ABL Transcripts Research Articles

Nilotinib for the Frontline Treatment of Chronic Myeloid Leukemia Carrying the p230 Transcript: Dream or Reality?

OPINION article Front. Oncol., 03 February 2014Sec. Hematologic Malignancies https://doi.org/10.3389/fonc.2014.00017

What Does a Deep Molecular Response Signify?

What Does a Deep Molecular Response Signify?

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

Clinical Efficacy and Safety Of Dasatinib For Newly Diagnosed Chronic Myeloid Leukemia In Chronic Phase In Japan

BackgroundDasatinib is a second-generation BCR-ABL inhibitor that has a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. The previous report from the global DASISION trial showed dasatinib resulted in significantly higher and faster rates of complete cytogenetic response (CCyR) and major molecular response (MMR) compared with imatinib. We conducted a phase II study to evaluate the efficacy and safety of dasatinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) in Japan. MethodsEighty newly diagnosed CML-CP patients were include in this study. Patients received dasatinib 100mg once daily. Treatment was continued until disease progression or unacceptable toxicity. Primary end point was the rate of major molecular response (MMR) by 12 months. MMR defined as a BCR-ABL transcript level of 0.1% or lower on the International scale by means of a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in peripheral blood. Secondary end points were the rate of complete cytogenetic response (CCyR) by 12 months, the rate of MR4.5 (either (i) detectable disease with <0.0032% BCR-ABL1 IS or (ii) undetectable disease in cDNA with >32,000 ABL1 transcripts in the same volume of cDNA used to test for BCR-ABL1) by 12 months and adverse events of dasatinib (UMIN #000006358). ResultsEighty newly diagnosed CML-CP patients were included in this study. All except one patient administered dasatinib 100 mg once daily. One patient was withdrawal before administration of dasatinib. So far, there were 71 patients with 6 months follow-up and 51 patients with 12 months follow-up. The estimated MMR rates were 69.5 % (95%CI, 58.7-80.3 %) by 6 months and 82.7% (95%CI, 73.0-92.4 %) by 12 months. The estimated MR4.5 rates were 27.1 % (95%CI, 16.7-37.5 %) by 6 months and 48.9% (95%CI, 36.0-61.7 %) by 12 months. Only 6 patients were withdrawal because of adverse event (5 patients) and ineffectiveness (1 patient). ConclusionDasatinib treatment results in higher rates of molecular responses in newly diagnosed CML-CP patients in Japan. Dasatinib as the first-line agent might be acceptable for CML-CP patients because of better clinical efficacy and less toxicity. Disclosures:No relevant conflicts of interest to declare.

The e13a2 BCR-ABL1 Fusion Transcript Is a Candidate Adverse Prognostic Factor In Chronic Myeloid Leukemia Patients Treated Frontline With Imatinib Mesylate

Background and aimsThe e13a2 and the e14a2 are the most frequent BCR-ABL1 transcripts in chronic myeloid leukemia (CML) patients. The aim of the present study was to assess the prognostic value of the e13a2 (b2a2) or the e14a2 (b3a2) fusion transcripts on the long-term outcome of early chronic phase (ECP) CML patients treated frontline with imatinib mesylate (IM). Few studies, mostly based on a small number of patients or with a relatively short observation, have been published, particularly in ECP setting. Due to partially conflicting data, the transcript type is not actually considered an established baseline prognostic factor. To our knowledge, this is the first long-term analysis conducted in the context of large prospective studies. MethodsA retrospective analysis of 3 GIMEMA CML WP prospective clinical studies (NCT00514488, NCT00510926, observational study CML/023) was performed. All the 559 enrolled patients were analyzed. A qualitative RT-PCR for BCR-ABL1 transcript was routinely performed at enrollment. Patients expressing rare transcripts or with both e13a2-e14a2 transcripts were excluded: 493 out of 559 patients were included, 203 (41%) with e13a2 transcript and 290 with e14a2 transcript (59%). Definitions. Major molecular response (MMR): BCR-ABL1IS ratio <0.1%. Deep molecular response (MR4.0): detectable disease ≤ 0.01% BCR-ABL1IS or undetectable disease with ≥10,000 ABL1 transcripts. Progression: transformation to accelerated or blastic phase. Failure: according to 2013 ELN recommendations. Event: failure or permanent discontinuation for any reasons. All the deaths, at any time and for any reasons, were included. ResultsThe median follow-up was 76 months; 95% of the patients had at least a 5-year observation. The 2 groups were comparable for demographic and hematologic characteristics, except for the proportion of male patients, that was higher in the e13a2 group (p = 0.05), and for the baseline percentages of eosinophils, that was lower in the e13a2 group (p = 0.009). The Sokal, Hasford and EUTOS risk score distributions and the proportion of patients with CCA in Ph+ cells were comparable. The complete cytogenetic response (CCyR) rate at 12 month was not significantly different (75% and 79% in e13a2 e14a2 patients, respectively, p=0.274), but the MMR rate at 18 months and the MR4.0 at 36 months were significantly lower in e13a2 patients (52% and 67%, p = 0.001; 20% and 30%, p = 0.013, respectively). As far as the rapidity of response, the time to MMR and the time to MR4 were significantly slower for patients with e13a2 transcript (12 and 6 months; 54 and 37 months, respectively), with an inferior overall estimated probability of MMR (85% and 92%, p < 0.001) and MR4 (57% and 71%, p = 0.002). The overall survival (83% and 86%, p = 0.023), the progression-free survival (81% and 86%, p = 0.007), the failure-free survival (54% and 71%, p < 0.001) and the event-free survival (46% and 61%, p = 0.003) were significantly lower in patients with e13a2 transcript (figure 1). In a multivariate analysis (Cox model), the transcript type retained its prognostic significance, when adjusted for other relevant variables. Analyzing separately the patients treated with IM 400 mg (n = 371; e13a2 and e14a2 transcript: 208 and 163 patients, respectively) or IM 800 mg (n = 122; e13a2 and e14a2 transcript: 82 and 40 patients, respectively), all the above mentioned response and outcome differences were confirmed, with one exception: in the high-dose IM group the overall survival probability was inferior for the e13a2 patients, but, probably due to the small number of patients, the difference was not statistically significant (83% and 86%, p = 0.268). [Display omitted] ConclusionsA long-term analysis in a large cohort of CML patients suggests that the e13a2 transcript is an adverse prognostic factor in ECP CML patients treated frontline with IM. An independent evaluation is required for confirmation. AcknowledgmentsUniversity of Bologna, BolognaAIL, COFIN, Fondazione Carisbo. Disclosures:Castagnetti:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Abruzzese:BMS, Novartis: Consultancy.

