ALBERT

Description: Background Chronic myeloid leukemia (CML) stem cells are inherently insensitive to tyrosine-kinase inhibitors (TKI). However, an important minority of CML patients was shown to discontinue TKI without experiencing molecular relapse. Underlying mechanisms are currently unknown. Plasmacytoid dendritic cells (pDCs) are critical regulators of immune responses. Following activation, pDC upregulate MHC-class II and other DC activation markers such as CD86 (also known as B7.2). CD86 is a co-stimulatory molecule during T-cell activation, but also ligand of the inhibitory immune checkpoint receptor CTLA-4, which counteracts T-cell activation. The origin and function of pDC in CML biology is unknown. Within a sub-study of the EUROSKI TKI discontinuation trial we prospectively tested the hypothesis that pDC counts and CD86 expression status govern relapse risk following TKI discontinuation. Methods: Using flow cytometry, cell sorting and fluorescence in situ hybridization (FISH), CD86 expression and BCR-ABL status were analyzed in PDCA-2+/CD123+ peripheral blood (pB) pDC of untreated CML patients (CML pDC), normal donors and 123 patients, who had stopped TKI therapy in deep molecular remission within the international EUROSKI study (EUDRACT 2011-000440-22). All 123 EUROSKI patients had given written informed consent to participate in the immunological sub-study of the EUROSKI trial. Fresh samples from 19 EUROSKI centers in Germany were centrally analyzed prior, as well as 1, 2, 3 and 6 months after TKI discontinuation. PB CD86+ pDC counts were calculated per 105 cells in the lymphocyte gate. Decision trees and 10-fold cross validation were employed to establish relapse prediction accuracy for this value. Results CML pDC were BCR-ABL-FISH positive (median: 81%; range, 57 to 100%). In contrast, the proportion of CD86+ CML pDC varied substantially (median: 25.9%, range 3.2% to 82.4%), suggesting that CD86 expression on CML pDC was not a direct consequence of oncogenic BCR-ABL signaling. This was confirmed experimentally in a murine CML model. In contrast to CML pDC, remission pDC were always BCR-ABL FISH negative (n=10), but still displayed a comparable high proportion of CD86 positive pDC (median: 21%; range, 2.2% to 62%). In contrast, normal donor pDC were rarely CD86 positive (median: 6.8%; range, 4.2% to 17%), reinforcing the aberrant, and BCR-ABL-independent nature of CD86 expression on CML and remission pDC. As a result, healthy donors displayed only between 26 to 84 CD86+ pDC per 105 lymphocytes, whereas EUROSKI remission patients exhibited between 6 to 309 CD86+ pDC per 105 lymphocytes. Based on the important role of CD86 as a high affinity ligand of the inhibitory immune checkpoint receptor CTLA-4, we next asked, whether CD86+ pDC counts are associated with relapse risk after TKI discontinuation. Strikingly, statistical models suggested that a CD86+ pDC count below or above 95 CD86+ pDC/105 lymphocytes optimally separated two relapse categories of EUROSKI patients. Whereas relapse free survival (RFS) (loss of MMR) for patients with more than 95 CD86+ pDC/105 lymphocytes was 30% (n=32), RFS was 69% for patients (n=91) with less than 95 CD86+ pDC/105 lymphocytes (p

