ALBERT

Description: The prognostic value of MRD detection in AML has been shown for PML-RARA, AML1-ETO, and CBFB-MYH11positive AML, a group which accounts for only 20–25% of AML. The largest group of AML is represented by cases with normal karyotype or unbalanced intermediate risk group karyotypes. In this group different molecular mutations occur, which are associated with heterogeneous clinical outcome. Thus, different MRD patterns can be anticipated. To expand the spectrum of molecular targets in intermediate risk AML, we used MLL-PTD, FLT3-LM, and NPM1 which are detectable in 45–50% of all intermediate risk group pts for MRD detection. In addition, overexpression of WT1 for MRD detection was used to even include pts. without detectable mutations. In total 996 samples (spl) of 234 patients (pts) were analysed by quantitative real time PCR. For MLL-PTD (321 spl of 78 pt), and WT1 (336 spl of 66 pts) universal assays were used. For NPM1 mutation specific assays (182 spl of 54 pts (42 x type A, 2 x type B, 7 x type D, 3 x rare not yet defined types)) and for FLT3-LM (161 spl of 18 selected pts) patient specific assays were used. All assays were RNA based. Sensitivity of the assays were between 1:100.000 to 1:1.000.000 for the FLT3-LM and NPM1, depending on pts specific assay. Sensitivity was 1:10.000 to 1:100.000 for MLL-PTD due to low background levels detectable in healthy controls. The sensitivity of WT1 was relatively low with 1:100 at most, as there was no high WT1 expressor at diagnosis in this cohort. With all markers the clinical course of the disease was clearly be reflected and all 84 relapses were detectable due to recurring high expression rates. In 17 cases, were samples 2–4 months before clinical relapse were available relapses were predictable based on increasing transcript levels. Five different follow up intervals (int) were defined: up to day 21; days 22–60; days 61–120; day 121–365, later. The log change from diagnosis to defined follow up intervals was analyzed. A rapid decline of median transcript ratios in the NPM1 group (int 1: 2 log; int 2 to 4: 4 log) was observed. Relapses in the NPM1 group occurred earliest after one year. AML with FLT3-LM similarly showed good responses with 1–2 log decreases in int 1 and 2 and 3 log in int 3 and 4. Also this group revealed the first relapses in int 5. The MLL-PTD group was characterized by slow response rates with only 0.2 log reduction in int 1 and 2 and hardly 3 log in int 3. In this group many relapses occurred in int 4 and 5. These data reflect the biological differences of these molecular subgroups: NPM1 as a favourable group, FLT3-LM as a slightly unfavourable group with good response rates but high relapse rates, and MLL-PTD as an entity with bad prognosis due to poor response rates and high relapse rates. Due to low sensitivity WT1 reflected only 0.2 log in interval 1 up to 2 log in interval 3. Seven cases were analyzed in parallel for WT1 and MLL-PTD, 6 for WT1 and FLT3-LM, and one for FLT3-LM and MLL-PTD. Although the correlation of parallel assessment was high (R=0.993) the median differences of log changes of FLT3-LM and NPM1 was one log larger than for MLL-PTD and three log larger than for WT1, depending on the initial sensitivity of the assays. In conclusion: MRD detection is feasible in the karyotypically intermediate risk group. it nicely reflects biological difference in this group NPM1, MLL-PTD and FLT3-LM are better MRD-markers than WT1, which may only be investigated in cases without any other available marker.

