Patients In Major Molecular Response Research Articles

Excellent Early and Major Molecular Responses Observed with Asciminib Treatment for CP-CML: Results from the ALLG CML13 Ascend-CML Study

ATP-competitive BCR::ABL1 inhibitors are effective in many patients with CP-CML patients. However, a significant minority experience treatment failure with currently available drugs due to resistance or toxicity. Asciminib, a first-in-class myristoyl site BCR::ABL1 inhibitor, is now indicated in many jurisdictions for CP-CML after ≥2 prior lines of treatment. The Australasian Leukaemia and Lymphoma Group (ALLG) CML13 ASCEND-CML trial assessed its tolerability and efficacy in the frontline setting, where data are scarce. ALLG CML13 is a prospective open-label phase II study that completed enrolment in Dec 2022 at 14 sites. Patients commenced asciminib 40mg twice a day (BID) at time of diagnosis, and were assessed according to optimal 2020 ELN targets ( BCR::ABL1 ≤10%, ≤1% & ≤0.1% at 3, 6, 12 months respectively); and ≤0.01% at 18 months. Patients in Major Molecular Response (MMR; BCR::ABL1 ≤0.1%) at 12 months were switched to 80mg QD for ease of administration. Patients with treatment failure ( BCR::ABL1 >10% at 3 or 6 months; BCR::ABL1 >1% at 12 or 18 months) continued asciminib, with additional imatinib or dasatinib or nilotinib, according to physician preference. Patients with a suboptimal response at 6, 12, or 18 months, but without treatment failure, continued asciminib but with the dose doubled to 80mg BID. This is a pre-specified analysis of the co-primary end points: i) final analysis of Early Molecular Response (EMR, BCR::ABL1 ≤10% at 3 months) and ii) interim analysis of MMR by 12 months. By data cut-off in May 2023, 101 patients had median follow-up of 20 (0-29) months (including 1 patient who enrolled but never started therapy due to elevated lipase at baseline). Grade 3/4 adverse events (AEs) included neutropenia and thrombocytopenia, (reported in 6% and 5% of patients, respectively); increased lipase/amylase reported in 8% (clinical pancreatitis reported in 2%), anaemia (2%), infection (2%) and 1% each of increased AST/ALT, back, abdominal and chest pain from pulmonary embolism. The only treatment emergent vascular event was a stroke, reported in a male patient aged 73 with pre-existing diabetes and hypertension, after 20 months of asciminib. Fifteen patients discontinued asciminib: 6 due to AEs (2 with persistent cytopenia requiring an allograft, 3 with recurrent asymptomatic lipase elevation, 1 with pancreatitis); 3 lost to follow-up; and 6 due to resistant disease (1 confirmed loss of MMR at 9 months, without ABL1 mutation; 1 transformed to lymphoid blast crisis at 6 months with myristoyl site mutations A337T/V and P465S; 1 loss of MMR at 6 months with A337T mutation; 1 loss of MR2 with V506L; and 2 with failure to achieve EMR - below). No other transformation occurred. During the first 3 months of treatment, 85% of patients had an assigned dose density of >90%; 9% of patients dose reduced to <66%. EMR was achieved by 94/101 patients (93.1%; 96% CI 86-97%): 1 patient never started treatment, 1 patient missed this assessment, 3 patients had withdrawn by this timepoint, and 2 had BCR::ABL1 of 24% and 210%, respectively. Neither of the EMR failure patients had myristoyl site mutations; both were subsequently intolerant of combination TKI/asciminib therapy because of cytopenia and headaches, respectively and are now on dasatinib monotherapy. Six patients dose escalated and remain on asciminib 80mg BID: 3 after failing to achieve MMR by 12 months, & 3 failing to achieve MR4 after 18 months; 2 have since achieved their targets. By 3 months, 48/101 (48%) patients achieved MMR and 12 (11.9%) achieved MR4. Considering only patients with >12 months of follow up, the interim rate of MMR by 12 months is 62/75 patients (82.7%; 98% CI 70-92%). With “off-protocol for any reason” as the competing risk, the cumulative incidences of MMR & MR4.5 by 12 months were 81.1% & 32.3% respectively (Figure). ASCEND is one of the first studies using frontline asciminib, with excellent achievement of EMR and MMR, and low transformation rate. Rates of MMR at 12 months are comparable to those reported with second generation TKIs, though achievement of deeper responses appears more rapid with asciminib. Treatment was well tolerated in general: majority of AEs were grade I/II events that did not result in treatment discontinuation, and only one vascular event was observed. Early achievement of MMR and MR4 may accelerate eligibility for, & increased success of, treatment-free remission attempts.