Description: Digital PCR (dPCR) generates an absolute read out that is largely tolerant to variations in PCR efficiency, reducing the requirement for standardisation like the conversion of the BCR-ABL/ABL ratio to international scale (IS). The aim of this study was to compare the results of dPCR to qPCR in blinded samples from two independent laboratories with respect to the observed rates of molecular response (MR) in CML patients (pts) having undergone 18 months of nilotinib treatment in the ENEST1st trial. A total of 230 cDNA samples from CML pts treated within the ENEST1st trial with e13 or e14/a2 BCR-ABL fusion genes were analysed in Leipzig (L, n=75) or Mannheim (M, n=155) with qPCR between 2012 and 2013. BCR-ABL levels were determined relative to those of ABL and standardization was achieved using plasmid DNA. Both labs are accredited by the European Treatment Outcome Study (EUTOS) collaboration. The cDNA samples were blinded for the qPCR results and re-analysed in L with a duplex dPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer's recommendations, samples yielding a minimum of 3 positive droplets in duplicates from the 12-19.000 routinely analysed were scored as positive (+). Depth of MR was scored using the EUTOS definitions used in the ENEST1st trial. For the whole cohort, the median copy number (CN) of BCR-ABL and ABL was 12 and 59350 by dPCR and 10 and 53537 by qPCR, respectively. Both methods detected similar numbers of BCR- ABL+ samples (dPCR 186, qPCR 189) with a median % BCR-ABL 0.022 by dPCR compared 0.013 by qPCR after conversion to IS. 90% of the BCR-ABL+ samples with dPCR were within an deviation of 4.06 -fold (median 1.22 fold) from qPCR for BCR-ABL, 1.77 fold (median 1.06 fold) for ABL and 6.43 fold (median 1.72 fold) for %BCR-ABLIS. Samples from L showed median CN with dPCR and qPCR for BCR-ABL (9 and 10) and ABL (29670 and 30734) with a correlation R2 = 0.95 and 0.84. The median % BCR-ABL was 0.02% by dPCR and 0.03% before and 0.01% after conversion to IS with qPCR. 90% of the BCR-ABL+ samples by dPCR were within a range of 2.9 -fold deviation (median 0.66 fold) from qPCR for BCR-ABL, 1.9 fold (median 0.91 fold) for ABL, 2.6 fold (median 0.74 fold) for %BCR-ABL and 7.5 fold (median 1.96 fold) for %BCR-ABLIS. Samples from M had higher median CN for BCR-ABL and ABL (16 and 80000) by dPCR compared to qPCR (10 and 66570). Correlation was better for BCR-ABL compared to ABL with R2=0.95 and R2 = 0.74. The median % BCR-ABL was 0.022 by dPCR compared to 0.017 and 0.015 with qPCR after conversion to IS. 90% of the BCR-ABL positive samples with dPCR were within a deviation of 4.7 -fold (median 1.6 fold) from qPCR for BCR-ABL, 1.7 fold (median 1.1 fold) for ABL, 4.9 fold (median 1.5 fold) for %BCR-ABL and 5.6 fold (median 1.4 fold) for %BCR-ABLIS. The cumulative rates of MR3, MR4 and MR4.5 or better @ 18 months of treatment in the ENEST1st trial were 83, 43 and 29% with qPCR. The distribution in MR classes was significantly different between dPCR and qPCR (p=MR4 when analysed by dPCR compared to qPCR (n=77 vs. 100, pMR3 (M/L) MR3 (M/L) MR4 (M/L) MR4.5 (M/L) MR5 (M/L) n (M/L) 〉MR3 37 (26/11) 1 (0/1) 1 (1/0) 0 0 39 (27/12) MR3 16 (7/9) 68 (48/20) 6 (4/2) 0 1 (1/0) 91 (60/31) qPCR (n) MR4 0 15 (10/5) 10 (6/4) 5 (3/2) 3 (3/0) 33 (22/11) MR4.5 0 15 (7/8) 12 (7/5) 9 (4/5) 8 (7/1) 44 (25/19) MR5 0 1 (1/0) 6 (5/1) 6 (6/0) 10 (9/1) 23 (21/2) dPCR n (M/L) 53 (33/20) 100 (66/34) 35 (23/12) 20 (13/7) 22 (20/2) 230 Disclosures Franke: Novartis: Other: Travel Costs; BMS: Honoraria; MSD: Other: Travel Costs. Frank:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Müller:Novartis: Honoraria, Other: CONSULTING OR ADVISORY ROLE, Research Funding; BMS: Honoraria, Other: Consulting or Advisory Role, Research Funding; ARIAD Pharmaceuticals Inc.: Honoraria, Other: Consulting & Advisory Role, Research Funding. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lange:Novartis: Research Funding.