Description: Abstract LBA-3 Introduction: IDH1 is the gene coding for the soluble isocitrate dehydrogenease 1 (NADP+), which catalyzes the oxidative decaroxylation of isocitrate to 2-oxoglutarate. The gene has been shown to be frequently mutated in high-grade gliomas at residue p.R132, which is located in the substrate binding site of IDH1. So far, several other tumors have been analyzed without detection of the respective mutation (Bleeker et al., Human Mutation 2009). However, recently a next generation sequencing project found IDH1 mutated in 8.5% of AML with normal karyotype (Mardis et al., NEJM, 2009). Aim: To further evaluate the importance of IDH1R132 (IDH1mut) in AML we have analyzed a cohort of 999 comprehensively characterized AML cases. Methods: The respective base exchange was analysed by a LightCycler based melting curve assay with subsequent sequencing of the positive samples. Results: The cohort was comprised of 536 male and 463 female patients (median age: 65.9 years; range: 17.1- 93.3 years). 833 had de novo AML (83.4%), 122 AML following MDS (s-AML,12.1%) and 44 AML after previous treatment of different malignancies (t-AML, 4.4%). Karyotype was available in all cases: 681 had a normal karyotype (NK) AML, and 319 had chromosomal aberrations (t(15;17): n=29; inv(16): n=12, t(8;21): n=23, t(11q23): n=10, t(6;9): n=4, inv(3): n=3; -7: n=27, +8: n=29, +13: n=11, -Y: n=4; complex aberrant: n=60, others: n=106). Overall, in 93 pts IDH1 p.R132 mutations were detected (9.3%). Five different amino acid exchanges were observed: R132C (n=49), R132L (n=22), R132 H and G (n=7, each), and R132S (n=5). With respect to history of the patient IDH1mut were found in 80/833 of de novo AML (9.6%), 11/122 (9.0%) of s-AML, and 2/44 (4.5%) of t-AML, respectively (n.s.). More females (57/463, 12.3%) than males (36/536; 6.7%) had IDH1mut (p=0.003). Age was slightly higher in the mutated cases (63.9 vs. 61.9 years, n.s.). No differences were found for WBC count. IDH1mut were distributed differently between karyotypes: in NK 69/681 (10.1%) and in aberrant karyotypes 24/318 (7.5%). However, IDH1 was never mutated in inv(16), t(8;21), t(6;9), t(11q23), inv(3), or in complex aberrant karyotypes (n=112). In 2 of 27 cases (7.4%) with t(15;17) an IDH1 mutation was detected. Thus, the IDH1 mututations clustered in the intermediate risk karyotype group in comparison to the good or poor risk groups (91/771; 11.8% vs 2/134 (1.5%), p

Description: Dose density during early induction has been demonstrated to be one of the prime determinants for treatment efficacy in acute myeloid leukemia (AML). The German AML Cooperative Group has therefore piloted a dose-dense induction regimen sequential high-dose AraC and mitoxantrone followed by pegfilgrastim (S-HAM) in which 2 induction cycles are applied over 11 to 12 days instead of 25 to 29 days as used in conventional double induction, thereby increasing dose density 2-fold. Of 172 de novo AML patients (excluding acute promyelocytic leukemia), 61% reached a complete remission, 22% a complete remission with incomplete peripheral recovery, 7% had persistent leukemia, 10% died (early death) resulting in an overall response rate of 83%. Kaplan-Meier estimated survival at 2 years was 61% for the whole group (patients with unfavorable karyotypes, 38%; patients with favorable karyotypes, 69%; patients with intermediate karyotypes, 75%) after S-HAM treatment. Importantly, the compression of the 2 induction cycles into the first 11 to 12 days of treatment was beneficial for normal hematopoiesis as demonstrated by a significantly shortened duration of critical neutropenia of 31 days compared with 46 days after conventionally timed double induction. (European Leukemia Trial Registry LN_AMLINT_2004_230.)