Somatic Mutations in Myeloid Transcription Factors and in Activated Signaling Pathway, but Not in Epigenetic Modifier Pathway, Predict the Risk of Treatment Failure and Progression to Advanced Phase in Chronic Myeloid Leukemia

Background Next-generation sequencing (NGS) is currently reforming contemporary clinical practice into precision medicine incorporating genomics-based knowledge into the clinic. Recently, multiple studies showed that mutations in epigenetic modifier genes are associated with poor treatment outcomes following tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML), particularly after Imatinib treatment. However, it is uncertain whether they either originated from clonal hematopoiesis or the CML clone. For example, ASXL1 mutation is the most recurrent mutation at CML diagnosis, and its presence is correlated with adverse prognosis in CML. Finding of rapid reduction in ASXL1 allele frequency following TKI therapy (Kim et al, Blood 2017) raises the question of whether it is a real driver of treatment failure in CML or not. In addition, it is of interest whether 2nd generation TKI (2G-TKI) therapies can abrogate the adverse impact of somatic mutations in CML. Accordingly, the present study attempted to evaluate the prognostic impact of mutation profiles on clinical outcomes in CML patients treated either with Imatinib or 2G-TKIs. Patients and Methods Our cohort includes a total of 394 samples including 254 samples collected at initial diagnosis and 140 samples taken during follow-up from 309 CML patients in two CML treating centers. Disease characteristics and treatment outcomes were evaluated and compared according to mutation profiles at initial diagnosis and during follow-up after TKI therapy. Treatment outcomes include any response with respect to major molecular response (MMR), molecular response (MR) with 2-log/deeper (MR2) or 4-log/deeper (MR4) with any lines of TKI therapy, failure-free survival (FFS), progression and overall survival (OS). Instead of conventional NGS, we applied smMIP-based NGS (limit of detection up to 0.2%) using our in-house CML-specific panel targeting 40 genes including epigenetic modifiers, myeloid transcription factors (TFs), tumour suppressors, genes in activated signaling, spliceosome and, cohesin complex. ResultsPatient group and treatment summary Summary of patient characteristics are as follows: median age of 54 years; male:female n=143/111 (56.3:43.7); disease status at presentation includes chronic- (n=233; 91.7%), accelerated- (n=15; 5.9%), blastic phase (n=4; 1.6%), or N/A (n=2; 0.8%); Sokal score, low (n=61, 24.0%), intermediate (n=84, 33.1%), high (n=74, 29.1%), or N/A (n=35, 13.8%). Frontline therapy includes Imatinib (n=190; 74.8%), or 2G-TKI (n=62) including Dasatinib (n=15; 6.0%), Nilotinib (n=37; 14.7%), Bosutinib (n=10; 4.0%), or other (n=2; 0.8%). Mutation profiles and their correlation with clinical outcomes following TKI therapy70 mutations were detected in 57 (22.4%) out of 254 diagnostic samples, while 64 mutations were detected in 39 (27.9%) out of 140 follow-up samples.Any mutation detected at initial diagnosis is associated with worse outcomes following TKI therapy. The adverse impact of somatic mutation was mainly from myeloid TF or activated signaling pathway mutations. Patients having those mutations at initial diagnosis showed higher risks of treatment failure (HR 2.8 95% CI [1.4-5.4], p = 0.003) and progression (HR 3.1 95% CI [1.1-9.1], p = 0.04), mainly when treated with Imatinib. On the other hand, the adverse impact of epigenetic modifiers was not apparent although a trend was observed toward a lower response rate for MR2 and MMR in patients carrying mutations in epigenetic modifiers.The adverse prognostic impact from these mutations seems to be abrogated by the use of 2G-TKI therapy, supporting the use of smMIP-based NGS for upfront TKI selection in newly diagnosed CML patients.In the cross-sectional analysis of follow-up samples, overall mutation frequency was significantly higher in the treatment failure or progression group compared to the optimal responders (p=0.012), suggesting longitudinal tracking of mutation profiles may help identify cases of impending treatment failure or progression early. Conclusion Our study shows that mutation profiling utilizing smMIP-based NGS can help identify patients with a potential adverse prognosis from initial diagnosis and may help upfront TKI selection. Further follow-up analysis is ongoing to evaluate the effect of TKI therapy on longitudinal kinetics of mutations in serially collected samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Natural killer cell dynamics and its association with response to imatinib-treated patients with chronic myeloid leukemia

