GIMEMA LAL Research Articles

INCB84344-201: Ponatinib and steroids in frontline therapy for unfit patients with Ph+ acute lymphoblastic leukemia

AbstractTyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.

Phase II trial with sequential clofarabine and cyclophosphamide for refractory and relapsed philadelphia-negative adult acute lymphoblastic leukemia. Results of the GIMEMA LAL 1610 protocol

Clofarabine (CLO) and cyclophosphamide (CY) combinations were tested in late stage refractory/relapsed (R/R) acute lymphoblastic leukemia (ALL) with disappointing results and high-grade toxicity. We designed a sequential 5-day combination of CLO 40 mg/m2/d plus CY 400 mg/m2/d as first salvage for Philadelphia-negative ALL patients refractory or relapsed until 24 months from complete remission (CR). Primary endpoint was an overall response rate (ORR) ≥ 40%. Among 26 study patients (median age 40.5 years) ORR was 57.6% (CR with complete [n = 8] or incomplete [n = 7] hematologic recovery). Despite severe myelotoxicity, no dose-limiting toxicity suggested de-intensification of CLO schedule. With a median follow-up of 17.0 months, median and 1-year overall and disease-free survival were 6.5 months and 28.6%, and 3.7 months and 28.1%, respectively. This association was tolerable and more effective in patients younger than 40 years with B-precursor ALL, longer first CR, not previously transplanted and achieving CR with full hematological recovery.

Comparative Analysis between RQ-PCR, Digital-Droplet-PCR and Next-Generation-Sequencing (NGS) of Immunoglobulin/T-Cell Receptor Gene Rearrangements to Monitor Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia Patients

▪Background. Minimal residual disease (MRD) is the strongest prognostic factor in both children and adults with acute lymphoblastic leukemia (ALL). Currently, it is most widely monitored by molecular methods based on real-time-quantitative-PCR (RQ-PCR). Digital-droplet-PCR (ddPCR) and next-generation-sequencing (NGS) represent advanced tools that have the potential to overcome some limitations of standard approaches and potentially provide additional benefits. We analyzed adult ALL follow-up (FU) samples by RQ-PCR, ddPCR and NGS in order to better define the discriminating power of these novel methods.Patients and Methods. Thirty adult ALL patients enrolled in the GIMEMA LAL 1913 protocol and their 83 FU bone marrow (BM) samples were studied. All patients received homogeneous induction/early consolidation chemotherapy, with concurrent MRD analysis at four time-points, to optimize risk classification and support risk/MRD-oriented therapy. RQ-PCR analyses followed the EuroMRD Consortium guidelines (van der Velden, 2007), ddPCR was performed as published (Della Starza, 2016; Cavalli, 2017) and NGS, as previously described (Faham, 2012; Kotrova M, 2017).Results. By MRD RQ-PCR analysis, 19/83 samples were positive and quantifiable (Q), 9/83 were positive not-quantifiable (PNQ) and 55/83 were negative (NEG). By MRD ddPCR analysis, 27/83 samples were Q, 1/83 sample was PNQ and 55/83 proved NEG. Comparing the results of the two methods, we observed that MRD detection was concordantly positive or negative in 81% (67/83) of FU samples, while 19% (16/83) samples were classified as discordant. Most of the discordances occurred in samples with low levels of disease, i.e. PNQ or NEG: 9/83 were RQ-PCR PNQ, 4 of which were Q by ddPCR and 5 were ddPCR NEG. In the remaining 7 discordant FU samples, 5 were RQ-PCR NEG/ddPCR Q, 1 sample was RQ-PCR Q /ddPCR NEG and 1 sample was RQ-PCR NEG/ddPCR PNQ. The use of ddPCR significantly reduced the proportion of PNQ samples if compared to RQ-PCR - 1/83 (3%) vs 9/83 (15%) - respectively (p=0.0179), increasing the proportion of Q samples: 27/83 (33%) vs 19/83 (23%). It is worth noting that ddPCR also quantified the levels of disease in 9% (5/55) of samples, that were RQ-PCR NEG (Table 1).MRD analysis was also performed by NGS in 41 samples from 15 patients: 18/41 samples proved Q and 23/41 were NEG. Comparing the MRD detection obtained by both ddPCR and NGS, we observed a concordant result in 98% (40/41) of samples; only 1 sample was ddPCR NEG and NGS Q with a MRD level of 1x10-5. The concordance between RQ-PCR and NGS was 78% (32/41 samples). Moreover, among these 41 samples 9 (from 7 patients) were discordant between RQ-PCR and ddPCR in the first comparative analysis: in 4 RQ-PCR-NEG FU samples, 3 were Q by both ddPCR and NGS, 1 was ddPCR NEG and NGS Q, with a MRD level of 10- 5; 1 subsequent relapse was observed; 4 FU samples that were RQ-PCR-PNQ/ddPCR-Q, were Q also by NGS; 1 subsequent relapse was observed. Finally, 1 RQ-PCR-PNQ sample was negative by both ddPCR and NGS, and no recurrence has so far been observed. Moreover, in the cohort of samples analyzed only by RQ-PCR and ddPCR, in 1 RQ-PCR NEG/ddPCR Q sample a relapse was observed, while the only case that was RQ-PCR Q/ddPCR NEG has so far not relapsed. Notably, 2 of the 3 relapses were documented in patients who were, at decisional treatment TPs, RQ-PCR PNQ or NEG and ddPCR/NGS Q.Conclusions. When MRD levels are very low, it can be difficult to dissect if the not-quantifiable signal observed by PCR is due to few residual leukemic cells or to a non-specific amplification of normal DNA. The superior sensitivity and accuracy of ddPCR and NGS could be instrumental to univocally define these samples, which presently represent a problematic gray area in the clinical practice of MRD-driven protocols and might be associated with clinical relapse: indeed, among 83 FU samples analyzed we observed 3 relapses, whose FU samples were classified as PNQ or NEG by RQ-PCR, but proved Q by ddPCR and/or NGS. At variance, no relapses were recorded in patients whose FU samples were defined RQ-PCR-PNQ, but proved ddPCR/NGS NEG. Moreover, in 2/3 relapsed cases the change of MRD status (PNQ or NEG vs Q) could have led to a switch in risk classification and therefore in a treatment change. Further studies with a larger number of discrepant cases and a longer FU time will allow to conclusively define the clinical application and implication of these new methods. [Display omitted] DisclosuresChiaretti:Shire: Consultancy; Pfuzer: Consultancy; Amgen: Consultancy; Incyte: Consultancy. Foà:NOVARTIS: Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; GILEAD: Speakers Bureau.