Description: Background: In the current ELN recommendations (Baccarani et al., Blood 2013) the optimal time point to achieve major molecular remission (MMR) is defined at 12 months after diagnosis of CML. MMR is not a failure criterion at any time point leading to uncertainties when to change therapy in CML patients not reaching MMR after 12 months. Aims: We sought to evaluate a failure time point for MMR using data of the CML-Study IV, a randomized five-arm trial designed to optimize imatinib therapy alone or in combination. In addition the optimal time-point to achieve a MMR should be evaluated. Methods: Patients with valid molecular analysis on MR4 level were divided randomly into a learning (LS) and a validation sample (VS). For the LS, MR2 (defined as BCR-ABL

Description: Introduction: Early prediction of outcome using response-related predictive landmarks has become a major paradigm in the clinical management of chronic myeloid leukemia (CML). Several studies have shown the predictive impact of 10% BCR-ABLIS at 3 and 6 months for different tyrosine kinase inhibitors. The question, which landmark should define treatment failure and determine treatment intervention has been discussed vividly. However, an objective analysis of quality criteria for different early prognostic landmarks is lacking up to now. Here we compare sensitivity, specificity and the proportion of later disease progressions predicted by 3-month and 6-month landmarks in imatinib-treated patients of the CML-study IV. Methods: A total of 1,303 newly diagnosed patients were assigned to an imatinib-based treatment arm of CML-Study IV by April 2010. Median follow-up was 7.1 years. The number of molecular assessments was as follows: n=789 (at 6 months), n=692 (at 3 months) and n=301 (at 3 months and at diagnosis, without pretreatment). Gene expression levels were determined by quantitative RT-PCR. At 3 and 6 months, a BCR-ABL ratio was calculated using ABL as reference gene and standardized according to the international scale (BCR-ABLIS). In addition, at 3 months and at diagnosis a BCR-ABL ratio was calculated using beta-glucuronidase (GUS) as reference gene in order to ensure linearity of measurement at diagnosis. The log reduction at 3 months was calculated from the BCR-ABL ratio at 3 months and at diagnosis. Due to the time-dependent nature of censored survival data, the sensitivity and specificity at eight years were calculated using the method by Heagerty et al. (Biometrics 2000). Overall survival (OS) is defined by the absence of death from any reason, progression-free survival (PFS) is defined as survival in the absence of progression to accelerated or blastic phase. Landmark analyses were performed to compare survival outcomes according to Kaplan-Meier. Results:Comparing the 10% BCR-ABLIS landmark at 3 and 6 months, 8-year OS and PFS rates are equal or comparable (table). In contrast, sensitivity and specificity differ substantially with an advantage in favor of sensitivity for the 3-month landmark and in favor of specificity for the 6-month landmark. This difference is paralleled by a smaller proportion of high-risk patients and less progressions identified by the 6-month landmark. From a clinical point of view the 6-month landmark is not only less than half as sensitive, moreover a treatment intervention at 6 months might also prevent less progressions due to the delay of 3 months. The half-log reduction landmark at 3 months is as sensitive as 10% BCR-ABLIS at the same time. However, it shows improved specificity and defines the smallest proportion of high-risk patients. Conclusion: The 10% BCR-ABLIS landmark, which is currently defining treatment failure at 6 months according to European LeukemiaNet (ELN) criteria, fails to detect the majority of patients with later disease progression. Less than a half-log reduction of individual baseline BCR-ABL transcript levels at 3 months on treatment identifies patients with later progressions as sensitive but with higher specificity as compared to 10% BCR-ABLIS. Abstract 156. Table Prognostic landmark 8-year OS (%) 8-year PFS (%) P-value for PFS Sensitivity to predict progression (%) Specificity to predict progression (%) High-risk patients Disease progressions classified as high-risk / total 3 months (n=692) 10% BCR-ABLIS 88 vs. 96 82 vs. 90 0.001 41.1 74.6 191 (28%) 32/74 (43%) 6 months (n=789) 10% BCR-ABLIS 88 vs. 96 84 vs. 95 0.001 18.2 93.8 95 (12%) 17/74 (23%) 1% BCR-ABLIS 90 vs. 97 89 vs. 97