The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

BackgroundChronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. AimsThe aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. MethodsWe analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). ResultsWe successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / ConclusionWe have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures:No relevant conflicts of interest to declare.

The Prospective Analysis Of Clinical Efficacy and Adverse Events In Chronic Myeloid Leukemia In Chronic Phase (CML-CP) Patients With Imatinib Resistance Or Intolerance, Evaluated By European Leukemia Net (ELN) 2013 Criteria

BackgroundDasatinib is a highly potent BCR-ABL inhibitor, with a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. Across a series of phase II and III trials with more than 2 years of follow-up, dasatinib has demonstrated durable efficacy in patients with CML following resistance or intolerance to imatinib. We conducted a phase II study to evaluate the efficacy and safety of dasatinib in Japanese patients with imatinib resistance or intolerance CML. MethodsFifty- five CML-CP patients from 2009 to 2011 with resistance (n=40) or intolerance (n=26) to imatinib were registered to dasatinib administration (100mg once daily). Eleven among 26 patients with intolerance also showed resistance to imatinib at the registration. Imatinib resistance was defined as a lack of partial cytogenetical response at 3 months, a lack of complete cytogenetic response at 6 months or a lack of a major molecular response at 12 months of imatinib treatment. This criteria was identical to the criteria in ELN 2013 recommendation for first line. In another words, the resistance in this study means non-optimal criteria (warning and failure) of definition of the response to TKIs, first line, in ELN 2013 recommendation. Efficacy and safety were assessed using rates of major molecular response (MMR)/ MR4.5 (either (i) detectable disease with<0.0032% BCR-ABL1 IS or (ii) undetectable disease in cDNA with >32,000 ABL1 transcripts in the same volume of cDNA used to test for BCR-ABL1) at 12 months and drug-related adverse events (AEs) respectively. All analyses were based on the modified to intension-to treat. ResultsOne patient was withdrawal before dasatinib administration. The median duration of imatinib therapy was 545 days. The overall incidence of MMR (primary endopoint) and MR4.5 at 12 months was 67.2% (95% confidence interval, 53.3-81.1%), and 20.2 % (95% CI, 8.4–32.0 %), respectively. Forty patients with resistance to imatinib, who were warning and failure patients judged by ELN 2013 criteria, were reassessed. Cumulative MMR and MR4.5 rate were 63.4% (95% CI: 46.3-80.6) and 18.0% (95% CI: 4.9-31.1) respectively at 12 months. Eleven patients, who showed more than 1% IS at 3 months of dasatinib treatment, did not reach to MMR at 12 months or discontinued dasatinib due to insufficient efficacy in this resistance cohort. However, progression to the accelerated or blastic phase had not been observed. When imatinib were changed to dasatinib, 5 patients showed the mutations, which were effective in dasatinib therapy. New mutations have not been observed during the treatment of dasatinib.Among 54 patients, most non-hematological AEs were in grade 1/2. including diarrhea (12.7%), rash (7.3%), myalgia (5.5%) and vomiting (3.6%). Grade 3/4 non-hematological AEs were infrequent, including decreased potassium (3.6%), and increased creatinine (3.6%). Grade 1/2 fluid retention AEs were shown in 9.1% of patients, including edema (3.6%). Pleural effusion, which was only in Grade 1/2, was shown in 32.7% of patients. Grade 3/4 hematological toxicities included anemia (7.3%) and thrombocytopenia (3.6 %). Only 3 patients have permanently discontinued dasatinib treatment due to AEs. ConclusionsThe patients with non-optimal responses (warning and failure) judged by ELN 2013 criteria should have early intervention to dasatinib, which is less toxicity in CML-CP patients. This intervention might induce good prognosis. The BCR-ABL1 IS less than 1% at 3 months of dasatinib administration will be the valuable landmark for outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Core Binding Factor Acute Myeloid Leukemia In Pediatric Patients Enrolled In The AIEOP AML 2002/01 Trial: The Impact Of Minimal Residual Disease On Patient Outcome

▪ IntroductionAcute Myeloid Leukemia (AML) accounts for 15% of pediatric leukemia. Improvements of prognosis achieved in the last twenty years are also due to a better stratification of patients into risk groups that allowed to deliver tailored treatment. The AIEOP LAM 2002/01 protocol classified patients with t(8;21)(q22,q22)AML1-ETO and inv(16)(p13;q22)CBFB-MYH11, who responded to induction therapy as belonging to the Standard-Risk (SR) group. These patients were considered at good prognosis; unfortunately, although they all reached morphological complete remission (CR) after the first induction course, they showed a high incidence of relapse (24%) (Pession A. et al., Blood 2013). At present, little is known about the kinetics of relapse or the development of resistance mechanisms; moreover, in many studies post-remission therapy in AML does not take into account the level of residual leukemia. Here, we evaluate the prognostic impact of molecular minimal residual disease (MRD) levels in terms of onset of relapse. MethodsWe studied bone marrow of 49 and 30 patients carrying either t(8;21) or inv(16) abnormalities, respectively, at time of diagnosis, and at the end of first and second course of induction therapy (ICE: Idarubicine, Citarabine and Etoposide). MRD was evaluated as number of fusion transcripts by quantitative RT-PCR using absolute quantification, and calculated as logarithmic (Log) disease reduction after the first and second ICE course as compared to diagnosis. Fusion transcripts were normalized to 10(5) ABL copies. The prognostic impact was assessed by the calculation of Overall Survival (OS) and cumulative incidence of relapse (CIR) according to the different MRD levels. ResultsAfter I ICE, t(8;21)-rearranged patients distributed equally in the four classes of MRD reduction. In particular, 10 patients reduced MRD levels of less than 1 Log, 11 patients reduced 1-2 Log, 17 patients reduced 2-3 Log and 11 patients reduced MRD levels of more than 3 Log as compared to diagnosis. After II ICE, the majority of patients increased the reduction of MRD levels (> 3 Log). We focused on the 10 patients who did not respond to the I ICE course and we found that 7 out of 10 reduced MRD levels of more than 1 Log after the II ICE, confirming a slow clearance of blasts for this subgroup of patients. We found that a reduction of MRD lower than 1 Log conferred a worst outcome to t(8;21)-rearranged patients (OS being 53.3% vs 85.2%, p=0.073 after I ICE; 33.3% vs 82.4 p= 0.062 after II ICE), and higher CIR after I and II ICE (52% vs 12.3%, p=0.0049, and 66.7 %, vs 17.2, p=0.024, at 6 years respectively). The group of inv(16)-rearranged achieved more than 1 Log reduction of MRD since I ICE course. After II ICE course, the majority of of patients reduced MRD of more than 3 Log, and 20% of them were completely negative. Inv(16) rearranged patients were all alive at last follow-up, and CIR never showed statistically significant differences for MRD levels in inv(16)-rearranged patients after the induction therapy. The impact of copy numbers (i.e. transcript levels) at diagnosis and after induction courses on relapse risk never showed significant results in both subgroups of CBF-rearranged patients. ConclusionsOur data indicate that MRD evaluation using quantitative RT-PCR is an important diagnostic tool that permits the assessment of response to CT and the identification of patients at greater risk of relapse after induction therapy for AML1-ETO rearranged patients. We propose that at the end of induction therapy the cut-off of MRD< 1 Log be used to guide therapeutic decisions for this subgroup of SR patients. On the contrary, MRD levels of CBFB-MYH11 rearranged patients do not have prognostic value on CIR after induction therapy, this suggesting that novel molecular features should be investigated for this subgroup of AML. Disclosures:No relevant conflicts of interest to declare.