PSY87 - Pegaspargase In The Treatment of Acute Lymphoblastic Leukemia: A CMA Model Demonstrates Its Economic Benefit For The Italian National Health System

Pegaspargase is a pegylated form of native E. Coli asparaginase used as an agent in the treatment of acute lymphoblastic leukemia (ALL). Compared to the native form, it presents two main benefits: (i) a longer half-life, thus reducing the number of administrations and (ii) a lower immunogenicity, both in terms of hypersensitivity/allergic reactions as well as antibody formation, thus avoiding to switch to second line costly treatment, such as that with Erwinia Chrisanthemi asparaginase. We present here a cost-minimization analysis (CMA) to show that pegaspargase leads to an economic benefit to the Italian National Health System (SSN). For the model, we constructed a decision tree based upon the clinical practice for the treatment of ALL, with inputs derived from Italian protocols AIEOP-BFM 2009 (for pediatrics) and GIMEMA LAL 1913 (for adults). The native E. Coli asparaginase has been selected as comparator since some patients still use this enzyme in the first-line treatment of ALL. Branch point probabilities representing immunogenic rates were obtained from the best available published literature. Direct costs (hospitalization, drug administration, allergic reactions management) were derived from Gazzetta Ufficiale. Our base-case analysis revealed that the cost-per-patient (both for pediatrics and adults) for pegaspargase was ~6000 € lower than that for native E. coli asparaginase mainly due to the reduced number of administrations and to the lower incidence of immunogenic reactions. The latter has in turn led to a lower hospitalization rate and to a lower switch to the more expensive second line treatment with Erwinia Chrisanthemi. The costs associated with the use of pegaspargase for the first line treatment of ALL are lower than those deriving from the native form of native E. coli asparaginase. Thus, pegaspargase is a cost saving option, with a potential beneficial budget impact for the Italian SSN.

A sequential approach with imatinib, chemotherapy and transplant for adult Ph+ acute lymphoblastic leukemia: final results of the GIMEMA LAL 0904 study.

In the GIMEMA LAL 0904 protocol, adult Philadelphia positive acute lymphoblastic leukemia patients were treated with chemotherapy for induction and consolidation, followed by maintenance with imatinib. The protocol was subsequently amended and imatinib was incorporated in the induction and post-remission phase together with chemotherapy. Due to the toxicity of this combined approach, the protocol was further amended to a sequential scheme based on imatinib plus steroids as induction, followed by consolidation with chemotherapy plus imatinib and, when applicable, by a hematopoietic stem cell transplant. Fifty-one patients (median age 45.9 years) were enrolled in the final sequential protocol. At the end of induction (day +50), 96% of evaluable patients (n=49) achieved a complete hematologic remission; after consolidation, all were in complete hematologic remission. No deaths in induction were recorded. Overall survival and disease-free survival at 60 months are 48.8% and 45.8%, respectively. At day +50 (end of imatinib induction), a more than 1.3 log-reduction of BCR-ABL1 levels was associated with a significantly longer disease-free survival (55.6%, 95%CI: 39.0-79.3 vs. 20%, 95%CI: 5.8-69.1; P=0.03), overall survival (59.1%, 95%CI: 42.3-82.6 vs. 20%, 95%CI: 5.8-69.1; P=0.02) and lower incidence of relapse (20.5%, 95%CI: 7.2-38.6 vs. 60.0%, 95%CI: 21.6-84.3; P=0.01). Mean BCR-ABL1 levels remained significantly higher in patients who subsequently relapsed. Finally, BCR-ABL1p190 patients showed a significantly faster molecular response than BCR-ABL1p210 patients (P=0.023). Though the study was not powered to evaluate the role of allogeneic stem cell transplant, allografting positively impacted on both overall and disease-free survival. In conclusion, a sequential approach with imatinib alone in induction, consolidated by chemotherapy plus imatinib followed by a stem cell transplant is a feasible, well-tolerated and effective strategy for adult Philadelphia positive acute lymphoblastic leukemia, leading to the best long-term survival rates so far reported. (clinicaltrials.gov identifier: 00458848).