Description: Abstract 417 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Here, we analyzed 81 CMML cases (45 CMML-1, 36 CMML-2). In chromosome banding analysis 59/76 (77.6%) patients showed a normal karyotype (data not availabel in 5 cases). Recurrent chromosome aberrations were trisomy 8 (n=6; 7.9%), monosomy 7 (n=3; 3.9%), and loss of the Y-chromosome (n=5; 6.6%). Fluorescence in situ hybridization (FISH) detected the deletion of one allele of the TET2 gene in 4/71 cases (5.6%). Thus, the majority of cases can not be genetically characterized by these techniques. Therefore, we applied next-generation sequencing (NGS) technology to investigate 7 candidate genes, represented by 43 PCR-products, at known mutational hotspot regions, i.e. CBL (exons 8 and 9), JAK2 (exons 12 and 14), MPL (exon 10), NRAS (exons 2 and 3), and KRAS (exons 2 and 3). In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2. NGS was performed using 454 FLX amplicon chemistry (Roche Diagnostics Corporation, Branford, CT). The median number of base pairs sequenced per patient was 9.24 Mb. For each target gene a median of 911 reads was generated (coverage range: 736-fold to 1606-fold). This approach allowed a high-sensitive detection of molecular mutations, e.g. detecting the JAK2 V617F mutation down to 1.16% of reads. In total, 146 variances were detected by this comprehensive molecular mutation screening (GS Amplicon Variant Analyzer software version 2.0.01). In 80.4% of variances consistent results were obtained after confirming NGS mutations with melting curve analysis and conventional sequencing. In the remaining discrepant variances (19.6%) NGS deep-sequencing outperformed conventional methods due to the higher sensitivity of the platform. After excluding 19 polymorphisms or silent mutations 127 distinct mutations in 61/81 patients (75.3%) were detected: CBL: n=21 point mutations and one deletion (18 bp) found in 20 cases (24%); JAK2: n=8 mutations (V617F) found in 8 cases (9.8%); MPL: no mutations found; NRAS: n=23 mutations found in 18 cases (22.2%); KRAS: n=12 mutations found in 10 cases (12.3%); RUNX1: n=6 point mutations and one deletion (14 bp) found in 7 cases (8.6%); and TET2: n=49 point mutations and 6 deletions (2-19 bp; 5/6 out-of-frame) found in 41 cases (50.6%). Furthermore, in 21 TET2-mutated cases 11 mutations previously described in the literature were detectable, whereas 28 cases carried novel mutations (n=28). In the cohort of TET2-mutated cases 17/41 (41.3%) patients harbored TET2 abnormalities as sole aberration. Interestingly, CBL mutations were found to be significantly associated with TET2 mutations (Fisher's exact test, p=0.008). In 17 of 20 (85.0%) CBL-mutated cases TET2 abnormalities were concomitantly observed. In contrast, no significant associations were found between any of the point mutations or deletions and the karyotype. There were also no associations observed between molecular aberrations and the diagnostic categories CMML-1 and CMML-2. With respect to clinical data a trend for better outcome was seen for patients that carried either or both TET2 and CBL mutations (median OS 130.4 vs. 17.3 months, alive at 2 yrs: 72.0% vs. 43.9%; p=0.13). In conclusion, 75.3% of CMMLs harbored at least one molecular aberration. In median 2 mutations per case were observed. Compared to limited data from the literature we detected not only a higher frequency of CBL mutations, but also add data on novel TET2 mutations. In particular, comprehensive NGS screening here for the first time has demonstrated its strength to further genetically characterize and delineate prognostic groups within this type of hematological malignancy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Description: Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal–related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Description: Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgVH status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.

Description: Abstract 3270 Poster Board III-1 In chronic myeloid leukemia (CML), the progress from chronic phase (CP) to accelerated (AP)/blast phase (BP) is frequently accompanied by cytogenetic evolution within the Philadelphia (Ph+) positive clone. It was shown that additional chromosomal alterations follow non-random patterns with frequent occurrence of “major route” (e.g. +Ph, i(17q), +8) and “minor route” changes (e.g. -Y, -7 or +21). Corresponding to the pre-imatinib era, Ph+ clonal cytogenetic evolution was still identified to worsen prognosis in patients receiving imatinib. However, whether the introduction of TKIs changed the pattern of additional chromosome aberrations, was not studied so far. We here compared two subgroups: 1.) 245 CML patients treated with tyrosine kinase inhibitors (TKIs) with clonal evolution in the Ph+ clone investigated in the Munich Leukemia Laboratory (MLL) between 2005–2009. 2.) 