Achievement Of a MR5.0 Within the Randomized CML-Study IV: Feasibility, Differences Between Treatment Arms, and Prognostic Implications

IntroductionDepth of molecular remission on tyrosine kinase inhibitor (TKI) treatment is of rising importance for chronic myeloid leukemia (CML) patients (pts) with regard to possible treatment discontinuation and competing TKIs available to improve molecular response. At present, it is unknown which level of deep molecular response is necessary for optimal prognosis and for successfully stopping therapy. The aim of this work is both to evaluate the technical feasibility of molecular monitoring at the mentioned level and to search for factors allowing to predict MR5.0 in pts on imatinib (IM)-based treatment. MethodsReal-time quantitative PCR on mRNA BCR-ABL transcripts in addition to total ABL transcripts as internal control has been performed on a LightCycler platform in 1,442 pts within the randomized CML-Study IV and adapted according to the International Scale (IS). In order to qualify for MR5.0 the BCR-ABLIS expression should meet one of the following criteria: a positive result ≤0.001% or a negative result with a minimum sample quality of 100,000 ABL copies (Cross et al., Leukemia 2012). Calculating cumulative incidences of remission or progression, the competing risks progression and/or death before possible progression were considered. Cox models were estimated for the multivariate analysis. ResultsIn 1,198 of the 1,442 molecularly examined pts at least one sample fulfilled the sensitivity criteria for a MR5.0 (8,266 of 24,101 samples, 34.3%). Cumulative incidence of MR5.0 was 51% at 8 years. The median time to MR5.0 according to randomized treatment arms differed as follows: IM 800mg 79.7 months (mos), IM 400mg 95.0 mos, IM 400mg + IFNα 98.0 mos, IM 400mg + AraC 103.3 mos, IM 400mg after IFN failure 112.9 mos. A Cox model examining the different treatment arms compared to IM 400mg revealed a significantly higher chance for MR5.0 in the IM 800mg arm (HR 1.305, 95% CI 1.003-1.698, p=0.048). Baseline factors like thrombocytosis >450/nl were associated with better responses (HR 1.701 compared to <450/nl, 95% CI 1.405-2.059, p<0.001) and higher leukocyte counts >100/nl (HR 0.503 compared to <50/nl, 95% CI 0.400-0.632, p<0.001) and 50-100/nl (HR 0.746 compared to <50/nl, 95% CI 0.591-0.942, p=0.014) with unfavorable responses. Other upfront factors like age, gender, blasts, eosinophils, hemoglobin, and EUTOS score did not significantly influence the probability for MR5.0. Taken all treatment arms together, our analyses have shown that the chance of achieving a MR5.0 by 8 years was considerably reduced if the pts had a BCR-ABLIS >10% at 3 mos (40.2% vs 58.0%), >1% at 6 mos (40.3% vs 68.7%), >0.1% at 12 mos (37.7% vs 72.0%), and >0.1% at 24 mos (21.5% vs 60.5%). ConclusionThis evaluation of a large randomized trial reveals feasibility of MR5.0 detection in the majority of pts underlining the benefits of standardized molecular monitoring on the IS with optimized highly sensitive technologies. Baseline low leukocyte count, high thrombocyte count and high dose IM treatment are predictors of future MR5.0. Further, early molecular landmarks qualify for excellent outcome giving hope to a rising number of pts to successfully discontinue treatment and avoid possible side effects or comorbidities. Disclosures:Müller:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Saussele:Novartis: Honoraria, Research Funding, Travel Other; BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria.

Consistency Of RQ-PCR Analysis Results In Peripheral Blood and Bone Marrow Samples In Chronic Myeloid Leukemia Patients: Relevance From The Practical and Logistic Point Of View