Multicenter Total Therapy Gimema LAL 1509 Protocol for De Novo Adult Ph+ Acute Lymphoblastic Leukemia (ALL) Patients. Updated Results and Refined Genetic-Based Prognostic Stratification

Introduction: Management of Ph+ ALL has changed since the introduction of tyrosine kinase inhibitors (TKI). We previously reported the preliminary findings of the GIMEMA LAL 1509 total therapy protocol, based on dasatinib plus steroids administration as induction therapy (Chiaretti et al, ASH 2014). The updated results on overall survival (OS), disease-free survival (DFS) and the impact of a genetic-based prognostic stratification are hereby provided.Methods: Steroids were administered from day -6 to day 31. Dasatinib (140 mg/day) was given between days 1 and 84. Patients reaching a complete molecular response (CMR, i.e. BCR/ABL1 to ABL1 ratio=0) at the end of induction (day 85) continued Dasatinib. Patients in complete hematologic remission (CHR), but not in CMR, underwent chemotherapy (clofarabine-cyclophosphamide) and/or an allogeneic transplant (HSCT), according to eligibility and donor availability. Dasatinib was administered until disease progression. Molecular testing was used to identify the presence of the BCR/ABL1 transcript on bone marrow samples, to define the fusion protein and to quantify BCR/ABL1 levels at baseline and follow-up (FU). Mutational screening was performed in relapsed cases, based on material availability. SNP array analysis was carried out using the Cytoscan HD arrays (Affymetrix, Santa Clara, CA) to identify genomic aberrations.Results: 60/63 enrolled patients were eligible. Median age was 41.9 years (range 18.7-59.1), 34 were males and 26 females; median WBC count was 12.5 x 109/l (range 1.4-178.0); the p190 fusion product was detected in 33 patients, p210 in 18 and p190/p210 in 9. Median FU is 28.4 months (range 4.2-43.7). After the steroid pre-phase, 38 patients (63%) had a blast reduction ≥75%. At day 85, 58 patients were in CHR (97%), while 2, in CHR at day 57, lost it: both harbored the p210 fusion transcript. They both returned into CHR following chemotherapy. A sustained CMR was obtained in 11 patients (18.6%): 72% had a p190 fusion transcript. No deaths in induction occurred. Among the CMR patients, only 1 experienced a hematologic relapse, which carried a T315I mutation. Of the 46 non-CMR cases, 14 relapses occurred, 8 of which in p210+ patients. Overall, there have been 12 deaths in CHR. OS is 58.3% (95%CI: 44.4-76.3) at 36 months and DFS at 30 months is 48.9% (95%CI: 36.8.0-64.9). A better DFS was observed in patients who obtained a CMR compared to cases with minimal residual disease (MRD) at day 85 (75% vs 44%, p=0.06), and in p190+ vs p210+ patients (57.1% vs 39.6%, p=ns). Mutational screening, performed in 7/15 cases at hematologic relapse detected mutations in 5: 3 T315I and 2 V299L, of which 1 with a concomitant F317I and F317L. SNP array analysis, performed in 39 cases with available DNA, showed that the most frequent aberrations were deletions of IKZF1 (85%), PAX5 (38%), CDKN2A/B (33%), MLLT3 (33%), RB1 (28%) and JAK2 (28%). While IKZF1 deletions alone did not impact on CHR or CMRachievement and DFS, a significantly worse DFS (p=0.01) and increased cumulative incidence of relapse (CIR, p=0.024) were observed in cases harboring deletions of IKZF1 plus CDKN2A/B and PAX5 (DFS: 40% vs 65% at 18 months; CIR: 40% vs 14% at 18 months (Fig. 1A and B). The relevance of this finding was further refined by stratifying patients according to the fusion protein: the impact of IKZF1 plus CDKN2A/B and PAX5 deletions is prognostically relevant in p190+, but not in p210+ patients, possibly because of the worse outcome of the latter group (Fig. 2). Finally, this analysis identified a set of genes specifically deleted in CMR cases; investigations are ongoing on additional cases to validate their potential role in predicting response to TKI.Conclusions: In this updated analysis of the GIMEMA 1509 trial, we confirm the effectiveness of a chemo-free induction in inducing CHR in almost all adult Ph+ ALL patients (97%) and CMR in a subgroup of cases (18.6%). OS and DFS at 36 months and 30 months, approaching 60% and 50%, are encouraging. More importantly, CMR achievement at day 85 is associated with extremely promising results, being 75% at 30 months, underlying that CMR should be regarded as a primary endpoint in Ph+ ALL. We confirm that p210+ patients may require an intensified approach, given the lower rate of CMR achievement and the higher relapse rate. Finally, we provide evidence that a broader genetic characterization at diagnosis allows a more refined prognostic stratification of Ph+ ALL patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresMartinelli:Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; MSD: Consultancy; Novartis: Consultancy, Speakers Bureau. Foà:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

First Results of the Multicenter Total Therapy Gimema LAL 1509 Protocol for De Novo Adult Philadelphia Chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Patients

