide molecule able to inhibit the enzyme complex 1,3‐d‐glucan synthetase; this action specifically damages the fungal cell wall. Caspofungin (CAS) is active, in vitro and in vivo, against most Candida species and Aspergillus species. We report on our experience with this drug as first‐line therapy for proven or probable pulmonary IFI in immunocompromised patients with hematologic malignancies. Thirty‐two consecutive patients (20 males and 12 females, with a median age of 52 yr) have been treated with CAS (27 acute leukemias, 1 chronic leukemia, 3 lymphomas and 1 multiple myeloma). Sixteen patients (50%) had a relapsed or resistant hematologic disease, while 12 patients were in complete remission and 4 were at onset of disease; 8/32 (25%) developed IFI after a hematopoietic stem cell transplant (HSCT) procedure. Seven out of 32 patients (22%) had a proven pulmonary IFI (7/7 Aspergillosis) and 25 (78%) had a probable IFI with pulmonary localization as defined according to international consensus. Thirty‐one patients (97%) had less than 1000 granulocytes/mL at onset of infection and at the start of CAS therapy. The CAS was given at the dose of 70 mg on day 1, followed by 50 mg/day. Median duration of CAS therapy was 20 d (range 8–64); all the 31 neutropenic patients received concomitant granulocyte colony‐stimulating factor (G‐CSF). The overall response rate was 56% (18/32) with 12/18 complete responses and 6/18 partial responses; two patients (6%) had a stable disease. Twelve out of 32 (38%) did not respond and seven died of mycotic infection. Univariate analysis showed that granulocytes recovery (>500/mL vs. <500/mL) and status of hematologic disease (remission/onset vs. refractory/relapsed) were significantly associated to favourable outcome. No clinical adverse events (AE) were reported and only a grades I and II transient increase of serum alkaline phosphatase and/or transaminases occurred in 4/32 (12%) patients. After CAS therapy six non‐responders and six cases with a partial or stable response were rescued with voriconazole. Two out of six patients (33%) in the former group and 6/6 (100%) in the latter obtained a complete resolution of IFI. Our experience suggests an efficacy of CAS, in combination with G‐CSF, as first‐line treatment of proven or probable IFI with pulmonary localization. The drug was well tolerated and there were no significant hepatic AE even in patients receiving CAS with cyclosporine after a HSCT. A significant proportion of non‐responders or partial responders to CAS can be rescued with a subsequent voriconazole‐based therapy.

Front-line treatment of Philadelphia positive chronic myeloid leukemia with imatinib and interferon-α: 5-year outcome

This study confirms the excellent response to imatinib front-line therapy in patients with chronic myeloid leukemia. By contrast, most patients discontinued pegylated interferon-α due to its side effects. In 2004, we reported the short-term results of a multicentric, phase 2 study of imatinib 400 mg daily and pegylated interferon-α in the treatment of 76 early chronic phase Philadelphia-positive chronic myeloid leukemia patients. In this report, we update the results with an observation time of five years. After two years of treatment, all but 10 patients (13%) had discontinued pegylated interferon-α. The complete cytogenetic response rate at five years was 87%, and 94% of complete cytogenetic responders maintained the complete cytogenetic response after five years. All but one complete cytogenetic response also achieved a major molecular response. These data confirm the excellent response to imatinib front-line and the stability of the complete cytogenetic response. Any possible additional benefit of the combination with interferon-α remains uncertain, due to low patient compliance.