Nicoletta Testoni

Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4–11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34+ cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K–Akt survival pathway.

Achieving a Major Molecular Response at the Time of a Complete Cytogenetic Response (CCgR) Predicts a Better Duration of CCgR in Imatinib-Treated Chronic Myeloid Leukemia Patients

Purpose: Most patients with chronic-phase chronic myeloid leukemia (CML) who receive imatinib achieve a complete cytogenetic remission (CCgR) and low levels of BCR-ABL transcripts. CCgR is durable in the majority of patients but relapse occurs in a subset. Experimental Design: To determine the potential of quantitative reverse transcription-PCR of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 97 CML patients with an imatinib-induced CCgR. Patients with late chronic phase CML after IFN-α failure were treated with imatinib (400 mg daily). Results: During the imatinib median follow-up time of 36 months (range, 12-54 months), disease monitoring occurred by cytogenetics and quantitative PCR. Twenty percent of patients experienced cytogenetic relapse at a median of 18 months after CCgR and a median of 24 months after starting imatinib. None of the possible prognostic factors studied in univariate and multivariate analyses seemed to predict for loss of cytogenetic response but the reduction of BCR-ABL transcript levels at the time of CCgR is an important prognostic factor. Conclusions: In our study, we showed not only that achieving a major molecular remission at 12 months is predictive of a durable cytogenetic remission but also that patients who achieved a major molecular remission (expressed both as the BCR-ABL/β2 microglobulin ratio % <0.0005 and as a 3-log reduction from median baseline value) already at the time of first achieving a CCgR have significantly longer cytogenetic remission durations than those without this magnitude of molecular response (P < 0.05).

Long-Term Outcome of Complete Cytogenetic Responders After Imatinib 400 mg in Late Chronic Phase, Philadelphia-Positive Chronic Myeloid Leukemia: The GIMEMA Working Party on CML

PURPOSEnImatinib mesylate (IM) has rapidly become the front-line treatment of Philadelphia-positive (Ph-pos) chronic myeloid leukemia, but the number of patients who were treated and are being treated with IM second-line is still substantial.nnnPATIENTS AND METHODSnWe have monitored and analyzed the cytogenetic and molecular response to IM 400 mg/d in a cohort of 277 late chronic phase (LCP) patients who were resistant or intolerant to interferon-alpha and were observed for 48 to 79 months (median, 72 months).nnnRESULTSnOne hundred fifty-three patients (55%) achieved a complete cytogenetic response (CCgR). Seventy-seven percent of them were still in CCgR after 5 years. The rate of response loss did not increase over time. The 6-year progression-free survival and overall survival of these 153 complete cytogenetic responders were 90% and 91%, respectively. Molecular response was less than major in 21%, major in 78%, and complete in one patient only.nnnCONCLUSIONnThese data confirm that, in LCP the CCgR rate to IM is 50% to 60%, and show that CCgR is stable and is associated with a prolonged survival, even if leukemia continues to be molecularly detectable.

Comparison Between Patients With Philadelphia-Positive Chronic Phase Chronic Myeloid Leukemia Who Obtained a Complete Cytogenetic Response Within 1 Year of Imatinib Therapy and Those Who Achieved Such a Response After 12 Months of Treatment

PURPOSEnImatinib mesylate is a potent inhibitor of BCR-ABL, the constitutively active tyrosine kinase protein critical for the pathogenesis of chronic myeloid leukemia.nnnPATIENTS AND METHODSnWe reviewed 284 patients with late chronic-phase Philadelphia chromosome (Ph) -positive chronic myeloid leukemia treated with imatinib 400 mg daily after interferon-alpha failure. In a retrospective study, we evaluated the pattern and rapidity of the response to imatinib, comparing the cytogenetic and molecular responses, progression-free and overall survival rates in patients who obtained a complete cytogenetic response within 1 year of treatment (early responders), and in patients where a complete cytogenetic response was detected after 12 months (late responders).nnnRESULTSnAfter 3 or 4 years of treatment, the molecular response of the late cytogenetic responders was similar to that of the early cytogenetic responders. At 36 months of treatment the amount of residual disease measured by standardized quantitative reverse-transcriptase polymerase chain reaction was 0.00047 in late responders versus 0.00022 in early responders, and at 48 months it was 0.00019 versus 0.00026 (median values, P value = nonsignificant). The estimated 4-year progression-free survival rate was 88% for early responders and 100% for late responders, while the estimated 4-year overall survival rates were 92% and 100% for early and late responders, respectively.nnnCONCLUSIONnThe sensitivity and the response (cytogenic and molecular) to imatinib may require 1 year or more. Long-term follow-up results continue to improve in terms of rates and durability of the complete cytogenetic response, major or complete molecular response, and progression-free and overall survival.

Multicentre phase III trial on fludarabine, cytarabine (Ara-C), and idarubicin versus idarubicin, Ara-C and etoposide for induction treatment of younger, newly diagnosed acute myeloid leukaemia patients.