▪Introduction: Ph+ ALL management has changed since the introduction of tyrosine kinase inhibitors (TKI). In the GIMEMA LAL 1509 protocol for adult Ph+ ALL, Dasatinib was administered in combination with steroids as induction treatment, and chemotherapy and/or allogeneic transplantation (HSCT) was given if patients did not reach a sustained complete molecular response (CMR) after induction.Methods: Steroids were administered from day -6 to day 31. Dasatinib (140 mg/day) was given between days 1 and 84. Patients reaching a confirmed CMR (i.e. BCR/ABL to ABL ratio=0) at the end of induction (day +85), continued Dasatinib for 6 additional months. Patients in complete hematologic remission (CHR), but not in CMR, were stratified according to HSCT eligibility: those with a promptly available donor underwent directly a HSCT; if the donor was not readily available, a consolidation cycle with clofarabine and cyclophosphamide was administered. HSCT non-eligible patients received two cycles of clofarabine and cyclophosphamide of 5 and 3 days, respectively. Dasatinib was administered until disease progression. Molecular testing was used to identify the presence of a BCR/ABL transcript, define the fusion protein and quantify BCR/ABL levels at baseline and at follow-up. Mutational screening was performed in relapsed cases.Results: Sixty of the 64 enrolled patients were eligible. Median age was 41.9 years (range: 18.7-59.1), 34 were males and 26 females; the median WBC count was 12.5 x 109/l (range 1.4-178.0); the p190 fusion product was detected in 33 patients, p210 in 18 and p190/p210 in 9. Median follow-up is 17.6 months (range 4.1-31.6). After the steroid pre-phase, 38 patients (63%) had a blast reduction ≥75%. At day 85, 58 patients achieved a CHR (97%) and 2 were non-responders (1 hematologic relapse and 1 disease progression): notably, both had a p210 fusion transcript. A CMR was obtained in 11 patients (18.6%): of these, 72% had a p190 fusion transcript. Overall, 24% of p190+ patients and 11% of p210+ or p190/210+ cases obtained a CMR. As previously reported in other GIMEMA trials, no deaths in induction occurred. Among the CMR patients, only 1 experienced a hematologic relapse and harbored a T315I mutation. Among the 46 non-CMR cases, 17/27 eligible patients underwent a HSCT, while 9 did not; 5 relapses occurred in the non-transplanted group, while 1 was observed in the HSCT group. Of the 19 HSCT non-eligible patients, 14 received the planned therapy: in this group, 2 relapses occurred. At 24 months, the overall survival (OS) is 69.1% (95%CI: 55.3-86.3), with a disease-free survival (DFS) at 18 months of 61.6% (95%CI: 47.6.0-79.8). A better DFS was observed in patients who obtained a CMR at day +85. Dasatinib exerted a rapid blast clearance, that was highly significant (p<0.0001) between the onset and day +22, and between day +22 and +45; a significantly more rapid clearance (p=0.0005, p=0.0013 and p=0.001 at days +22, +45 and +57, respectively) was observed in patients who achieved a CMR at day +85, underlining the heterogeneous sensitivity of Ph+ ALL blasts to TKI. Importantly, a significantly greater clearance was observed in p190 (n=33) vs p190/210 and p210 (n=27) cases (p=0.0008, p=0.0032, p=0.0031, p=0.0016, p=0.007 at diagnosis, days +22, +45, +57, and +85, respectively). Mutational screening was performed in 6/12 patients who relapsed and mutations were detected in 5: 3 T315I and 2 V299L, of which 1 with a concomitant F317L. Overall, 12 relapses (5 in the p190 and 7 in the p210 subgroup) and 7 deaths in CR occurred, 5 of which in allografted patients. The median time to relapse was 6.7 months (range 0.3-22.5).Conclusions: We confirm that a chemo-free induction approach for adult Ph+ ALL patients results in a very high CHR rate (97%), with no deaths in induction. In this Dasatinib-based protocol, a sustained CMR is achieved after induction in >18% of cases, suggesting that a subset of patients may be spared further intensive treatment. We witnessed an increase in p190/p210-p210+ cases compared to earlier studies; these patients showed a worse prognosis as underlined by a lower susceptibility to TKI, lower blast clearance and greater incidence of relapses (58%). At this first analysis, OS and DFS are at least comparable to those obtained in previous GIMEMA studies, despite the higher incidence of p210+ patients – which accounted for 45% of cases in this cohort – and which may require an intensified approach. DisclosuresMartinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ARIAD: Consultancy.

The therapeutic response and clinical outcome of adults with ALL1(MLL)/AF4 fusion positive acute lymphoblastic leukemia according to the GIMEMA experience

The clinical outcome of 21 adults with ALL1(MLL)/AF4 positive acute lymphoblastic leukemia enrolled in the GIMEMA LAL 2000 trial and of 25 patients entered into the previous 0496 study is reported. LAL 2000 included more intensive consolidation and transplants. Complete remission rates were 90% and 88% in the LAL 2000 and 0496 trials, respectively. Fifteen patients were transplanted (5 autologous, 10 allogeneic). At 36 months, overall and disease free survivals were 32.9%, 31.8%, 28% and 27.3%, in LAL 2000 and 0496 trials, respectively. Relapses remained the main reason of failure occurring in 10 and 16 of the 19 and 22 responding patients. In the LAL 2000 study, 4 relapses were observed before transplant. Thus, ALL1(MLL)/AF4 abnormality characterized a subset of patients with adverse prognosis in which the overall strategy adopted in the LAL 2000 study, rather than transplants per se, failed to improve the patient clinical outcome.