500 CML cases published in the Mitelman Database (http://cgap.nci.nih.gov/Chromosomes/Mitelman) before the year 2000 (i.e. before the introduction of imatinib into the treatment of CML). The 245 patients from our cohort were selected for this study based on the occurrence of Ph+ clonal evolution at diagnosis or during the course of CML. Patients received imatinib or 2nd generation TKIs. First, analysis was performed for the Mitelman cohort and for the MLL cohort. Then, analysis was separated for those patients from the MLL cohort who showed Ph+ clonal evolution already at diagnosis of CML before start of TKIs (n=91), and for those patients who developed Ph+ cytogenetic alterations during TKIs (n=154). The patterns of chromosomal gains and losses were analyzed with the support of the CyDAS cytogenetic data analysis system (http://www.cydas.org/OnlineAnalysis/). All 4 cohorts showed comparable patterns of chromosomal gains/losses in addition to the Philadelphia translocation: Most frequent were +8, +Ph chromosome, +19, and i(17)(q10), and -Y. Less frequent were -7 and +21. Therefore, no difference between the patterns of abnormalities dating from the pre-imatinib era (data from the Mitelman database) or after the introduction of TKI (MLL cohort) was obvious. Also, there was no difference in cytogenetic patterns between patients who showed Ph+ clonal evolution at diagnosis of CML already and those who acquired them during TKI treatment. In conclusion, the patterns of cytogenetic alterations in the Ph+ clones in CML are similar as investigated in the pre-TKI and the TKI eras and therefore are independent, i.e. of the time point of analysis at diagnosis or during follow-up and also treatment of CML. Disclosures: Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Description: Abstract 3482 Poster Board III-419 About 25% of patients with severe hemophilia A develop neutralizing antibodies to factor VIII (FVIII), termed inhibitors, within a median of eleven exposure days to factor VIII containing therapeutic replacement products. Effective monitoring of inhibitor development in young hemophilic children is hampered by their frequently difficult venous access and their limitations on blood sample size, often making it a challenge to obtain samples suitable to carry out standard PTT based Bethesda assays. We evaluated the performance of the Factor VIII Antibody Screen (GTI Diagnostics, Waukesha, WI), an ELISA-based assay to detect FVIII antibodies in a cohort of hemophilia A patients as they were first exposed to FVIII replacement therapy for treatment of hemorrhages. FVIII antibodies were detected in the assay by incubation of duplicate patient samples and controls in microtiter wells coated with recombinant FVIII. After washing, bound FVIII antibody was detected with alkaline phosphatase conjugated goat anti-human IgG, and a colorimetric endpoint (optical density [OD] read at 405or 410 nm by spectrophotometry) determined in an ELISA plate-reader after incubation with p-nitrophenyl phosphate. Samples were considered positive if the average of the sample ODs was higher than the positive controls or negative if the average of the sample ODs was lower than the negative controls. Thirty consecutively identified patients with severe hemophilia A, who were enrolled in a longitudinal inhibitor study, and had samples of serum or plasma banked from the time of their first exposures to FVIII-containing therapeutic products were included. Patients were followed a median of 15.5 years (range 2 – 23 years). Nineteen (63%) of the patients never developed clinical or laboratory evidence of inhibitor development during follow-up. Eleven of the thirty (37%) developed an inhibitor during follow-up; one of these occurred in a 5 year-old after more than 650 exposure days to factor VIII concentrate. There were no differences in the time-to-first-exposure or pattern of hemorrhages in the two groups with the exception that all post-circumcision hemorrhages (N=6) occurred in the non-inhibitor group. In the non-inhibitor group, banked samples were selected corresponding to 0, 5, 10 and 〉50 factor VIII exposure-days; none of these samples had a positive result in the FVIII antibody screen ELISA. In the 11 inhibitor patients, banked samples were selected that corresponded with the earliest available sample, a sample obtained prior to the first positive Bethesda assay, the first Bethesda positive sample, a sample obtained at the initiation of immune tolerance induction (ITI), the peak Bethesda titer sample, the first negative Bethesda titer sample during ITI, and the most recent sample. All eleven of those who developed an inhibitor underwent successful immune-tolerance therapy with high dose (100 units/kg/day) factor VIII infusions. All Bethesda positive samples were positive by the FVIII antibody screen ELISA with one exception, a sample from one of the inhibitor patients just prior to development of a recurrent inhibitor. There were 5 Bethesda assay negative/FVIII antibody screen ELISA positive samples in the inhibitor patients; each of these samples had been obtained during ITI at 24-48 hours post factor VIII concentrate infusions, and were concordant with lower than expected factor VIII recoveries. We conclude that the ELISA-based FVIII antibody screen is sensitive and specific for the detection of factor VIII antibodies in patients with hemophilia A who develop inhibitors. Because it can be carried out with small serum as well as plasma samples, it provides a convenient method to obtain results in small patients with poor venous access, although quantification of the antibody titer in positive samples would require additional sample to carry out a PTT-based Bethesda assay. Unlike the Bethesda assay, this ELISA-based assay was able to detect antibodies in transfused patients undergoing ITI without the need for a prolonged washout period. Prospective studies to determine the utility and cost-effectiveness of this method are warranted. Disclosures: Gill: GTI Diagnositcs: Consultancy. Stapleton:GTI Diagnostics: Employment. Kern:GTI Diagnostics: Employment.