Fludarabine plus cytarabine (Ara‐C) and idarubicin (FLAI) is an effective and well‐tolerated induction regimen for the treatment of acute myeloid leukaemia (AML). This phase III trial compared the efficacy and toxicity of FLAI versus idarubicin plus Ara‐C and etoposide (ICE) in 112 newly diagnosed AML patients <60u2003years. Fifty‐seven patients received FLAI, as the first induction–remission course, and 55 patients received ICE. Post‐induction treatment consisted of high‐dose Ara‐C (HDAC). After HDAC, patients in complete remission (CR) received a second consolidation course (mitoxantrone, etoposide, Ara‐C) and autologous stem cell transplantation (auto‐SCT) or allogeneic (allo)‐SCT, according to the age, disease risk and donor availability. After a single induction course, CR rate was 74% in the FLAI arm and 51% in the ICE arm (Pu2003=u20030·01), while death during induction was 2% and 9% respectively. Both haematological (Pu2003=u20030·002) and non‐haematological (Pu2003=u20030·0001) toxicities, especially gastrointestinal (i.e. nausea, vomiting, mucositis and diarrhoea), were significantly lower in FLAI arm. In both arms, relapses were more frequent in patients who were not submitted to allo‐SCT. After a median follow‐up of 17u2003months, 30% and 38% of the patients are in continuous CR in FLAI and ICE arm respectively. Our prospective randomised study confirmed the anti‐leukaemic effect and the low toxic profile of FLAI as induction treatment for newly diagnosed AML patients.

Treatment of Philadelphia-Positive Chronic Myeloid Leukemia with Imatinib: Importance of a Stable Molecular Response

Purpose: The achievement of a major molecular response (MMolR) at 12 months is a surrogate marker of progression-free survival in chronic myeloid leukemia patients treated with imatinib. Experimental Design: We evaluated the prognostic value of the long-term evolution of the molecular response based on a retrospective analysis of 130 late chronic phase chronic myeloid leukemia patients who achieved a complete cytogenetic response (CCgR) with 400 mg/d imatinib and have now a median follow-up of 72 months (range, 48-77). Results: In 71 (55%) patients, molecular response was consistently major (stable MMolR); in 19 (15%) patients, molecular response was occasionally less than major (unstable MMolR); in 40 (30%) patients, MMolR was never achieved (never MMolR) during all the course of CCgR. Patients with stable MMolR had a longer CCgR duration and a significantly better progression-free survival compared with patients with absent or unstable MMolR. The achievement of a MMolR, if maintained continuously, conferred a marked long-term stability to the CCgR: patients with a stable MMolR have a significantly lower risk of losing the CCgR than patients with unstable and never MMolR (4% versus 21%, P = 0.03, and 4% versus 33%, P < 0.0001, respectively). Finally, if a MMolR is not maintained consistently, the risk of losing the CCgR is higher but not significantly than if it is never achieved (33% versus 21%, P = 0.5). Conclusions: These data confirm that achieving a MMolR is prognostically important but point out that the prognostic value of achieving a MMolR is greater if the response is confirmed and stable.

Poor outcome of adult acute lymphoblastic leukemia patients carrying the (1;19)(q23;p13) translocation.

The (1;19)(q23;p13) translocation, leading to the production of the E2A/PBX1 fusion transcript, is one of the most common translocations in pediatric B-lineage acute lymphoblastic leukemia (ALL). It was assumed to be associated with a poor clinical outcome, although intensive therapy and bone marrow transplantation have been shown to be able to overcome the negative prognostic impact. Only few data are available concerning t(1;19)(q23;p13) in adult ALL. In particular, the prognostic significance of this genetic aberration is not yet clear. We describe three cases of adult ALL carrying the t(1;19)(q23;p13), who were all characterized by an aggressive clinical course and short survival, and discuss the molecular features of the disease as recently identified by gene expression profiling.

Case-control study of multidrug resistance phenotype and response to induction treatment including or not fludarabine in newly diagnosed acute myeloid leukaemia patients

One hundred and six patients aged ≤60u2003years with newly diagnosed acute myeloid leukaemia (AML) treated with fludarabine‐based regimens (cases) were matched with 106 AML patients treated with conventional non‐fludarabine‐based regimens (controls). The cases and controls were matched by expression of the multidrug resistance P‐glycoprotein (MDR‐Pgp), measured by flow cytometry as mean fluorescence index (MFI), cytogenetics, and age. The complete remission (CR) rate of the cases was 61% among the MDR‐Pgp‐positive (posve) patients (MFIu2003≥u20036) vs. 75% among the MDR‐Pgp‐negative (negve) ones (MFIu2003<u20036) (Pu2003=u20030·16). Conversely, in the controls, the CR rate was 44% among the MDR‐Pgp‐posve patients vs. 67% among the MDR‐Pgp‐negve ones (Pu2003=u20030·02). The 4‐year disease‐free survival (DFS) and overall survival (OS) of MDR‐Pgp‐posve cases were significantly longer than those of MDR‐Pgp‐posve controls (DFS, 28·1% vs. 6·5%, Pu2003=u20030·004; OS, 33·5% vs. 9·6%, Pu2003=u20030·01). This difference was not found among the MDR‐Pgp‐negve patients. By univariate (Pu2003=u20030·007) and multivariate (Pu2003=u20030·007) analysis, the MDR‐Pgp‐posve phenotype was negatively correlated with CR and it emerged as the most important independent negative prognostic factor, after cytogenetics. Our study confirms the prognostic impact of the MDR phenotype in AML and strongly suggests fludarabine‐based induction treatments as a promising strategy for MDR‐Pgp‐posve AML patients. In this setting of patients, large prospective randomised studies should be planned.

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.

Molecular and cytogenetic characterization of a new case of t(5;17)(q35;q21) variant acute promyelocytic leukemia

Molecular and cytogenetic characterization of a new case of t(5;17)(q35;q21) variant acute promyelocytic leukemia