BackgroundConsensus has been achieved that standardized molecular quantitative analysis (RQ-PCR) on peripheral blood (PB) is a suitable method for monitoring residual disease in chronic myeloid leukemia (CML). However, BM is still obtained at specific timepoints, and in a number of cases, only bone marrow (BM) sample collected for cytogenetic analysis is available. Being one of the laboratory involved in the standardization process of molecular monitoring for CML patients, we decided to perform a comparative analysis of BM and PB samples in order to evaluate the consistency of the results. MethodsBetween March 2009 and January 2013, 230 consecutive RQ-PCR tests to assess BCR-ABL transcript levels from simultaneously collected PB and BM samples were performed (for a total of 460 analysis) on 77 patients affected by Ph+ CML in chronic phase treated in our center. All samples were analyzed in the same laboratory following international guidelines (Cross N, Leukemia 2012) and results were expressed according to the International Scale; ABL1 was used as control gene. Time from blood-drawn to processing was within 3-4 hours. ResultsAmong the 230 pairs, 3 were considered as not evaluable because of inadequate material; for the purpose of this study, the remaining 227 pairs were considered as “evaluable”. 204 pairs were classified as “fit” when both BM and PB ABL amplification resulted in more than 10.000 copies; 23 pairs were considered unfit for ABL1 <10.000 in either one of the two samples (21) or both (2). The mean number of ABL1 copies in all evaluable samples was 35.639 for BM (SD 21.465) and 30.958 for PB samples (SD 18.696). Correlation analysis was performed on the whole population and in 4 subgroups: No Complete Cytogenetic Response (CCyR, 22%), CCyR without Major Molecular Response (MMR), (21.6%), CCyR with MMR (excluding patients with MR4 or better,19.8%), and CCyR with MR4 – MR4.5 (32,6%). Cytogenetic response was not available in 9 BM samples (4%), not included in the subgroup analysis. Spearman correlation of BCR/ABL ratio values between PB versus BM paired samples resulted in a statistically significant correlation in all groups, both for evaluable and fit pairs. Correlation was stronger in samples that were not in MMR or better (table 1 and figure 1). The Wilcoxon test showed that the mean difference of BCR/ABL values between paired PB and BM samples was not significantly different from zero (in evaluable and fit pairs by considering the whole population). Concordance was further analyzed by the K test which resulted in a coefficient equal to 0.627, corresponding to a notable degree of concordance. For patients in CCyR, agreement on classification of response (MMR, MR4, MR4.5) between paired PB and BM samples was observed in 125/168 evaluable pairs; 22 out of the 43 evaluable cases of disagreement were due to technical failures (in 10 BM and 12 PB samples). In 14 of the remaining 21 cases, PB was more sensitive.Table 1Spearman correlation of % is between paired PB and BM samples / fit samplesCoefficientP-value%IS Total+0.966<0.001%IS – Subgroup No CCyR+0.921<0,001%IS – Subgroup CCyR-No MMR+0.752<0.001%IS – Subgroup CCyR-MMR+0.4980.003%IS – Subgroup: CCyR-MR4, MR4.5+0.478<0.001 [Display omitted] ConclusionsIn a single center experience of molecular analysis, BCR/ABL ratio was highly consistent in BM and PB samples. In less than 10% of the cases a single test did not reach the required sensitivity of 10.000 ABL copies and the double testing allowed to obtain a valid result. This may be especially valuable in evaluating an early response (i.e. at 3 months), when the amount of disease has prognostic relevance. The analysis will be expanded to include samples coming from different centers to evaluate a possible role of timing and transport on data consistency. Disclosures:Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Long Term Follow-Up After Imatinib Cessation For Patients Indeep Molecular Response: The Update Results Of The STIM1 Study

BackgroundImatinib treatment significantly improves survival in patients with CML. We previously demonstrated that Imatinib could be safely discontinued in pts with deep molecular response (DMR) i.e., with undetectable minimal residual disease (UMRD) of at least 2 years (Lancet oncology, 2010;11: 1029-1035).Around 40% of CML patients with stable DMR on imatinib for at least 2 years are likely to remain in a prolonged treatment-free remission (TFR) after treatment is stopped and this rate has been safely confirmed by the recent Australasian TWISTER study (Blood, 2013; 122:515-22). MethodsIn this multicenter STIM study, imatinib (of >3 years duration) was prospectively discontinued in CML pts with who were in DMR with UMRD sustained for at least 2 years (ClinicalTrials.gov,NCT00478985). The UMRD was defined with a sensitivity> to 4.5 log with ≥ 50,000 ABL transcripts amplified as internal control). Importantly, the molecular relapse was defined as positivity of BCR–ABL transcript in quantitative RT-PCR confirmed by a second analysis point indicating the increase of one log in relation to the first analysis point, at two successive assessments, or loss of MMR at one point. Quantitative RT-PCR for BCR–ABL transcripts from peripheral blood was performed every month during the first year and every 2 months thereafter. Beyond 2 years, molecular biology FU was performed every 3 months.In cases of molecular relapse, the treating physician was recommended to reintroduce TKI treatment. ResultsFrom July 9, 2007, to Dec 17, 2009, 100 pts (48 male, 52 female) with CML (51 pts previously treated with IFN) and a median age of 63 years (range 29–80) were recruited in the STIM trial. Eight years after the first inclusion, the median follow-up of the STIM study is now 50 months (range 9-72) with a mean of 51 months. Sixty-one patients met the definition of molecular relapse: 58 relapses occurring during the first 7 months and 3 late relapses at month 19, 20 and 22, respectively). No other molecular relapse was observed at the last update (July 2013). However, 4 extra-hematological deaths were observed: 1 case in DMR after 9 months of imatinib cessation (due to myocardial infarction) and 3 cases belonging to the molecular relapse group (due to respectively stroke, mesothelioma, and gastric carcinoma). So, considering the death in DMR without any relapse (n = 1) as a competing event the cumulative incidence of molecular relapse remains 60%. Among the 58 patients alive, we confirmed that all patients were sensitive to TKI re-challenge, and were re-treated with imatinib (n=48), nilotinib (n=5) and dasatinib (n=5). One patient stopped again for side effects. A Second attempt of TKI discontinuation was proposed for 15 patients in sustained DMR and 5 cases of molecular relapse were reported at the last update after this second attempt of TKI cessation.Among the initial 38 patients alive who are still in DMR without any treatment the median follow up is 54 months (range : 24- 71).Taking into account the cost of imatinib and the number of months without treatment in the total study population at last analysis, the savings within the STIM trial were estimated at 5.5 millions Euros. ConclusionImatinib can be safely discontinued in pts with a DMR of at least 2 years duration and we continue to recommend proposing discontinuation only in clinical trials with close molecular monitoring. In spite of the fact that we did not observed other molecular relapse beyond 2 years a long-term follow-up of the different cessation studies will be necessary to affirm cure. Because the life expectancy of de novo CML is now closed to the healthy population, the long-term medical costs and quality of life have become very important and depend on the possibility to cease safety TKI in long term. Disclosures:Rea:Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Etienne:novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Rousselot:BMS, Ariad, Pfizer: Honoraria from BMS, Ariad, Pfizer. Grants from BMS Other.