ObjectiveThis study aims to assess the effect of natural killer (NK) cells and natural killer T (NKT) cells on response to imatinib (IM) therapy in patients with chronic myeloid leukemia (CML).BackgroundNK cells and NKT cells play a vital role in innate immunity against tumors. NKT cells recognize tumor cells by receptor-ligand interaction. After contact with the ligand, NKT cells activate NK cells through inflammatory cytokines [interferon-gamma (IFN-γ) and interleukin 2].Patients and methodsA total of 48 newly diagnosed patients with CML (chronic phase) were included. They were followed up for 1 year after the start of IM therapy and were categorized into IM-responder patients with CML (n = 23) and IM-resistant patients with CML (n = 25). Flow cytometry was used to measure NK and NKT cells. Serum levels of IFN-γ were determined by enzyme-linked immunoassay.ResultsWe found that NK cell% and NKT cell% were increased in the IM-responder group after 1 month of IM therapy. IFN-γ was increased at both presentation and 1 month after the start of IM therapy. Major molecular response was achieved earlier in the IM-responder group, with increased NK cell%, absolute count of NK cell, and IFN-γ.ConclusionWe conclude that NK cells have a critical role in the response of patients with CML to IM therapy and are a predictor of major molecular response in patients with CML.

Half: A Prospective Multi-Centre Phase II Clinical Trial Evaluating the Efficacy and Safety of Tyrosine Kinase Inhibitors' Discontinuation after Two-Step Dose Reduction in Patients with Chronic Myeloid Leukemia in Deep Molecular Remission

Background.Treatment-free remission (TFR) has become a new treatment goal for chronic myeloid leukemia (CML) patients. However, usually abrupt tyrosine kinase inhibitors (TKIs) therapy discontinuation has been successful only in about half of eligible patients and it can cause burdening TKI withdrawal syndrome (TWS) in about 30% of them. Moreover, any robust clinical or biological factor predictive for successful TFR has not been identified yet. On top of that, sustainable deep molecular response (DMR) as the main prerequisite for TKI discontinuation attempt is achieved only in 20-40% of patients. The majority of CML patients, therefore, need to be treated with the effective and well-tolerated drug for a long time or even life-long.Study design and methods.With the recognition of all these aspects, we designed a nationwide prospective investigator-initiated phase II clinical trial HALF (ClinicalTrials.gov NCT04147533) in order to evaluate efficacy and safety of TKI discontinuation after previous two-step dose reduction in patients with CML in DMR (Fig. 1). Step-wise TKI dose reduction, i.e. half of the standard during the first 6 months after study entry, and the same dose given alternatively (every other day) during the next 6 months, was derived from pharmacokinetics and experimental data as well as from clinical trials' results. We assume that the step-wise and eventually meaningful TKI dose reduction enables a higher rate of patients achieving successful TFR with less pronounce TWS, or even would represent a more reasonable and safer alternative to the complete and sudden TKI interruption. This unique nationwide academic project has been facilitated by hematological patients care centralization in the Czech Republic.A primary study objective is to evaluate the proportion of patients in major molecular response (MMR) at 6 and 12 months and in TFR at 18, 24, and 36 months after the study enrollment, respectively, and molecular recurrence-free survival at all mentioned time points as well. Main secondary and exploratory objectives are: to evaluate the proportion of patients loosing MMR and in whom MMR and MR4.0 would be re-achieved after TKI re-introduction, time to MMR and MR4.0 re-achievement, FFS, PFS, OS, TWS, and QoL assessment, predictive factors for successful TFR identification, quantification of BCR-ABL1 using digital droplet PCR at both the DNA and mRNA levels, immunological profiling, BCR-ABL1 kinetics mathematical modeling, assessment of TKI pharmacokinetics, clonal hematopoiesis and pharmaco-economics.Results.The study was launched in December 2019; however, due to the COVID-19 outbreak, patients' recruitment started on June 16, 2020. Here, characteristics of the first 74 patients included in the study until April 2021 are presented. There were 37 males and 37 females, with median age at the time of diagnosis of 53 years (range, 23-74) and at the time of the study entry of 67 years (range, 35-86). A median time of CML disease, TKI treatment, and DMR duration before the study initiation was as follows: 9.9 years (range, 4.4-22.5), 9.8 years (range, 4.2-20.2), and 7.3 years (range, 3.2-18.3), respectively. The ELTS score was low, intermediate, high and unknown in 62.2%, 21.6%, 13.5%, and 2.7% of patients, respectively. At the time of study entry, 58 patients (79.5%) were treated with imatinib, 10 (13.7%) with nilotinib, and 5 (6.8%) with dasatinib, respectively, whereas in 63 patients (86.3%) it was in the first line of therapy. With almost half of patients (48.6%), the TKI dose was already reduced at the time of study entry. With 10 (13.5%) patients, interferon-α treatment preceded TKI administration. At the time of this abstract preparation, on July 26, 2021, altogether 102 patients (from planned 150) have been enrolled in the study; 48 of them (47.1%) have already moved to the second de-escalation phase and 9 (8.8%) patients to the TFR phase. There were 2 cases of confirmed MMR loss (both in month 8 after the study entry) and no patient experienced symptoms resembling TWS.Conclusions.Despite the COVID-19 pandemic, the HALF study was successfully launched and initiated in the majority of centers, with 102 already included patients and continuing intensive enrolment. Based on our very preliminary results, the step-wise dose reduction seems to be an effective and safe approach. More included patients, longer follow-up and further analyses are needed in order to reach all set up objectives. [Display omitted] DisclosuresŽácková: Angelini: Consultancy, Speakers Bureau; Novartis: Speakers Bureau. Faber: Angelini: Consultancy, Other: conference fees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Other: conference fees, Research Funding, Speakers Bureau; Novartis: Consultancy, Other: conference fees, Research Funding, Speakers Bureau; Pfizer: Other: conference fees; TERUMO: Other: conference fees. Bělohlávková: Novartis: Consultancy; BMS/Celgene: Consultancy. Horňák: Angelini: Honoraria. Svobodník: Roche: Speakers Bureau; Janssen-Cilag: Speakers Bureau. Machová Poláková: Incyte: Consultancy; Angelini: Consultancy; Novartis: Research Funding. Mayer: Principia: Research Funding.