Advanced Philadelphia chromosome positive acute lymphoblastic leukemia patients relapsed after treatment with tyrosine-kinase inhibitors: successful response to clofarabine and cyclophosphamide

Advanced Philadelphia chromosome positive acute lymphoblastic leukemia patients relapsed after treatment with tyrosine-kinase inhibitors: successful response to clofarabine and cyclophosphamide

Line Treatment of Adult Ph+ Acute Lymphoblastic Leukemia (ALL) Patients. Final Results of the GIMEMA LAL1205 Study

Background: Dasatinib is a potent, oral inhibitor of the BCR-ABL, c-KIT and SRC kinase family, which has proven to be a more active inhibitor of BCR-ABL and c-KIT than imatinib in preclinical studies. Clinically, it has been shown to be effective in chronic myeloid leukemia and in Ph+ ALL patients resistant or intolerant to imatinib.Aims: The primary objective of the study was to assess the activity of dasatinib in de novo adult Ph+ ALL patients in terms of complete hematological remission (CHR); the secondary objectives were treatment toxicity, rate of immunophenotypic and molecular responses, disease-free survival (DFS), relapse rate and overall survival (OS).Methods: The GIMEMA LAL 1205 protocol was designed for patients ≥18 years (no upper age limit) who receive dasatinib po, 70 mg BID. A steroid pre-phase is started 7 days prior to dasatinib administration and continued up to day 31, and then tapered. The pre-phase allows identification of the BCR/ABL transcript. Dasatinib is given for 12 weeks. Two intrathecal methotrexates are administered at days +22 and +43. All cases are analyzed through a central handling of samples at presentation for morphology, immunophenotype, cytogenetics and molecular biology at the coordinating center in Rome. Minimal residual disease (MRD) is also centrally investigated by flow-cytometry and Q-RT-PCR at days +22, +43, +57 and +84. The protocol was designed for 48 patients.Results: Recruitment started in November 2006 and was completed in August 2008. The median age of the 48 enrolled BCR/ABL+ ALL patients was 54 years (range 24-76), 26 were females and 22 males. The median WBC count was 20.1 (range 2.2–133). The last analysis has been conducted on 36 patients. One patient stopped treatment after 14 days due to intestinal toxicity and 1 patient refused treatment. Thus, to date 34 patients are evaluable for response. Nineteen cases were p190+ and 15 p210+ (5 were p190/p210+). A >75% response to the steroid pre-phase was recorded in 82.7% of patients. All 34 patients (100%) have witnessed a CHR: 32 (94.12%) at the 1st determination at day +22, 1 at the 2nd at day +43 and 1 at the 3rd at day +57. No fatalities have been observed. In 13 patients, at least 1 severe adverse event (SAE) has been recorded, for a total of 26 SAEs. Overall, the compliance has been good; only 1 patient stopped treatment at day 67 due to toxicity while in CHR. The median follow-up is so far 11.2 months. The OS at 10 months is 80.7%. Immunophenotypic and BCR/ABL Q-RT-PCR monitoring of MRD has shown a very marked clearance of leukemic cells by day +22, progressively strengthened at the subsequent timepoints. In p190+ cases the minimal MRD value was reached at day +43, while in p210+ cases this was observed at day +84, documenting a lower susceptibility to dasatinib by this molecular subgroup. So far, 9 patients have relapsed, at a median of 72 days from the end of induction; in agreement with the MRD data, 7/9 relapses occurred within the p210+ cases and 2/9 within the p190+ cases. The presence of mutations has been investigated in 8/9 relapsed samples: 5 showed a T315I mutation, 1 an E255K mutation and 2 were wt. Cloning experiments allowed to detect in 2 cases low levels of T315I mutations already at presentation and on MRD cells at the end of the induction treatment. The first multivariate analysis has been conducted for DFS and the degree of PCR reduction − ≤10−3 vs ≥10−3 - has so far emerged as the only significant prognostic factor.Conclusions: This study demonstrates that in adult Ph+ ALL dasatinib monotherapy is capable of inducing a CHR in virtually all patients, irrespective of age, without important toxicities and with no fatalities. The hematological response is associated with a very marked and rapid debulking of the neoplastic clone documented at the MRD level, particularly for p190+ cases. The degree of PCR response is of prognostic relevance. No relapses have been observed during the induction phase. The optimal post-induction treatment strategy, which was not part of the study, remains to be defined, particularly in light of the genetic instability of Ph+ ALL and of the selection/induction of mutations. These results, together with those previously reported by our group with imatinib alone for elderly patients, question the use of chemotherapy, with or without a TK inhibitor, for the remission induction of Ph+ ALL.

High Levels of Tumor Load Reduction after Intensive Chemotherapy Favorably Affect the Prognosis of BCR/ABL+ ALL Adult Patients Receiving Imatinib Mesylate Alone as a Post-Consolidation Treatment According to the Italian GIMEMA LAL 0201/A Protocol.