Frontline Treatment With Imatinib Mesylate in Chronic Myeloid Leukemia Patients in Early Chronic Phase: a Very Long-Term Analysis by the GIMEMA CML Working Party

BackgroundThe chronic myeloid leukemia (CML) therapeutic scenario has been enriched by the approval of second generation TKIs as frontline treatment of early chronic phase (ECP) patients, but imatinib mesylate (IM) still represents the standard for many patients. The long term outcome is extremely important to assess the treatment efficacy and to decide on the allocation of resources. The phase 3 trials comparing second generation TKIs versus standard-dose IM have not still demonstrated a clear improvement in terms of progression-free survival and overall survival. In the IRIS trial, at 8 year, 55% of patients were still on IM and the overall survival (OS) was 85%. Other published reports have shorter follow-up. Aims and MethodsTo assess the very long-term outcome of CML patients treated frontline with IM, we analyzed 559 patients enrolled within 3 multicentric prospective studies conducted by the GIMEMA CML Working Party (NCT00514488, NCT00510926, observational trial CML023). Definitions: major molecular response (MMR), BCR-ABLIS ratio <0.1%; deep molecular response (MR4.0), detectable disease ≤ 0.01% BCR-ABLIS or undetectable disease with ≥10,000 ABL transcripts; progression, transformation to accelerated or blastic phase; failure, according to 2013 ELN criteria; event, treatment discontinuation for any reason or lost to follow-up. Information on survival and progression were regularly collected. All deaths, at any time and for any reason, were included. All the analysis have been made according to the intention-to-treat principle. ResultsBaseline demographics characteristics: median age: 52 years (extremes 18-84 years); male sex: 60%; high Sokal, high Euro and high EUTOS scores: 22%, 7% and 7%, respectively; clonal chromosomal abnormalities (CCA) in Ph+ cells: 4% (not evaluable in 32% of patients for insufficient number of metaphases); e13a2 BCR-ABL transcript: 36%. Median follow-up: 76 (7-99) months. The cumulative incidence of complete cytogenetic response (CCyR), MMR and MR4 was 88%, 85% and 61%, respectively. The median time to CCyR, MMR and MR4 was 6, 7 and 42 months, respectively. Patients with high Sokal, high Euro and high EUTOS scores had significantly lower overall estimated probability of CCyR and MMR with respect to low and intermediate risk patients. A high Sokal score also predicted a significantly inferior probability of MR4; patients with high Euro and high EUTOS score had lower overall estimated probability of MR4, but the difference were not statistically significant. The reasons for IM discontinuation were: lack of efficacy (19%), toxicity or death (9%), withdrawal of informed consent (3%); 4% of patients were lost to follow-up. The 8-year event-free survival (EFS), failure-free survival (FFS), progression-free survival (PFS) and OS were 55% (95% CI: 51-60%), 66% (95% CI: 61-70%), 84% (95% CI: 78-89%) and 85% (95% CI: 79-90%), respectively. A high Sokal and a high Euro scores were able to identify patients with significantly lower probability of EFS, FFS, PFS and OS with respect to the other patients. High EUTOS score patients had significantly poorer EFS and FFS, but PFS and OS differences were not significant. Age, performance status and e13a2 transcript resulted independent prognostic factors on PFS and OS. ConclusionsUntil now, the available data on the very long-term outcome of newly diagnosed chronic phase CML patients treated frontline with imatinib are limited to a company sponsored study (IRIS study). The GIMEMA CML Working Party provided an unbiased overview of the long-term imatinib therapeutic effects in a multicentric nationwide experience. These results should be taken into consideration to make treatment decision concerning the choice of the first line TKI, particularly in low risk patients. AcknowledgmentsUniversity of Bologna, BolognaAIL, COFIN, Fondazione Carisbo. [Display omitted] Disclosures:Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Abruzzese:BMS, Novartis: Consultancy. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Preliminary Report Of The STIM2 Study: A Multicenter Stop Imatinib Trial For Chronic Phase Chronic Myeloid Leukemia De Novo Patients On Imatinib

BackgroundIn the Stop imatinib study (STIM)1, we previously demonstrated that Imatinib could be safely discontinued in patients with a sustained deep molecular response (DMR) (undetectable BCR-ABLtranscripts (UMRD) for at least 2 years) (Lancet oncology, 2010;11: 1029-1035). Recently, these results were confirmed by the Australasian TWISTER study using criteria of imatinib cessation very similar to those used in the STIM1 study (Blood, 2013; 122:515-22). However in the both studies, half of the patients were previously treated with IFN leading to a non-homogenous cohort of patients. So we conducted prospectively a second trial (STIM2) where cessation of imatinib treatment was proposed for patients in sustained DMR who were treated only with imatinib. MethodsIn this new multicenter, non-randomized STIM2 study (ClinicalTrials.gov, NCT01343173), imatinib was discontinued in CML pts with the same criteria as those we reported previously for the STIM1 i.e. sustained DMR defined as remission lasting more than 2 consecutive years and confirmed on five datapoints of BCR–ABL analyses by quantitative RT-PCR during these 2 years. The UMRD was defined with a sensitivity> to 4.5 log with ≥ 50.000 ABL transcripts amplified as internal control. The molecular relapse was defined as positivity of BCR–ABL transcript in Quantitative RT-PCR confirmed by a second analysis point indicating the increase of one log in relation to the first analysis point, on two successive assessments, or loss of MMR at one point. Quantitative RT-PCR for BCR–ABL transcripts from peripheral blood was performed every month during the first year and every 2 months thereafter. Beyond 2 years, molecular biology FU was performed every 3 months. ResultsFrom April 2011 to June 2013, 124 CML pts were included (62 men,62 women) with a median age of 61 years recruited in the 23 French centers. At the last update, the median follow up of the first 124 pts enrolled is 12 months (range 1-25). After discontinuation of imatinib, a molecular relapse occurred in 48 pts (45 relapses during the first 6 months and 3 relapses between 6 and 12 month), and 76 patients were still free of treatment at the last update with DMR. However 41 experienced a BCR-ABL RQ-PCR fluctuation without clear molecular relapse. In this so-called-fluctuation group of patients, 7 were found positive once, 6 twice, 12 patients between 3 and 5 times, 10 patients between 6 and 10 times and 6 patients more than 10 times confirming that BCR-ABL reappearance does not mean automatically clinical relapse.We confirmed also that all patients were sensitive to a TKI re-challenge. Actually, 33 patients were retreated with imatinib 5 with nilotinib and 3 with dasatinib. The median time to achieve again a CMR from the molecular relapse was 7 months (range 4-16 months) and from re-initiation of TKI was 4 months (range 2-14). Taking into account the cost of imatinib and the number of months without treatment in the total study population at last analysis, the savings within the STIM2 trial were estimated at 2.8 millions Euros. ConclusionWe confirm that Imatinib can be safely and prospectively discontinued in pts with DMR of at least 2 years duration in patients only treated with imatinib. To stop treatment it may not require the complete eradication of residual leukemic stem cells since positive fluctuation PCR results do not lead to CML relapse or progression. These intriguing results, even for imatinib treated patients since disease onset (already observed after IFN therapy), are comparable to those reported with the more sensitive PCR on DNA in the TWISTER study and are under investigations in our laboratory. Disclosures:Mahon:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Brisol Myers Squibb: Consultancy, Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Nicolini:Novartis, Ariad, Teva, BMS and Pfizer: Honoraria from Novartis, Ariad, Teva, BMS and Pfizer. Grants from Novartis. Other. Rea:Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Etienne:novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees.