Description: Introduction The impact of the type of therapy on cytogenetic evolution in chronic myeloid leukemia (CML) regarding the occurrence of additional cytogenetic aberrations (ACA) at the time point of blast crisis (BC) may be critical. The aim of this analysis was to elucidate whether patients (pts) treated with imatinib (IM) had ACA less frequently than pts treated with BU and other therapies used prior to IM as hydroxyurea (HU) and interferon alpha (IFN). We comparatively analyze the BC karyotype of CML pts treated in consecutive trials of the German CML Study Group (Studies I, II and IV) to answer the following question: Does CML therapy influence the occurrence or even induce ACA or do these alterations rather reflect the natural history and the biology of the disease and are independent of therapy? Materials and methods Cytogenetic data of 157 pts with Philadelphia chromosome and BCR-ABL positive CML in BC were analyzed from a total of 2,380 pts randomized to CML study I (BU vs. HU vs. IFN, recruitment 1983 – 1991), CML study II (IFN + HU vs. HU, recruitment 1991 – 1994), and CML study IV (IM 400 mg vs. IM 800 mg vs. IM 400 mg +IFN vs. IM 400 mg + AraC vs. IM 400 mg after IFN failure recruitment 2002 – 2012). Cytogenetic analysis was reported according to ISCN 2005. ACA were divided into major route (+8, i(17)(q10), +19, +der(22)t(9;22)(q34;q11)) and minor route alterations (reciprocal translocations other than the t(9;22)(q34;q11), e.g. t(1;21), t(2;16), t(3;12), t(4;6), t(5;8), t(15;20) (Fabarius et al., Blood 2011). Confirmatory testing of pairwise comparisons of therapies with regard to their frequency of major ACAs was performed using two-sided chi-square test. To keep the level of significance at 0.05 despite multiple testing, a priori hypotheses were hierarchically ordered: First, frequency of major route ACA of pts on IM was compared with that on BU, then, with HU and with IFN. Next, the comparisons of IFN vs. BU and IFN vs. HU were planned. Cumulative incidences were estimated under consideration of death before BC as a competing risk. Results 115 of 188 pts randomized to BU (CML study I), 117 of 194 pts randomized to HU (CML study I only) and 159 of 360 randomized to IFN-based therapy (CML studies I+II) progressed to BC. Eight-year cumulative incidence probability of BC was 0.63 [95%-confidence interval (CI): 0.56; 0.69], 0.60 [95%-CI: 0.53; 0.66], and 0.49 [95%-CI: 0.43; 0.54] in pts randomized to BU, HU, and IFN-based therapy, respectively and 0.06 [95%-CI: 0.04; 0.07] in pts on IM (CML-study IV). Three-year survival probabilities after BC were 0.009 [95%-CI: 0.001; 0.043] with BU, 0.017 [95%-CI: 0.003; 0.055] with HU, 0.013 [95%-CI: 0.003; 0.042] with IFN, and 0.252 [95%-CI: 0.157; 0.368] with IM. Cytogenetic data at BC with banding analysis were available from 21 pts on BU, 31 on HU, 56 on IFN and 49 on IM. 81% of pts treated with BU, 52% with HU, 38% with IFN and 55% with IM showed major route ACA. All other pts had minor route ACA or translocation t(9;22)(q34;q11) and variant translocation (t(v;22)) without ACA (Table 1). The difference in major route ACA between BU and IM was significant (p = 0.04, two-sided chi-square test). There was no statistically significant difference in ACA between pts on HU and IFN in comparison to IM. According to the testing order, further comparative testing was not possible. However, the differences of induction of major route ACA between HU and BU and IFN and BU were even more pronounced than the difference between IM and BU. The most frequently observed major route ACA was trisomy 8 in all studies and therapy arms. Conclusions The type of cytogenetic aberrations in CML BC after different therapies is comparable. The characteristic major route ACA after various therapies points to a CML BC-related chromosomal pattern rather than a therapy-induced effect. Pts treated with IM showed a significantly lower rate of major route ACA than BU. IM not only reduces the frequency of BC and increases survival probabilities but appears to moderately change the biology of BC as compared to BU Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Travel Other. Müller:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding; Ariad: Honoraria. Kolb:Pierre Fabre, Therakos: Honoraria; Kolb Consulting UG: Consultancy, Equity Ownership. Saussele:BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Travel Other.