Accumulation of DNA Damage and Alteration of the DNA Damage Response in Chronic Myeloid Leukemia

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

Monocytic Myeloid Derived Suppressor CELLS (M-MDSC) As Prognostic Factor in Chronic Myeloid Leukemia Patients Treated with Dasatinib

INTRODUCTION. Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in most cancer patients. Recently, we and another group demonstrated that MDSC play an important role of immune escape in chronic myeloid leukemia (CML) patients inducing T cell tolerance. The aim of this study was to investigate the effect of the tyrosine kinase inhibitors (TKI) therapy on MDSC and possible correlation with clinical response.METHODS. MDSCs were analyzed in peripheral blood of 20 healthy donors (HD) and 30 CML patients at diagnosis. MDSCs were also measured during TKI treatment (18 patients treated with imatinib and 10 patients with dasatinib). Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (M-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of MDSCs with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. Exosomes were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation.RESULTS. G-MDSCs and M-MDSCs percentages in CML patients were greater than HD (respectively 82.5±9.6% vs 56.2±5.4% and 33.6±19% vs 5.9±4%, p<0.0001). Both isolated subpopulations showed expression of BCR/ABL and were able to inhibit T cells proliferation in comparison to positive control (from 48±7.6% to 25±5% for G-MDSC, p=0.0057 and 16.7±0.6% for M-MDSC, p<0.0001). No suppressive effect was observed in co-cultures with G-MDSC and M-MDSC obtained from HD. In addition, M-MDSC percentage correlated with BCR-ABLABL transcript levels in patients at diagnosis (r=0.5816, p=0.0006). Evaluating the effect of TKI therapy on MDSC levels, we found that both imatinib (IM) and dasatinib (DAS) induced a significant reduction of G-MDSC percentage at 6 months (from 82.5±9.6% to 55±17.3% after IM and 48.7±13% after DAS, p<0.0001) and 12 months (61.2±9.7% after IM and 33.4±14% after DAS, p<0.0001) of treatment. The levels of M-MDSCs significantly decreased only after DAS therapy (from 33.6±19% to 6.8±12.6% at 6 months, p=0.014 and 11.4±12.3% at 12 months, p=0.008); while there was a mild reduction after IM treatment (22.2±24.5% and 22.3±21.7% respectively at 6 and 12 months) although a great variability was observed among patients. Subsequently, correlation of MDSC reduction and clinical response to TKI therapy was investigated. We found that in DAS, but not in IM treated patients, a correlation between percentage of Major Molecular Response (MMR) and number of persistent M-MDSCs was found.. In fact, a significant difference was recorded by comparing M-MDSC levels in the MMR group (n=6) versus no MMR (n=4) at 6 or 12 months (p=0.0034).In addition, to evaluate if leukemic cells are able to expand MDSC by releasing soluble factors or exosomes, we incubated monocytes obtained from HD with sera or exosomes from CML patients at diagnosis or healthy subjects. M-MDSCs percentage significantly increased only in conditions with CML serum ( 29±13%; p=0.0006) or exosomes (8 ±2,8%; p=0.01) while no effect was observed on G-MDSC percentage. CONCLUSIONTherapy with TKI reduces the percentage of MDSCs and levels of the monocytic subset correlates with MMR in patients treated with dasatinib, suggesting their importance in clinical investigation as prognostic factor. Moreover, our data suggest the possible development in CML patients of a circuit primed by tumor cells that, through the release of soluble factors and exosomes, are able to expand M-MDSCs, creating an immunotolerant environment that results in T cell anergy and facilitates tumor growth. DisclosuresNo relevant conflicts of interest to declare.