Thirty-six adult patients aged between 20 and 60 years (median age: 44) with BCR-ABL+ ALL were prospectively monitored by Q-RT-PCR between December 2001 to November 2005. All patients were treated according to the GIMEMA LAL 0201/A protocol, in which Imatinib alone, was administered as post-consolidation therapy, in responding patients after high-dose induction and consolidation treatment, at the dosage of 400 mg x 2 daily for at least six months and continued on the basis of the opinion of physicians. Twenty-three (64%) were p190+ and 13 (36%) p210+. At presentation, the median BCR-ABL/ABL ratio was 1.33 (range 0.23 to 7.8). Before Imatinib, all patients were in 1st CR (34 after the first induction and consolidation course; 2 after a salvage treatment). At this time point, the median reduction from baseline in BCR-ABL transcripts, calculated for each individual patients, was 2.1 log (range: 0 to 6.1). In particular, 18 of the 36 cases showing a log reduction of the BCR-ABL/ABL ratio > 2.1 (p190=14 cases; p210 = 4 cases) were considered as good responders to the antecedent chemotherapies, whereas 18 cases (p190=9; p210=9 cases) showing a log reduction ≤2.1 were considered as bad responders. The median reduction from baseline in BCR-ABL levels was 2.49 log (range: 0.00–6.1) and 1.45 log ( range: 0.19–5.34) in p190 and p210 positive cases, respectively (p=.16). During Imatinib treatment, 10 (56%) of the 18 poor responders patients presented a relapse of disease versus 5 (28%) ( 3 =BM; 2=CNS) of the 18 good responders.At last follow-up 16 patients were in first CR; of these patients, only 4 were allo-transplanted (2 good, 2 bad responders), therefore 12 (9 good, 3 bad responders) persisted in first CR maintained by Imatinib alone after a median follow-up of 24 months (range: 7–48). At 24 months, the cumulative incidence of relapse was 29% vs 44% for good and bad responders (p=.034), respectively, whereas, the DFS and OS rates were 65% vs 33% ( p=.0032) and 76% vs 32% (p=.0056) for good and poor responder patients, respectively. Monitoring of MRD was prospectively evaluated at the starting of Imatinib therapy and afterwards at the 3rd, 5th and 6th month. At these time points, the median BCR-BCR/ABL ratio were 0.015, −0.006, −0.008 and −0.009, respectively. In addition to the classification as good/ bad responders, the current variation of BCR-ABL ratio (used as time-dependent covariate in a Cox model), was associated with an increase of the hazard of failure (hazard ratio of an increase by 2 log of the BCR-ABL levels compared to a constant ratio equal to 2.1). In conclusion, Imatinib mesylate is a highly effective post-consolidation treatment for adult Ph+ ALL patients. However, the clinical outcome after Imatinib was significantly affected by the level of molecular response evaluated before the starting of Imatinib, resulting excellent for the good responders but remaining dismal for patients who responded poorly to the initial chemotherapy.

A New Method To Evaluate the Prognostic Value of Response to Prednisone Pre-Treatment in Adult (15–60 Yrs) Patients with Acute Lymphoblastic Leukemia: Results of the GIMEMA LAL 2000 Study.

The prognostic significance of the response to initial prednisone treatment in adult ALL has been recently emphasized. Prednisone response is usually defined on the basis of the peripheral leukemic blast count. The threshold value for the defintion as good or poor prednisone response is 1000 blasts/mmc on day 8 of prednisone pre-treatment. The drawback of this definition is the difficulty of classifying patients with less than 1000 blasts at diagnosis. In the LAL2000 GIMEMA study we therefore evaluated whether the blast reduction rate, which is not affected by the initial blast level, could be a factor with comparable prognostic value. The protocol design provided a 7-day (−6 to 0) pre-treatment phase with an escalating dose of prednisone up to 60 mg/sqm. On day 1 before starting the induction the response was assessed both according to the absolute blast count (< versus ≥ 1000/mmc) (criterion 1) and according to the blast reduction rate ≥ 75% (criterion 2) in the peripheral blood. The induction included high dose Daunorubicin; for patients in complete remission (CR) this was followed by consolidation with high dose ARA-C, chemo and radio prophylaxis of the central nervous system, and periodical reinduction over a three years maintenance period. Patients with adverse cytogenetic features [i.e. t(9;22), t(4;11), t(1;19)] who achieved a CR were treated according to the HAM protocol that included high dose ARA-C and Mitoxantrone followed by Imatinib for Ph+ ALL and by allogeneic or autologous hemopoietic stem cells transplantation for the others. Between September 2000 and December 2003 a total of 368 patients were evaluable for response to induction. The median age was 35 years (15–60) and median WBC count 15′109/L (0.3–872); 72 (20%) were T ALL and 121 (33%) had cytogenetic high risk features (104 (86%) Ph+, 4 (3%) t(4;11) and 13 (11%) t(1;19)). Eighty-seven percent of the patients were evaluable for response to steroid pre-treatment: 'responders' were 75% according to criterion 1 (blast <1′109/L on day 0), and 80% according to criterion 2 (blast reduction rate ≤75% on day 0). The overall CR rate was 83%. The probability of response was significantly higher in prednisone responders with respect to non responders according to both criteria: 87% versus 63% (p<0.0001) according to criterion 1, 85% versus 68% (p=0.001) according to criterion 2. Also the post CR outcome was better in steroid responders, regardless of the definition. Using criterion 1, median disease-free survival (DFS) was 24 months in responders and 11 months in non responders; using criterion 2, median DFS was 23 months in responders and 12 months in non responders. Both criteria were significantly related to DFS in a multivariate analysis adjusted for cytogenetic risk and WBC count at diagnosis (>=/<50). In conclusion, our study confirms that the sensitivity to steroids is an independent prognostic factor for the outcome of adult ALL; moreover, we propose an alternative method of its evaluation with respect to the one currently used. This method has the advantage of allowing to classify all patients, regardless of the initial blasts level, and shows a comparable prognostic value.

Breast Cancer Resistance Protein (BCRP) Expression Affects Event-Free Survival in Adult Acute Lymphoblastic Leukemia Patients Uniformly Treated According to the GIMEMA LAL 2000 Protocol.