Description: Introduction Early assessment of BCR-ABL transcript levels at 3 months allows the prediction of survival and may serve as a trigger for treatment intensification in CML patients with slow response to imatinib. The exact decline of BCR-ABL transcript levels within the first 3 months of treatment is defined by the ratio BCR-ABL transcripts at 3 months to BCR-ABL transcripts at baseline. This ratio might better reflect the individual biology of disease and its susceptibility to tyrosine kinase inhibition. Methods A total of 408 chronic phase CML patients (pts) with baseline and 3 month blood samples available in one single laboratory were investigated. Pts with pre-treatment before first blood sampling were excluded (imatinib with or without hydroxyurea, n=58; hydroxyurea only, n=49). A total of 301 evaluable pts (median age 52 years, range 18-85, 41% female) were treated with an imatinib-based therapy within the CML-Study IV. Median follow-up was 4.8 years. Transcript levels of BCR-ABL, total ABL, and beta-glucuronidase (GUS) were determined by quantitative RT-PCR. Exploratory landmark analyses were performed with regard to overall and progression-free survival (OS, PFS) to evaluate the prognostic significance of (i) BCR-ABL/GUS before treatment, (ii) the individual reduction of transcripts given by (BCR-ABL/GUS at 3 months) / (BCR-ABL/GUS before treatment), and (iii) the 3-month 10% BCR-ABLIS landmark. Results The median BCR-ABL/GUS ratio was 15.5% at diagnosis (0.06-107) and 0.63% at 3 months (0-84) reflecting a decline to the 0.04-fold (1.4 log reduction). i) No prognostic cut-off could be identified for BCR-ABL/GUS before treatment. ii) A reduction to the 0.35-fold of the initial BCR-ABL transcript level at diagnosis (0.46 log reduction) was identified as best cut-off according to a hazard ratio of 5.6 (95%-CI 2.3-13.4, p

Description: Introduction The emergence of next generation RNA sequencing (RNA-Seq) technologies will likely advance diagnostic, prognostic and therapeutic strategies for patients (pts) with various cancers.Novel fusions have recently been described in AML and solid tumors using RNA-Seq, and many were out-of-frame.It is not known whether novel fusions are generated at diagnosis (Dx) of CML and if so, their impact on treatment outcome. We used RNA-Seq coupled with whole exome sequencing to identify and characterize novel fusions at Dx of CML and at blast crisis (BC). A highly complex pattern of genomic rearrangements of chromosome (chr) 9 and 22 was found in some pts at Dx that generated novel fusions associated with multiple genomic breaks, multiple non-contiguous deletionsand inversion of genomic sequences, including BCR and ABL. Method RNA-Seqwas performed on Dx samples of chronic phase pts treated with first line TKI representing 2 extreme response groups: 14 pts with BC at a median of 6 months (mos), range 3-25 (group A, poor response), and 16 pts with rapid major molecular response by 3mosof imatinib (group B, optimal response). RNA-Seqwas also performed for 9 of 14 pts at BC (group C).The TruSeq Stranded Total RNA-RiboZero Gold Sample Prep Kit (Illumina) was used. This method enables computation of transcription direction and detection of genomic breaks from precursor RNA. Fusions were identified usingthe STAR