The Role of Therapeutic Drug Monitoring of Imatinib in Patients with Chronic Myeloid Leukemia and Metastatic or Unresectable Gastrointestinal Stromal Tumors

Imatinib mesylate is a tyrosine kinase inhibitor used as first-line treatment in Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) and metastatic or unresectable gastrointestinal stromal tumors (GIST). Therapeutic drug monitoring (TDM) for imatinib has been suggested to improve efficacy, assess compliance, and evaluate drug-drug interactions. Imatinib has proven efficacy in improving treatment response and survival in patients with Ph+ CML and GIST. Several analytical methods are available to quantify total plasma imatinib concentrations. A good relationship exists between total imatinib plasma concentrations and pharmacologic response. Clinical evaluation of pharmacologic response to imatinib alone may be insufficient given the long duration of therapy before clinical response in patients with Ph+ CML and GIST. Thus, the authors have used a previously published 9-step decision-making algorithm to evaluate the utility of TDM for imatinib. The suggested trough concentrations for improved complete cytogenetic or major molecular response in patients with Ph+ CML and improved time to progression for patients with GIST are >1000 and >1100 ng/mL, respectively. Imatinib exhibits interindividual pharmacokinetic variability. Increased apparent clearance of imatinib has been observed in chronic phase chronic myeloid leukemia and increased body weight. Decreased apparent clearance has been observed in renal impairment and patients on concomitant medications with potent inhibition of cytochrome P450 3A4. Duration of therapy in patients with Ph+ CML and GIST is lifelong. Based on the available evidence, TDM for imatinib may provide additional information on efficacy, compliance, and safety than clinical evaluation alone. Patients with suboptimal response to treatment, treatment failure, rare adverse events, drug interactions, or suspected nonadherence will attain the greatest benefit from TDM.

Supportive Methods for Testing CML Progression in Addition to BCR-ABL Transcript Monitoring – What to Do If BCR-ABL Monitoring doesn’t Work?

Abstract 4902BCR-ABL expression level is the exclusive and irreplaceable marker for monitoring the response to treatment in patients with chronic myeloid leukemia (CML). CML therapy by tyrosine kinase inhibitors (TKIs) is very succesfull, nevertheless resistance to imatinib treatment develops in about 25 –30% of patients. This resistance can have different reasons, both BCR-ABL-dependent and independent. According to our experience, CML patients exhibiting suboptimal response or therapy failure represent a highly heterogenous group in terms of relaps emergence and the molecular monitoring of BCR-ABL transcript not always provides sufficient evaluation of the risk of relaps. Additional markers are thus required to detect the arising resistance early before disease deterioration.In our previous studies we found supportive markers that can describe the disease status and the progression probability in addition to commonly used methods for CML characterization (hematological, cytogenetical and BCR-ABL transcript monitoring). We analysed Hsp90 protein level in CML pacients with various therapy responses –HSP90/HSP70 protein levels tested by western blots indicate the probability of disease deterioration if the ratio is higher than 0,26 (EHA2011 – Zackova et.al.). Second possible marker, WT1 transcription level (real-time PCR according to Cilloni et al. 2006, B2M was applied as control gene), also correlated with relaps emergence during further course of the disease - expression levels above 0,1 (2ΔΔct) suggested higher (90%) probability of relapse. (Lopotova et.al. EHA 2011). Another method we used is in vitro cultivation of patient leukocytes with TKIs and the subsequent analysis of Crkl phosphorylation status as an indicator of BCR-ABL inhibition. Testing of the in vitro sensitivity to TKIs can give clues to current sensitivity to treatment and partially answer the further course of disease development (Zackova et. al. ASH2010).In the current study we tested these three methods in CML patients. Up to now we have included a) at diagnosis (DG) (8), b) nonresponders - with stable BCR-ABL transcript level (10) and c) patients in major molecular response (MMR) (3).Ad a) All patients tested at diagnosis with 100% sensitivity to TKIs and with low levels of HSP90 protein and WT1 transcript achieved MMR (5/8). Patients without 100% inhibition of BCR-ABL kinase by TKIs and with high levels of HSP90 protein or WT1 transcript didn't respond or relapsed (3/8).Ad b) Patients with BCR-ABL transcript fluctuating about a constant level could relapsed or stay without relapse. The testing of HSP90 protein or WT1 transcript levels could predict probability of relaps. High levels of both markers preceeded all relapses (4/10).Ad c) All patients in MMR had low levels of HSP90 protein and WT1 transcript, sensitivity to TKIs was 100%.Our results suggest that WT1 transcript level, HSP90 protein level and in vitro tests of sensitivity patients leukocytes to TKI can help to characterise the real state of disease and to predict the disease evolution. Disclosures:No relevant conflicts of interest to declare.