There are currently no reports concerning the role of the transporter breast cancer resistance protein (BCRP) in adult acute lymphoblastic leukemia (ALL). The aim of this study was to evaluate the frequency of BCRP expression, its correlation with other MDR-related proteins and their prognostic role in 118 untreated adult ALL patients uniformly treated according to the GIMEMA protocol LAL2000. BCRP protein expression was detected by flow cytometry using the MoAb BXP-34 (Kamiya, WA); results were analyzed according to the KS statistic test (D-value). Detection of BCRP in the cell lines MCF7pcDNA3 and MDA231pcDNA3 showed a D-value of 0.12±0.11 and 0.09±0.06, respectively. In contrast, the cell lines MCF7pcDNA3clone 8 and MDA231pcDNA3clone23 that overexpress BCRP were characterized by a D-value of 0.44±0.21 and 0.33±0.11, respectively. Analysis of primary ALL samples showed a BCRP expression (D-value≥ 0.20) in 86/118 (72.9%) cases, with a mean value of 0.36±0.22 (range 0.00–0.87, median 0.37) in the overall population. BCRP expression resulted associated with age (P=0.054). No significant difference was found with other clinical characteristics. The multidrug resistance associated protein (MRP1) (MoAb MRPm6) resulted expressed in 70.1% of samples (D-value≥ 0.20); the MDR1/Pglycoprotein-170 (MDR1) (MoAb MRK16) was present in 28.2% of cases (D-value≥ 0.05). Samples analyzed for both BCRP and MRP1 expression (115/118) showed a significant correlation (R=0.56; P=0.0001): 15.6% of cases were negative for both proteins, while 61.7% expressed both BCRP and MRP1. BCRP expression did not correlate with MDR1 expression. None of these proteins separately influenced the achievement of complete remission (CR). In contrast, BCRP/MRP1 co-expression was associated (P=0.091) with failure to respond to induction treatment: 31% (18/58) of BCRP+/MRP1+ patients failed to achieve CR, while 84.2% (32/38) of cases negative for only one protein or for both responded to induction treatment. Kaplan-Meier analysis was then performed for event-free survival (EFS): among 99 evaluable patients a significant difference (P=0.03) was observed with a median EFS of 8.4 months (95%CI, 5.63–11.28) in BCRP+ cases (72) compared to BCRP- patients (27) for whom median EFS has not been achieved. Multivariate analysis confirmed the unfavorable prognostic role of BCRP positive cases on EFS (P=0.072;OR1.87, 95% CI, 0.94–3.70). In summary, our study shows that adult ALL frequently express BCRP. In patients treated with the GIMEMA LAL 2000 protocol EFS was unfavorably affected by BCRP expression, while co-expression of MRP1 and BCRP was associated with failure to induction treatment. The expression of these proteins may contribute to explain the overall poor outcome of adult ALL patients, suggesting that therapeutic strategies based on overcoming drug resistance remain an important goal to be pursued.

Role of Extracellular Signal-Regulated Kinase-1/2 (ERK) on Complete Remission Achievement of Primary Adult Acute Lymphoblastic Leukemia Patients Enrolled in the GIMEMA Protocol LAL 2000: In Vitro Activity of the MEK Inhibithor PD98059.

The mitogen-activated protein kinase (MAPK) pathway contributes to the proliferative and differentiative control of hematopoietic cells. Extracellular signal-regulated kinase-1/2 (ERK) is one of the members of the MAPK pathway; its constitutive activation (p-ERK) has been found in a broad spectrum of hematological malignancies, particularly in acute myeloid leukemia (AML). A molecular therapeutic approach based on MAPK inhibitors has been therefore attempted. The aim of this study was to assess the expression and the clinical role of p-ERK in adult acute lymphoblastic leukemia (ALL) evaluating, in addition, the in vitro effects of the MEK inhibitor PD98059, used alone or in combination with chemotherapy, on the proliferation and apoptosis of ALL blast cells. Primary samples from 131 uniformly treated de novo patients enrolled in the GIMEMA LAL 2000 protocol were evaluated, using a p-ERK1/2-specific MoAb (clone E10), by Western blot analysis and flow cytometric assay which allowed single cell phospho-protein determination by the Kolmogorov-Smirnov statistic test (D-value). Lymphoid cell lines (Jurkat, CEM, RAJI and RPMI8866), as well as normal PMA-activated PBL expressed p-ERK, whereas resting PBL exhibited minimal levels (D-value <0.10). In clinical ALL samples p-ERK ranged between 0 and 0.83, constitutive activation (D-value ≥ 0.10) was found in 45/131 samples (34.5%) and resulted significantly associated with higher WBC values (>50x109/L; P=0.0128) and lower RNA-index of G1 phase (P=0.0296). Age, leukemia phenotype and molecular characteristics did not associate with p-ERK expression, while phospho-protein levels, measured as a continuous variable, correlated with failure to achieve CR (P=0.069). According to cell availability, we evaluated the effects of the MEK inhibitor PD98059 at 25μM on 30 fresh ALL samples, cultured in vitro for 24 hours. Eight of them were characterized by constitutive ERK activation, which was efficiently abrogated in 6; however, this effect did not induce cell cycle changes or apoptosis either alone or in combination with chemotherapeutic agents (Ara-C and/or Dexametasone). In summary, our study demonstrated that p-ERK is expressed in one third of adult ALL samples at presentation. Patients with p-ERK expression are characterized by higher leukemic mass, with a trend to induction treatment failure. MEK inhibition by PD98059 is capable of modulating p-ERK in primary ALL cells, though it does not result in cell cycle/apoptotic changes. In conclusion, ERK signaling appears only partially involved in the control of proliferative signals in adult ALL and modulation of additional pathways may result in more effective therapeutic strategies.

Intensification of Post-Remissional Treatment in Adult Acute Lymphoblastic Leukemia (ALL) with t(4;11) or t(1;19): Results of the GIMEMA LAL 2000 Study.