algorithm and those detected in 4 normal controls were filtered out. Fusions with a high unique read count, supporting genomic breaks or detection atDxand BC for individual pts were prioritized for validation and their somatic status confirmed by RT-PCR.Correspondingwhole exome sequencingwas conducted for 30 samples. Copy number variation was detected usingSequenzaand exon level resolution ofdeletionswas achieved using an in-house sequence read normalization method. Results BCR-ABL fusions were detected by RNA-Seq in 29/30 pts at Dx and all pts at BC. In addition, novel fusions were identified in eachptgroup. GroupA(poor response). AtDx, 8 cytogenetically cryptic novel fusion transcripts were detected in 4/14 pts, Fig A pts1-4. All fusions involved genes or sequences onchr9 and/or 22 and all 4 pts had concomitant genomic inversion events. Fusion partners included inverted ABL intronic sequences and an inverted intergenic region on chr 22, potentially derived from the generation and activation of cryptic splice sites. BCR was a frequent fusion partner (5/8 fusion transcripts). Genomicdeletionswere detected adjacent to some fusions (3deletionsin 1pt),indicatingdeletionsmay have contributed to fusion formation, Fig B. All 4 pts with novel fusions and inversions had very rapid BC (within 5mosofDx). Group B (optimal response).AtDx, only 1/16 pts had a fusion detected in addition to BCR-ABL: TNRC6B (chr22)-NEK6 (chr9), Fig Apt5. Thisptalso had multiple non-contiguousdeletions: 2 each onchr9 and 22 associated with fusion formation, but no inversions, Fig B. Group C (BC). At BC, 3/9 pts gained fusions. No inversions were detected. Two pts had MLL fusions; MLL-BCAT1 (novel) and MLL-MLLT6. The MLL gene is a known fusion partner in acute leukemia, associated with poor prognosis. Both pts had sudden onset BC after a complete cytogenetic response. These fusions were supported by translocation events detected by cytogenetic analysis;t(11;12)(q23;p12) and t(11;17)(q23;q21). The thirdptgained an out-of-frame ANKRD11-UBQLN1 fusion at BC. Indeed, ANKRD11 expression was reduced by 3-fold at BC. Interestingly, thispthad a germline gain of function TP53 mutation. ANKRD11 is a p53 coactivator and loss of expression defined poor prognosis in breast cancer pts that harbored gain of function p53 mutations (Noll, 2012). The ANKRD11 fusion detected at BC in CML may have been selected with disease progression in the context of mutant p53. Conclusion We identified a subset of pts with novel fusions and inversion events at Dx involvingchr9 and 22. These inversions were detected among the pts studied with very rapid BC. The biological effects of the novel fusions remain to be determined. Our data support the presence of novel fusions, additional to BCR-ABL in CML and add a further layer of genetic heterogeneity associated with the Philadelphia translocation. Whether genomic inversions identify a small subset of CML pts with very poor prognosis requires expanded analysis. Disclosures Yeung: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Mueller:Ariad: Honoraria; Institute for Hematology and Oncology GmbH: Employment; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Dietz:Institute for Hematology and Oncology GmbH: Employment. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy.