Clinical Outcome of CML Patients with Philadelphia Chromosome Complex Variants.

AbstractAbstract 4499 INTRODUCTION:The presence of translocation between chromosomes 9 and 22 that characterizes the chronic myeloid leukemia (CML) is occasionally accompanied by more complex variations involving additional exchange of genetic material with other chromosomes. This variants of Philadelphia chromosome have no worse prognosis than those others with the common translocation. MATERIAL:Since 1997 we have diagnosed in our hospital 5 CML patients who didn't show any of these variants. Three women 17, 23 and 76 years old and two men of 36 and 65. All of them diagnosed in chronic phase. Risk stage at diagnosis by Sokal were 1 high, 1 intermediate and 3 low. By Hasford 2 were intermediate end 3 low.Philadelphia chromosome variants involve a third chromosome in 4 cases (translocations 2;9;22, 9;22;12, 9;22;9 and 6;9;22) and in one case involving four chromosomes (translocation 1;2;9;22).Three patients diagnosed before the imatinib approval started treatment with IFN and Ara-C and subsequently changed to Imatinib treatment. Two others started treatment with imatinib directly. RESULTS:All patients had a good outcome with treatment being the current state of 2 patients in complete molecular response (105 and 60 months of follow-up) and 2 patients in Major Molecular Response (146 and 143 months). The 5th patient, a 17 years old woman, has been treated for three months with Imatinib and showed complete haematological response at first month and major cytogenetic response at third month. It is striking in this last patient the fact that two years earlier had been referred for study of myeloid moderate leukocytosis (20,000 leukocytes with circulating myeloid progenitors without anemia, thrombocytosis, or splenomegaly). The patient didn't come to clinic when Bone Marrow Test was cited and two years later resumes the visit continuing with the same leukocytosis in peripheral blood and without splenomegaly unchanged despite not having received any treatment. CONCLUSION:Our experience confirm that this type of patients with complex translocation variants have no worse prognosis than normal translocation under imatinib treatment and suggests that may have a more benign clinical behavior. Disclosures:No relevant conflicts of interest to declare.

Prognostic Significance of the Lost of a Major Molecular Response In Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia (Ph+CML).

AbstractAbstract 3435 Background:Albeit of well-known, dramatic improvements, there remain some questions to be solved around Ph+CML in treatment with tyrosine kinase inhibitors (TKI). Among these, the significance of the amount of minimal residual disease (MRD) measured by RT-PCR. For instance, loss of a so-called major molecular response (MMR) is claimed to be a Òsuboptimal responseÓ and following the ELN recommendations, a change in treatment should be considered in these patients. Aims:To evaluate the relevance of a loss of MMR in patients with complete cytogenetic response (CCR). Study Group and Methods:We have analized 81 patients treated with imatinib for CML in chronic phase with a median follow up of 66 months. 36 patients started imatinib after interferon failure and 45 as front line therapy. Major Molecular Response (MMR; BCR-ABL/ABL ratio<0.1% IS) at any time was achieved by 63 patients. Results:22 patients (34%) lost MMR (documented al least twice). The risk of losing MMR was higher in late MMR (>18 months) compared with those cases whose MMR came much earlier (<18 months): 70% vs 18% (p=. 000). We have found no correlation among the lost of MMR and classical prognostic factors (Sokal-Index, mutations at the TK domain or imatinib plasma levels). Of these 22 patients, 7 (32 %) recovered MMR later with no therapy changes, 8 (36%) experienced fluctuations in the BCR-ABL transcript-levels without losing CCR, 4 (19%) did not attain a MMR but remained in stable CRR, and 3 (13%) lost CCR. These regained MMR after being treated on second generation TKI. The results show how the stability of the early MMR is greater than late MMR (table1). Conclusions:In our experience, one third of the patients who lost MMR recovered it later on the same treatment. And only 13% went on to treatment failure. Perhaps some similar cases (after first losing MMR) should be closely monitored before a change in treatment. Also of note is, of course regarding only our experience, that the risk of a loss of MMR seems to be maximal in patients who achieve a late MMR.Table 1Evolution of patients after lossing MMREarly MMR (n= 9)Late MMR (n=13)Recover MMR45% (4)23% (3)Fluctuations33% (3)38% (5)Remained in Suboptimal Response11% (1)23% (3)Lost CCR11% (1)15% (2) Disclosures:No relevant conflicts of interest to declare.

The Combination of Interferon-Alpha with Imatinib in Early Chronic Phase Chronic Myeloid Leukemia Patients Induces a Significant Improvement of the Molecular Responses in the First Two Years of Treatment: Results From Three Studies From the GIMEMA CML Working Party.