Low WT1 Expression At Diagnosis Is a Strong Predictor On Poor Outcome In Patients With t(8;21) Acute Myeloid Leukemia

IntroductionAcute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease. The dynamic patterns of AML1-ETO transcript levels during treatment and clinical outcome of patients vary greatly. Our AML05 trial revealed that minimal residual disease (MRD)status and treatment strategy were independent risk factors for outcome and that risk stratification treatment directed by MRD may improve the outcome of t(8;21) AML in CR1 (Blood 2013; 121:4056). As for pretreatment parameters, c-KIT mutation is a well-established adverse predictor on survival. However, a subset of t (8;21) AML patients without c-KIT mutation still showed poor clinical outcome. The prognostic value of WT1 transcript levels at diagnosis in AML has been investigated and the results were controversial. We wondered if WT1 expression associated with outcome in t (8;21) AML patients. MethodsA total of 101 patients were included. They all were eligible cases who enrolled into AML05 trial from June 2005 to December 2012, and had available bone marrow samples at diagnosis. After 1 or 2 induction therapy and 2 cycles of intermediate-dose cytarabine-based consolidation therapy, fifty-seven patients continued cytarabine-based consolidation chemotherapy or received autologous-hematopoietic stem-cell transplantation (auto-SCT) and were defined as CT group, the remaining 44 patients received allogeneic SCT (allo-SCT) and defined as SCT group.WT1 and ABL transcript were tested by real time quantitative PCR, and WT1 transcript levels were calculated as WT1copies/ABL copies in percentage. The upper limit of normal bone marrows (NBMs) was 0.5%. c-KIT mutations in exon 8 and 17 were screened by direct sequencing. ResultsThe median follow-up time was 25 (6-93) months for 73 alive patients. The cumulative incidence of relapse (CIR) at 3 years was 35.3%. The 3-year disease free survival (DFS) and overall survival (OS) rates were 57.2% and 62.8%, respectively. The median WT1 transcript levelsssof all patients were 9.1% (0.02%-99.3%). c-KIT mutation was detected in 31 patients. Receiver operating characteristics (ROC) curves revealed that WT1 transcript levels of 5.0% (1-log increase compared to the upper limit of NBM) were the best cutoff values to discriminate patients with different outcome. WT1 transcript levels of ≤5.0% were significantly associated with c-KIT mutation (23/42 vs 8/59, P<0.001), but didn't related to other pretreatment parameters (all P>0.05). In CT group, patients with WT1≤5% (n=19) had significantly higher CIR rate at 3-year, lower 3-year DFS and OS rate than those with WT1>5% (n=38), respectively (89.5% vs 27.9%, P<0.0001; 10.5% vs 66.1%, P<0.0001; 28.5% vs 66.5%, P=0.0062). Furthermore, patients with WT1≤5% and c-KIT mutation (-) had similar CIR, DFS and OS rate to both patients with WT1≤5% and c-KIT mutation (+) and patients with WT1>5% and c-KIT mutation (+) (all P >0.05, Figure 1). Therefore, they were merged into one group (n=24). Thus, patients with WT1≤5% and/or KIT mutation (+) had significantly higher CIR rate at 3-year, lower 3-year DFS and OS rate than those with WT1>5% and c-KIT mutation (-) (n=33) in CT group, respectively (89.4% vs 27.4%,P<0.0001; 10.6% vs 72.6%, P<0.0001; 26.1% vs 72.2%, P=0.0013). For patients ith WT1≤5%, allo-SCT significantly lowered CIR rate at 3-year, improved 3-year DFS rate and tended to improve OS rate compared to chemotherapy/auto-SCT by landmark analysis, respectively (11.5% vs 88.2%, P<0.0001; 65.4% vs 11.8%, P=0.0001; 60.6% vs 32.6%, P=0.10. Figure 2). Multivariate analysis revealed that WT1 transcript levels (≤5% vs >5%) and treatment (chemotherapy/auto SCT vs allo-SCT) instead of other pretreatment parameters were independent prognostic factors for relapse (hazard ratio (HR) 0.20, 95% CI 0.093¨C0.44; 0.096, 95% CI 0.033¨C0.28. all P<0.0001) , DFS (HR 0.22, 95% CI 0.11¨C0.44; 0.23, 95% CI 0.11¨C0.49. all P<0.0001) and OS (HR 0.30, 95% CI 0.14¨C0.66, P=0.003; 0.37, 95% CI0.16¨C0.82, P=0.014).▪▪ ConclusionLess than 1-log increase of WT1 transcript levels at diagnosis is a strong predictor on poor outcome in patients with t (8;21) AML, and allo-SCT could significantly improve outcome of such patients. Grant SupportBejing Municipal Science & Technology Commission(Z111107067311070) and Nature Science Foundation of China (81170483). Disclosures:No relevant conflicts of interest to declare.

Development and Validation Of Highly Sensitive Mrdx BCR-ABL Test For Monitoring Deep Molecular Response In Patients With Chronic Myeloid Leukemia

Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKI) has led to progressively lower levels of disease burden and higher rates of complete molecular responses. Very few assays with an analytical sensitivity of 4.5-logs (0.00316% BCR-ABL/ABL) have been validated, and assay standardization at these levels has been problematic (EUTOS, 2007). In order to have an International Scale (IS) standardized assay that can precisely and accurately detect 4.5 log reductions in BCR-ABL/ABL ratios, the test system design must be tightly controlled to ensure this critical performance metric can be reproducibly met. The MolecularMD MRDxTM BCR-ABL Test was developed and validated to be an accurate, reproducible, and highly sensitive IS-standardized solution to measure minimal residual disease (MRD) in CML patients using either PAXgene Blood RNA or EDTA blood collection tubes.The MRDx Test is a one-step quantitative real-time polymerase chain reaction (RT-qPCR) test that allows for quantitation of BCR-ABL e13/14a2 (b2/3a2) transcripts (covering ≥ 95% of CML patients) and ABL transcripts in RNA extracted from peripheral blood. To achieve a sensitivity of 4.5 logs, several one step enzyme systems were evaluated against RNA samples that spanned the potential dynamic range of the assay (MR1.0 to MR5.0: MR1.0=1 log molecular response (MR), or 10% IS; MR5.0=5 log MR, or 0.001% IS). The one step enzyme system with the best sensitivity and precision was chosen based on the performance of three distinct lots. In addition, in order to better control for the variation of the reverse transcription, in vitro transcribed RNA calibrators were developed to create a standard curve for copy number determination rather than the more commonly used plasmid calibrators that may not accurately reflect the same processing as the patient sample RNA.The limit of detection (LOD) for blood drawn into PAXgene Blood RNA tubes was validated by testing a dilution series created from a baseline CML patient blood sample diluted into non-diseased subject blood. Creating this type of dilution series allows for the determination of the LOD in the actual clinical sample matrix as compared using cell line dilution series that have much higher BCR-ABL and ABL copy numbers than routine patient specimens. Based on the analysis of each level of sample tested over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.7 (Mean=MR4.9, 95% CI=MR5.2 to MR4.7) using the more conservative upper bound of the 95% confidence interval. The precision was evaluated based on the standard deviation of the log10 BCR-ABL/ABL ratio and was found to be ≤ 0.20 SD at MR5.0 and above. The LOD for blood drawn into EDTA blood collection tubes was validated by creation of a dilution series from a baseline CML patient RNA sample diluted into RNA isolated from blood of non-diseased subjects. Based on the number of samples with detectable BCR-ABL in at least 95% of replicates over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.9 (Mean=MR5.0, 95% CI=MR5.1 to MR4.9) using the more conservative upper bound of the 95% confidence interval with precision being ≤ 0.22 SD at MR5.0 and above.The accuracy of the BCR-ABL copy numbers, ABL copy numbers, and BCR-ABL/ABL ratio using the MRDx BCR-ABL Test was evaluated for ten patient samples using one step droplet digital PCR (ddPCR) as a reference method. For the BCR-ABL copy numbers, ABL copy numbers, and the BCR-ABL/ABL ratio, the bias of the MRDx Test relative to ddPCR by Bland-Altman method was 0.053, 0.030, and 0.023 respectively. The MRDx BCR-ABL Test reports on the IS with a conversion factor of 1.0 by use of the WHO International Standard 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR; secondary standards were created for bi-annual monitoring of the assay conversion factor to ensure accurate reporting of patient results on the IS. Confirmation of the conversion factor was done by an independent laboratory using different real-time PCR instruments and multiple technicians.Based on the validation data, the MRDx BCR-ABL Test is an accurate, reproducible, and highly sensitive IS-standardized solution to the growing need for a reliable and robust quantitative BCR-ABL assay that can be used for the monitoring of minimal residual disease in CML patients. Disclosures:Toplin:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Eibl:MolecularMD: Employment. Fjeld:MolecularMD: Employment. Yager:MolecularMD: Employment. McAdams:MolecularMD: Employment. Hawkins:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

Scoring Systems For Predicting Outcome Of Chronic Myeloid Leukemia In Adults Are Poorly Informative In Pediatric Patients Treated With Imatinib

IntroductionImatinib (IM) front-line treatment impressively improved survival of children with chronic myeloid leukemia (CML). In contrast to adult CML, specific scoring systems predicting the treatment response in individual pediatric patients (pts) are still lacking. Here we analyzed a cohort of pediatric pts with CML applying the established prognostic scores for adults in a comparative fashion. We question the value of four scoring systems (Sokal-, Sokal young-, Hasford-, Eutos-Score) especially with regard to grouping individual children differently or homogeneously into a defined risk category. In addition, we analyzed which scoring system would classify most specifically the prognosis of pediatric CML with regard to early molecular response (MR) on IM. MethodsA total of 90 pts (male/female: 57/33; median age: 11.6 yrs, range: 1-18) with CML-CP enrolled in the prospective trial CML-PAED-II were included in this analysis. Registry data were collected on standardized forms filled in by the treating physicians. On this basis the Eutos-, Sokal- and Hasford-Scores were calculated using internet resources of the ELN (www.leukemia-net.org/content/leukemias/cml/cml_score), whereas the Sokal young Score (Sy) – a score described specifically for adolescents and younger adults (Sokal JE, Blood 1985;66:1352) – was manually calculated. Pts were grouped using the original three risk categories (low=LR, intermediate=IR, high=HR) or two categories, respectively, for the Eutos-Score (LR or HR). Evaluation of therapeutic response was performed by assessing the MR by measurement of the transcript ratio BCR-ABL1/ABL1 in blood specimen sent to the central reference laboratory at month 3 after start of IM treatment. Measurements were expressed according to the International Scale. ResultsBy Sokal-Score 59/90 pts were classified as LR, 20/90 pts as IR and 11/90 pts as HR. By Hasford Score 57/90 pts were classified as LR, 25/90 pts as IR, and 8/90 pts as HR. By Eutos Score 73/90 pts were classified as LR and 17/90 pts as HR. As the hematocrit value was not collected systematically at diagnosis, this necessary parameter for calculating the Sy-Score was applicable only in 46/90 pts and thus 44/46 pts were classified as LR, 2/46 as IR, and 0/46 as HR. Comparing results of individual pts only 25/46 pts (54%) were categorized homogeneously as LR by applying all 4 scoring systems, while 54/90 pts (60%) were classified as LR if Sy-Score was excluded. Thus, the remaining 21/46 pts (46%) were grouped heterogeneously by applying each of the 4 prognostic scores, and correspondingly 33/90 pts (37%) were classified heterogeneously within different risk categories by the Eutos, Hasford and Sokal Score. Only 3 pts were categorized homogenously as HR by each of the Sokal, Hasford, and Eutos Score and by applying all 4 scoring systems no patient was concordantly classified as HR. When comparing only the Sokal-Score to the Sy-Score, discordant results were obtained in 19/46 (41%) pts.BCR-ABL1/ABL1 transcript ratio could be analyzed quantitatively in 72/90 pts at month 3 after treatment initiation. In this cohort we identified 46/72 good responders (ratio BCR-ABL1/ABL1 <10%) and 26/72 poor responders (ratio >10%). Although the Eutos-score performed best in in a logistic regression analysis with an Odds Ratio OR=3.02 to predict an unfavorable course of IM-treated CML in the HR group, the discrimination did not reach statistical significance (p=0.08). However, by reducing the cut-off point for the Eutos Score from 87 to 64 an OR=4.8 with p=0.004 was achieved, thus indicating that a refined risk categorization appears beneficial. ConclusionComparing risk categorization by all four scores in individual pediatric pts, results may vary considerably. Keeping in mind that the number of pts analyzed is still small, especially applying the Sy-Score seems not to provide benefit in this cohort with a median age of only 11 years. Contrasting results in adults, in this pediatric cohort the Sokal- and Hasford-Scores did not predict a poor IM treatment response at month 3 while the Eutos Score achieved borderline significance. Thus, there is an urgent need for the development of a more specific pediatric risk score. Disclosures:No relevant conflicts of interest to declare.