Description: Introduction: The clonal selection of a mutant BCR-ABL positive clone can be observed in about one of two patients with imatinib-resistant chronic myeloid leukemia (CML). The early detection of BCR-ABL kinase domain mutations is crucial, since it allows to change the tyrosine kinase inhibitor (TKI) regimen in a timely manner and may therefore prevent disease progression and the accumulation of further genetic lesions. European LeukemiaNet (ELN) recommendations suggest a mutation analysis if optimal response criteria are not achieved at 3, 6, 12 or 18 months, or whenever a loss of optimal response occurs (Soverini et al., Blood 2011). Several attempts have been made to derive this indication from a specific increase of BCR-ABL levels. Here we report on the correlation of a rise in BCR-ABL transcript levels and the prevalence of BCR-ABL kinase domain mutations in imatinib-treated patients of the CML-Study IV. Methods: A total of 1,173 patients were enrolled until 2009 and randomized to one of four imatinib-based treatment arms. BCR-ABLIS of 988 patients was determined in 7,876 samples by quantitative RT-PCR in the central laboratory (median sample number per patient: 8.4, range 1-37; median follow up: 34 months, range 0-86), representing the eligible patients for the study. Thereby, the estimated intra-laboratory variance is assumed to be about 20%. A first rise of BCR-ABLIS to at least two-fold and 〉0.1% between two samples of a patient's molecular course defined a sample suspected of bearing a mutant BCR-ABL positive clone. A mutation analysis was performed on this critical sample by direct sequencing of ABL exons 4 to 10. Results: A critical rise in BCR-ABLIS was observed in 231 of 988 patients (23%) after a median of 15.2 months on treatment (range 2.8-59.4). In the corresponding sample 33 mutant clones could be detected in 31 patients (13%). Thereby a steeper rise of BCR-ABLIS was correlated with a higher incidence of BCR-ABL mutations in the respective group (table). A total of 18 different mutations could be detected, the most frequent were: M244V, n=7 (21%); E255K, n=4 (12%); T315I, n=3 (9%); L248V, G250E, L387M and F486S, n=2 (6%), respectively. Mutations occur in a substantial proportion (8%) of patients with an only 2 to 3-fold rise of BCR-ABLIS transcript levels (table). Therefore, the most sensitive cut-off should be applied and mutation analysis may be triggered by a doubling of BCR-ABL transcripts at levels 〉0.1% IS. Conclusion: BCR-ABL kinase domain mutations occur already in a substantial proportion of patients with a doubling of BCR-ABL transcript levels, which should determine mutation analysis. Table 1. Rise of BCR-ABL expression Patients (n) Patients with BCR-ABL mutations (n) Patients with BCR-ABL mutations (%) Inter-sample interval(median, days) 2 to 3-fold 72 6 8.3 98 3 to 5-fold 50 3 6.0 100 5 to 10-fold 39 4 10.3 99 10 to 100-fold 49 10 20.4 98 〉 100-fold 21 8 38.1 125 〉 2-fold (total) 231 31 13.4 101 Disclosures Hanfstein: Novartis: Research Funding; Bristol-Myers Squibb: Honoraria. Hehlmann:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding. Saussele:Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Neubauer:MedUpdate: Honoraria, Speakers Bureau. Kneba:Novartis: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pfirrmann:Novartis: Consultancy; Bristol-Myers Squibb: Honoraria. Hochhaus:Pfizer: Consultancy, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Müller:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Description: During the course of chronic myeloid leukemia (CML) progression to blast crisis (BC) is thought to be caused by genetic instability such as cytogenetic aberrations in addition to the translocation t(9;22)(q34;q11). We have shown previously that major route ACA indicate an unfavorable outcome (Fabarius et al., Blood 2011). We now investigate whether there is a correlation in time between appearance of major route ACA and increase in blast count. Methods: Cytogenetic data and blast count in the peripheral blood were available from 1,290 CML patients recruited to the German CML-studies III (621 patients) and IIIa (669 patients) from January 1995 to January 2004. Treatments were interferon-alpha-based or related allogeneic stem cell transplantation (HSCT). Presence of ACA and major route ACA was considered as a time-dependent covariate. Multivariate proportional hazards models were estimated taking Euro CML score, study III vs. IIIa and stem cell transplantability into account. Cumulative incidences of blast increases were calculated starting at the date of the first ACA or major route ACA, respectively, regarding death as a competing risk. Patients were censored at the date of HSCT with an unrelated donor. Results: 1,287 patients were evaluable with median observation times of 13 and 12 years and a 10-year survival of 48% and 61% in CML studies III and IIIa, respectively. 258 patients progressed to BC with a cumulative 10-year incidence of 20%. 195 patients displayed ACA during the course of disease. 45 patients (15.7%) showed ACA already at diagnosis. 44 patients showed unbalanced minor route, 29 balanced minor route aberrations, 23 -Y. 109 patients showed major route aberrations including 10 with other prior ACA. In a multivariate analysis on 1,257 patients, patients with ACA had a hazard ratio (HR) for a blast increase of between 2.0-2.2 (p