In the GIMEMA LAL 0496 trial, which has been conducted in Italy between 1996 and 2000, patients with adult ALL received an induction therapy with VCR, DNR and ASP until they reached complete remission (CR). Consolidation therapy with Ara-C and CTX was then administered and, if still in CR, patients received a 3-year maintenance therapy. Ph+ ALL patients were considered at high risk and after the induction phase a stem cell transplant (SCT) was planned, allogeneic for patients with an HLA identical sibling or autologous. A total of 498 patients have been enrolled by 50 GIMEMA Centers. Of these, 29 patients (5.8%) with either t(4;11) or t(1;19), who received the standard post-remissional approach, showed a statistically significant worse prognosis after CR: the projected probabilities of DFS and OS at 2 years were 19% (C.I. 95%: 4.1–34.4) and 31% (C.I. 95%: 14.2–47.9), respectively. Thus, in the new protocol - GIMEMA LAL 2000 - these patients received an intensive post-remissional treatment including SCT. Between 2000 and 2003, a total of 383 patients have been enrolled in this new protocol. Of these, 23 (6.0%) were positive either for t(4;11) or for t(1;19). The clinical characteristics of these patients are comparable to those of the patients enrolled in the previous study and the overall CR rate is also super imposable.GIMEMA LAL 0496 vs GIMEMA LAL 2000Fig. 1 shows the comparison in DFS at 2 years between LAL 0496 and LAL 2000; though not statistically significant, a trend towards a better prognosis is clearly seen. The DFS probability projected at 2 years is 52% (95% C.I. 26–78) compared to 19% (95% C.I. 4–35). The OS, projected at 2 years, is 42% (95% C.I. 14–70) compared to 31% (C.I. 95%: 14.2–47.9).Despite the relatively limited case series, these results suggest that for t(4;11)+ and t(1;19)+ adult ALL intensification of the post-remissional treatment with SCT is a feasible approach that may improve the outcome in this subset of patients at high risk of relapse with standard post-remissional therapies. [Display omitted] GIMEMA LAL 0496GIMEMA LAL 2000pPatients t(4;11) or t(1;19)29230.7626Sex (M/F)14/158/150.3280Age yrs, median (min-max)37.8 (14.9–59.4)39.4 (19.6–59.2)0.1481WBC x 109/L median (min-max)41.2 (2.2–663.0)50.3 (2.4–300.0)0.9193Phenotype (B/T)29/023/0-CR rate92.8%85.7%0.6392

Clinical Impact of Q-RT-PCR Monitoring of Minimal Residual Disease in Newly Diagnosed Adult Patients with BCR-ABL+ Acute Lymphoblastic Leukemia Receiving Imatinib Mesylate Alone as a Post-Consolidation Treatment According to the Italian GIMEMA LAL 0201/A Protocol.

Twenty-six adult patients (pts) aged between 22 and 60 years (median age: 46) with BCR-ABL+ acute lymphoblastic leukemia (ALL) were prospectively monitored by Q-RT-PCR between August 2001 and July 2004. All pts entered the GIMEMA LAL 0201/A protocol, in which Imatinib alone, at the dosage of 400 mg x 2 daily for at least six months, was administered as post-consolidation therapy in responding pts after intensive chemotherapy (CHT). Eighteen pts (69%) were p190+and 8 (31%) p210+ with/without the p190. In these two groups, the mean number of BCR-ABL copies at diagnosis (ie: BCR-ABL/ABL x 104) was 13,052 (range: 1,466–35,449) and 22,487 (range: 7,315–78,000) (p=ns), respectively. All pts were in 1st complete hematologic remission (25 pts after the first induction-consolidation course; 1 pt after a salvage treatment). Before Imatinib, 8 of the 18 p190+ pts (44%) showed a BCR-ABL copy reduction of ≥ 3 log compared to the levels at diagnosis, (mean BCR-ABL copies 3.6; range: 0–10), and they were defined as good responders to CHT. The remaining 10 pts (56%), defined as poor responders, showed a reduction of < 3 log (mean copies 2,825.8; range: 12 – 25,245). In the 10 poor responders, BCR-ABL copies constantly increased over time and this was predictive of an hematologic relapse in 8/10 patients. By contrast, 7 of the 8 good responder pts during Imatinib treatment persistently showed levels of BCR-ABL below 10. These 7 pts are in CCR maintained by Imatinib alone at 6, 9, 13, 13, 17, 21 and 23 months, respectively. In 1 pt, at 6 months from the start of Imatinib, a CNS relapse preceded a marked increase in the BCR-ABL copy numbers in BM cells. Therefore, after a median follow-up of 6 months (range 3 – 23), for poor responders, and of 13 months (range 6 – 30), for good responders, the actuarial probability of relapse was 100% and 12.5%, at 24 months (p=.027), respectively. p210+ pts achieved a molecular response rate slightly different respect to the p190+ cases. In fact, before starting Imatinib, only 2 of the 8 cases (25%) analyzed showed a reduction in BCR/ABL copies ≥ 3 log whereas in the remaining 6 pts, 1 relapsed at 6 months, but the other 5 showed a decrease of the BCR-ABL copies that in 2 cases fell below the 3 log reduction after 8 and 12 months, respectively. Altogether, after a median follow-up of 6 months (range: 2–28), 7/8 p210+ pts were in CCR maintained by Imatinib alone without transplant at 2, 4, 5, 6, 19, 24 and 28 months, respectively. In conclusion, Imatinib is a highly effective post-consolidation treatment for adult Ph+ ALL pts being able to maintain and/or to induce a minimum level of BCR-ABL expression, without the high morbidity and mortality relating to allografting procedures. In addition, in the p190+ cases an unsatisfactory molecular response rate after CHT was a powerful predictor of a subsequent clinical resistance to Imatinib.