Description: Abstract 273 AML with inv(3)(q21q26) or t(3;3)(q21;q26) have been defined as a distinct entity in the WHO classification in the category of “acute myeloid leukemia with recurrent genetic abnormalities”. Whereas cases with t(8;21)(q22;q22), inv(16)(p13q22/t(16;16)(p13;q22) or t(15;17)(q22;q12) are considered as acute leukemias regardless of the percentage of blasts in the bone marrow, it is not clear whether cases with inv(3)(q21q26)/t(3;3)(q21;q26) should be categorized as such if blast cell count is

Description: The TP53 gene is the most frequently mutated gene in human tumors identified so far. In a prior study we demonstrated that 78% of AML with complex aberrant karyotype show a mutation of the TP53 gene. The aim of this study was to determine the frequency of TP53 mutations in an unselected cohort of AML and to analyze the relation to cytogenetic and molecular genetic aberrations. In total 149 AML cases were examined by chromosome banding analysis and screened for FLT3-length mutations (FLT3-LM), MLL partial tandem duplication (MLL-PTD), NPM1 mutations, and TP53 mutations. The cohort included cases with t(8;21) (n=10), t(15;17) (n=6), inv(16) (n=4), 11q23/MLL-rearrangement (n=6), trisomy 8 sole (n=13), AML with normal karyotype (n=46), AML with complex aberrant karyotype defined as showing 3 and more clonal abnormalities but no balanced rearrangement leading to a leukemia specific fusion gene (n=26), and AML with other abnormalities (n=38). FLT3-LM were observed in 21, MLL-PTD in 4, and NPM1-Mutations in 26 cases. TP53 mutation screening of exons 3–9 was performed by denaturing high performance liquid chromatography (DHPLC). All mutations detected were verified by direct sequencing. Overall, TP53 mutations were detected in 20 of the 149 cases (13.4%). Within this cohort of TP53 mutated cases, coincidences of FLT3-LM and MLL-PTD, respectively, with TP53 mutation were detected in one case each. A complex aberrant karyotype was present in 17 of 20 cases (85%) with TP53 mutation. The remaining 3 cases with TP53 mutation showed a normal karyotype, a trisomy 8, and t(8;21) as the sole abnormality, respectively. Therefore, we confirmed a high incidence of TP53 mutations in AML with complex aberrant karyotype (17/26, 65.4%). On the other hand TP53 mutations are very rare in AML without a complex aberrant karyotype (3/123, 2.4%). Furthermore, we divided AML with complex aberrant karyotype into two subgroups:AML with “typical” complex aberrant karyotype showing a deletion of at least one of the following regions: 5q31, 7q31, 17p13 (definition according to Schoch et al. GCC, 2005) andAML with “untypical” complex aberrant karyotype comprizing all others. Interestingly, the frequency of TP53 mutations within the “typical” complex aberrant karyotype group was 75% (15/20) while in the “untypical” group it was 33% (2/6) (p=0.138). In conclusion, the overall incidence of TP53 mutations is low in AML. TP53 mutations are highly associated with AML and complex aberrant karyotype and occur very infrequently in all other cytogenetic subgroups (p