Description: Introduction Current evidence indicates that acquired genetic instability in chronic myeloid leukemia (CML) as a consequence of the balanced reciprocal translocation t(9;22)(q34;q11) or the variant translocation t(v;22) and the resulting BCR-ABL fusion causes the continuous acquisition of additional chromosomal aberrations (ACA) and mutations and thereby progression to accelerated phase and blast crisis (BC). At least 10% of patients in chronic phase (CP) CML show ACA already at diagnosis and more than 80% of patients acquire ACA during the transformation process into BC. Therefore, alterations at diagnosis as well as acquisition of chromosomal changes during treatment are considered as a poor prognostic factor. Differences in progression-free survival (PFS) and overall survival (OS) have been detected depending on the type of ACA. Patients with major route ACA (+8, i(17)(q10), +19, +der(22)t(9;22)(q34;q11)) and with other alterations like -X, del(1)(q21), del(5)(q11q14), +10, -21 at diagnosis resulting in an unbalanced karyotype have a worse outcome. Patients with minor route ACA (for example reciprocal translocations other than the t(9;22)(q34;q11) (e.g. t(1;21), t(2;16), t(3;12), t(4;6), t(5;8), t(15;20)) resulting in a balanced karyotype show no differences in OS and PFS compared to patients with the standard translocation, a variant translocation or the loss of the Y chromosome (Fabarius et al., Blood 2011). Here we compare the type of chromosomal changes (i.e. balanced vs. unbalanced karyotypes) during the course of the disease from CP to BC aiming to provide a valid parameter for future risk stratification. Patients and Methods Clinical and cytogenetic data available from 1,346 out of 1,524 patients at diagnosis (40% females vs. 60% males; median age 53 years (range, 16-88)) with Philadelphia and BCR-ABL positive CP CML included until March 2012 in the German CML-Study IV (a randomized 5-arm trial to optimize imatinib therapy) were investigated. ACA were comparatively analyzed in CP and in BC. Results At diagnosis 1,174/1,346 patients (87%) had the standard t(9;22)(q34;q11) only and 75 patients (6%) had a variant t(v;22). Ninety-seven patients (7%) had additional cytogenetic aberrations. Of these, 44 patients (3%) lacked the Y chromosome (-Y) and 53 patients (4%) had ACA. Regarding the patients with ACA thirty-six of the 53 patients (68%) had an unbalanced karyotype and 17/53 patients (32%) a balanced karyotype. During the course of the disease 73 patients (out of 1,524 patients) developed a BC during the observation time (5%). Cytogenetic data were available in 52 patients with BC (21 patients with BC had no cytogenetic analysis). Three patients had a normal male or female karyotype after stem cell transplantation. Nine patients showed the translocation t(9;22)(q34;q11) or a variant translocation t(v;22) (six and three patients, respectively) only and in 40 patients ACA could be observed in BC (40/49 (82%)). Out of these 40 patients with ACA, 90% showed an unbalanced karyotype whereas only 10% of patients had a balanced karyotype. No male patient in BC showed the loss of the Y chromosome pointing to a minor effect of this numerical alteration on disease progression. Conclusion We conclude that patients with CML and unbalanced karyotype at diagnosis are under higher risk to develop CML BC compared to patients with balanced karyotypes or compared to patients without ACA. In BC, 90% of CML patients showed unbalanced karyotypes (only 68% of CML patients at diagnosis have unbalanced karyotypes) supporting the hypothesis that the imbalance of chromosomal material is a hallmark of disease progression, representing the natural history of the disease from CP to BC and indicating therefore a strong prognostic impact. Consequently, different therapeutic options (such as intensive therapy or stem cell transplantation) should be considered for patients with unbalanced karyotypes in CP CML at diagnosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. 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. Müller:Ariad: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Saussele:Pfizer: Honoraria; BMS: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other.