Clinical application of catalytically cleavable fluorescence probe technology for multiplexing quantification of BCR–ABL1 fusion transcripts

BackgroundAccurate measurement of BCR–ABL1 fusion transcripts is critical for therapeutic stratification in patients with chronic myelogenous leukemia (CML). Previous studies have reported the variable performance of the existing quantitative reverse transcription polymerase chain reaction (RQ-PCR). Here, we developed a one-step multiplex RQ-PCR method based on the catalytically cleavable fluorescence probe technology for quantification of BCR–ABL1 transcripts. MethodsPerformance was evaluated with respect to the limit of detection (LoD), linearity, precision, and comparison on the VIIA7 Real-Time PCR system. Multiplex RQ-PCR was performed by the one-step and one-well reaction without the hands-on time. ResultsOur assay showed a LoD of 1.5pg with linearity in the range of more than 4 logs of dilution. Intraassay, interassay, and total percent CVs at the concentration of 150ng were 12.8%, 22.6%, and 28.0%, respectively. The assay correlated well with Asuragen's BCR/ABL1 Quant™ kit over a 6 log concentration range (r=0.9967). ConclusionOur assay demonstrated comparable performance characteristics in comparison with previous RQ-PCR based on the TaqMan probe technology. We conclude that our method could be a reliable tool in the clinical setting.

Philadelphia chromosome positive precursor T acute lymphoblastic leukemia.

To the Editor, 26 year old male presented with generalized bulky lymphadenopathy, raised white blood cell count, anemia and thrombocytopenia in December of 2009. The peripheral smear and bone marrow were morphologically consistent with acute lymphoblastic leukaemia. Immunophenotype showed that the blast cells were positive for CD3 and CD7. All the B lineage markers were absent. In addition the blast cells were positive for CD13 and CD 33, but negative for CD117 and cytoplasmic myeloperoxidase. Cytoplasmic CD 3 was positive. We therefore made a diagnosis of acute T lymphoblastic leukemia with aberrant myeloid antigen expression. Conventional cytogenetics showed 9;22 translocation. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the presence of minor BCR–ABL transcript at high levels which is shown in the Fig. 1. Fig. 1 Band at 106 bp is patient’s sample which is positive for minor break point e1a2 (lane 5) corresponding to p190KD protein. The previous lane (lane 4) showing the band at 330 bp is internal control for the ABL region of the patient’s ... T lymphoblastic leukemia with Philadelphia positivity is very rarely reported in the literature [1–4]. There is a debate whether it is de novo leukemia or blastic phase of chronic myeloid leukemia [5]. Our patient did not have a palpable spleen. Absence of splenomegaly and the presence of minor BCR–ABL transcript probably favours an acute lymphoblastic leukemia of precursor T cell lineage. The presence of aberrant myeloid markers makes our case more interesting. The patient was treated with a combination of chemotherapy and imatinib. He achieved morphologic remission but has not achieved molecular remission and hence switched over to second line tyrosine kinase inhibitor dasatinib. There was no BCR–ABL mutation detected. Bone marrow transplantation could not be offered as the patient does not have a histocompatible sibling and an alternative donor transplantation was not feasible due to various logistic constraints.

Cleavage of BCR–ABL transcripts at the T315I point mutation by DNAzyme promotes apoptotic cell death in imatinib-resistant BCR–ABL leukemic cells

The BCR-ABL fusion transcript encodes the BCR-ABL tyrosine kinase (TK), which causes chronic myelogenous leukemia (CML). Although the TK inhibitor imatinib mesylate, which targets the BCR-ABL protein, has been proven to be effective in controlling leukemic growth, imatinib resistance has been observed with disease relapse because of point mutations in the ABL gene that inhibit imatinib efficacy. In this study, we designed oligodeoxyribozymes (DNAzymes) that specifically target and cleave both the junction sequence and the site of the point mutation (T315I), conferring imatinib resistance in BCR-ABL mRNA. DNAzymes significantly induced apoptosis and inhibited proliferation in wild-type and T315I-mutant BCR-ABL-positive cells. Selective cleavage of T315I-mutant ABL mRNA by DNAzyme (T315I Dz) led to cell cycle arrest in G0/G1 phase, with induction of caspase-3/-7 in imatinib-resistant BCR-ABL-positive cells harboring the T315I mutation. Moreover, cotreatment with the DNAzyme targeting the T315I mutation and imatinib resulted in enhanced inhibition of proliferation and induction of apoptosis in T315I leukemic cells as compared with imatinib alone, thereby antagonizing imatinib resistance in CML cells bearing T315I-mutant BCR-ABL. Therefore, cleavage of T315I-mutant ABL mRNA by DNAzyme combined with imatinib treatment may be an alternative approach to overcoming imatinib resistance in leukemic cells.

IK6 isoform with associated cytogenetic and molecular abnormalities in Chinese patients with Philadelphia chromosome-positive adult acute lymphoblastic leukemia

The IK6 isoform plays an important role in Philadelphia chromosome-positive adult acute lymphoblastic leukemia (Ph + ALL). This study was designed to monitor the expression of the IK6 isoform with associated cytogenetic and molecular abnormalities. The IK6 isoform, cytogenetic and molecular abnormalities were detected in 100 Chinese patients with de novo Ph+ adult ALL. Expression levels of the IK6 isoform and BCR–ABL1 transcripts were monitored during treatment. BCR–ABL1 mutation was identified in 45 paired samples. Strong correlations were found between the expression status of the IK6 isoform and blast cells, additional cytogenetic abnormalities, BCR–ABL1 transcripts, increased risk of relapse, shorter relapse-free survival and overall survival at diagnosis. Higher frequencies of single IK6 expression and ABL mutation, including the types and shifts thereof, were confirmed in relapsed patients. Furthermore, expression of the IK6 isoform was dynamically consistent with BCR–ABL1 transcript levels during treatment in the single expression group, whereas no such correlation was observed in the co-expression group. The expression pattern of the IK6 isoform was altered in three patients from the co-expression group. The findings of this study in Chinese patients with Ph+ adult ALL exhibit some discrepancies with data reported in other countries, thereby enhancing current knowledge on the therapeutic response and prognosis of this disease.