Abstract 2192Poster Board II-169Thanks to its striking effectiveness, imatinib (IM) is the current standard of therapy in chronic myeloid leukemia (CML). However, Interferon-alpha (IFN-alpha) has induced a low but reproducible curative effect in some CML patients, inducing durable remissions lasting even after therapy discontinuation. For this reason, the interest on the possible use of IFN-alpha in the treatment of CML is still substantial, and very recently some newer prospective studies from the French and the German Groups have proposed the re-introduction of IFN-alpha in the front-line treatment of early chronic phase CML (ECP-CML), in association with IM (Rousselot et al, Haematologica 2009;94:abs.1093; Hehlmann et al, Haematologica 2009;94:abs. 0478). We compared the response of 495 ECP-CML patients, enrolled into three different prospective studies promoted by the GIMEMA CML WP (419 treated with imatinib 400mg and 76 treated with imatinib 400mg plus IFN-alpha) (study protocols NCT00511121, NCT00514488 and NCT00511303). Cytogenetic analysis was performed by standard banding techniques and cytogenetic response was rated according to the European LeukemiaNet guidelines. Molecular response was assessed on peripheral blood cells by a standardized quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) method on an ABI PRISM 7700 Sequence Detector (Perkin Elmer, Faster City, CA). BCR-ABL transcript levels were expressed as a percentage according to the IS.Patients were equally distributed by Sokal risk in the two cohorts. Median follow-up was 43 mos (range, 12- 67) in the IM group and 54 mos (range, 11-63) in the IM+IFN-alpha group. Compliance to IM was excellent in both groups, with 90 to 95% of patients receiving IM 400 mg/daily throughout the follow-up. Conversely, the proportion of patients continuing IFN-alpha dropped from 41% at 12 mos to 18% at 18 mos, 13% at 24 mos, 3% at 36 mos; by the end of the fourth year, all patients were off IFN-alpha.The number of patients in CCgR was higher in the IM+IFN-alpha than in the IM group at 6 mos (60% vs 42%, p=0.002) but not from 12 mos on (Table 1). The number of patients in MMolR was higher in the IM+IFN-alpha than in the IM group at 6, 12 and 24 mos, but not later on (Table 2). Also the number of patients with undetectable Bcr-Abl transcript levels was higher in the IM+IFN-alpha group at 12 months (15% vs 5%, p=0.003) but not later on (19% vs 18% at 48 mos).Table 1Number of patients in complete cytogenetic response at different time-pointsno of patients in CCgR, (%)IM (419 patients)IM+IFN-alpha (76 patients)6 months175 (42%)46 (60%)12 months284 (68%)53 (70%)24 months336 (80%)62 (82%)36 months342 (82%)66 (87%)48 months346 (82%)62 (82%)Table 2Percentage of patients in major molecular response (MMolR) evaluated on the entire study group.MMolRp valueIM (n.419)IM+PegIFN (n.76)6 months34%58%<0.000112 months46.5%67%0.00124 months62%64.5%0.736 months58%65%0.5248 months57%65%0.26These data support the preliminary analysis of the French SPIRIT group, reporting a higher rate of MMolR after initial treatment with IM and IFN-alpha as compared to IM alone. The loss of the difference from 24 mos on could be explained by the fact that almost all patients had discontinued IFN-alpha during the first two years, pointing out that the low compliance to the combination may limit its utility in practice. ACKNOWLEDGEMENT The Italian Association Against Leukemia-lymphoma and myeloma (BolognAIL), The Fondazione del Monte di Bologna e Ravenna, The Italian Ministery of Education (PRIN 2005, No. 20050 63732_003, and PRIN 2007, No 2007F7 AE7B_002), The University of Bologna, The European Union (European LeukemiaNet). Disclosures:Pane:Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding. Saglio:Celgene: Honoraria; Novartis: Honoraria.

Efficacy of low-dose imatinib in chronic-phase chronic myelogenous leukemia patients

Imatinib mesylate is very effective in the treatment of chronic myelogenous leukemia (CML) and 400 mg/day imatinib is considered the standard treatment dose for chronic-phase (CP) patients. However, despite its relative tolerability, some patients require a lower dosage or temporary cessation of treatment because of severe adverse events. It remains unclear whether reduced-dose imatinib is as effective as the standard dose in achieving and maintaining a major molecular response (MMolR), an important goal of imatinib therapy. In this study, Japanese patients with CML-CP, as classified by their Sokal scores, were treated with imatinib. MMolR was observed in all patients with low Sokal scores who were treated with 300 or 400 mg/day imatinib, suggesting that low scores predict favorable response to treatment even at lower dosages, which is preferable since it is associated with fewer adverse events. Low-dose therapy also achieved MMolR in patients with intermediate or high scores. However, loss of complete cytogenetic response (CCgR) or an increase in BCR-ABL transcripts was noted shortly after these patients achieved CCgR, suggesting that a CCgR short of MMolR still indicated a risk of loss of response in these risk categories. MMolR was maintained in these patients by timely increases in dosage. Achieving CCgR within 12 months and maintaining it without an increase in BCR-ABL transcripts might indicate that low-dose imatinib therapy can produce acceptable outcomes without excess toxicity.

Allogeneic Stem Cell Transplantation (ASCT) in CML with Partial T-Cell Depletion, No Prophylaxis for Graft-Versus-Host Disease (GvHD) and Preemptive DLI for Patients with Post-Transplant Minimal Residual Disease (MRD): An Alternative Approach in CML Transplantation with Low Morbidity and Long Term Disease Free Survival.

The introduction of imatinib mesylate has revolutionized the treatment of CML, but despite significant cytogenetic responses primary therapy failure and emergence of resistance pose a major obstacle to successful treatment. ASCT still has a pivotal role in the treatment of CML and reduction of morbidity and mortality remains an important focus of ongoing studies in CML. We conducted a prospective study with ablative conditioning and partial T cell depleted ASCT from a matched sibling donor with no post-transplant GvHD prophylaxis. This was followed by an escalated donor lymphocyte infusion (DLI) administered if there was evidence for the presence of MRD, as determined by FISH and PCR/RQ-PCR for bcr-abl. Patients and methods: 40 consecutive patients underwent ASCT for CML between 1999–2005. The transplant regimen contained busulfan (12mg/kg), cyclophosphamide (120 mg/kg), anti-thymocyte globulin (ATG-Fresenius) (25mg/kg) and fludarabine (200mg/kg) followed by an infusion of ≥5×106/kg of G-CSF-mobilized and positively selected CD34 cells and 1×105/kg T cells. No post-transplant GvHD prophylaxis was given. The presence of MRD was determined every 3 months. DLI was administered in escalated doses starting with 3×106/kg and escalating in increments up to 1×108/kg. Results: 40 patients, median age 38 (19–63), 35 transplanted in first chronic phase (CP), 1 in second CP and 4 in accelerated phase. Patients were transplanted at a median of 4 months from diagnosis (1–66). The median of positively selected CD34+ cells was 9×106 cells/kg (5–24). 39 patients engrafted neutrophils at a median of 11 days (9–25) and platelets at a median of 15 days (9–134). Primary non-engraftment occurred in one patient and late graft failure in two, all salvageable by back-up marrow in two patients or donor stem cell boost in a single patient. The median follow-up of the entire cohort is 44 months (6–84).Overall Survival80%Transplant Related Mortality (TRM)day 1005%day > 10010%Acute GvHDPost transplant (n = 38)Post DLI (n= 24)grade I-II21%21%grade II-III08%grade IV00Chronic GvHDPost transplant (n = 38)Post DLI (n= 24)limited2.6%16%extensive00Need for DLIOne dose63%Two doses31%≥ Three doses16%Disease-Free SurvivalMajor cytogenetic response6%Major molecular response6%Complete cytogenetic and molecular response88%Disease Progression10%None of the patients developed significant VOD. Post transplant acute GvHD developed in only 21% of patients and was confined to grade I–II, all responding to a short course of immunosuppression. Additional 7 out of the 24 patients receiving DLI developed acute GvHD. There was no grade IV or mortality from GvHD. At the time of this analysis all apart from 1 patient were able to stop immunosuppressive treatment for GvHD. There were no significant infectious complications in this group. All late TRM were secondary to autoimmune complications, unrelated to GvHD, and occurred in 5 patients (13% of all) at 6–14 months post transplant. These complications included: ITP, AIHA, autoimmune pneumonitis, nephritic syndrome, TTP and Kaposi sarcoma. The mortality from these complications was 80% (4 out of 5 patients). 4 patients developed blast crisis post transplant; 2 patients have died; and the others are currently in CR post salvage treatment, DLI and imatinib. Twenty five percent of patients attained complete molecular remission without ever needing DLI, while 75 % responded after DLI, mostly following a single dose. Two patients are in major cytogenetic response, one of whom received back-up marrow and for the other a DLI donor is not available. The two patients in major molecular response are awaiting DLI. Conclusions: Matched ASCT for CML can be performed with significantly reduced TRM and morbidity using partial T cell depletion, no GvHD prophylaxis and preemptive DLI. This regimen offers > 80% long-term curative potential. Programmed DLI is safe and associated with a low risk of GvHD. The late immunological complications pose a major risk. This appears to be related to an imbalance between B and T cell reconstitution and the precise mechanisms of this need to be elucidated. In the imatinib era ASCT using this approach may be reserved for patients not responding or resistant to the imatinib mesylate.