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Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor-or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions. © 2012 Macmillan Publishers Limited.

Telomerase reverse transcriptase gene (TERT) promoter mutations are identified in many malignancies but not in hematological malignancies. Here we analyzed TERT and protection of telomeres 1 gene (POT1) mutations, and four different TERT SNVs in 226 acute myeloid leukemia (AML) patients and 806 healthy individuals in a case referent design, where also overall survival was assessed. A significant association for increased risk of AML was found for TERT SNVs, rs2853669 (OR = 2.45, p = 0.00015) and rs2736100 (OR = 1.5, p = 0.03). The overall survival for patients with CC genotype of rs2853669 was significantly shorter compared to those with TT or TC genotypes (p = 0.036 and 0.029 respectively). The influence of TERT rs2853669 CC on survival was confirmed in multivariable Cox regression analysis as an independent risk biomarker in addition to high risk group, higher age and treatment. No hot spot TERT promoter mutations at -228Cgreater thanT or -250Cgreater thanT or POT1 mutations could be identified in this AML cohort. We show that rs2853669 CC may be a risk factor for the development of AML that may also be used as a prognostic marker to identify high risk normal karyotype -AML (NK-AML) patients, for treatment guidance. Funding Agencies|Swedish Research Council; Swedish Cancer Society; County Council of Ostergotland; AFA Insurance; FORSS

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi-drug transporter P-glycoprotein, and overall survival (OS) in normal karyotype (NK)-AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK-AML patients (n = 201) were analysed for 1199Ggreater thanA, 1236Cgreater thanT, 2677Ggreater thanT/A and 3435Cgreater thanT, and correlations to outcome were investigated. FLT3 wild-type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0.017. There was also an inferior outcome in FLT3 wild-type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0.039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236Cgreater thanT and 2677Ggreater thanT may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild-type group, which may contribute to future individualizing of treatment strategies. Funding Agencies|Swedish Cancer Society; County Council of Ostergotland; AFA Insurance; Stockholm Cancer Society; Karolinska Institutet; Swedish Research Council

De novo acute myeloid leukemia with normal karyotype (NK-AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single-nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and −451C>T rs532545), 5′-nucleotidase (cN-II 7A>G rs10883841), and deoxycytidine kinase (DCK 3′UTR 948T>C rs4643786) in 205 de novo NK-AML patients. In FLT3-internal tandem duplication (ITD)-positive patients, the CDA 79C/C and −451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, P

Telomerase reverse transcriptase gene (TERT) promoter mutations are identified in many malignancies but not in hematological malignancies. Here we analyzed TERT and protection of telomeres 1 gene (POT1) mutations, and four different TERT SNPs in 256 acute myeloid leukemia (AML) patients. A significant association for increased risk of AML was found for TERT SNPs, rs2853669 (OR = 2.45, p = 0.00006) and rs2736100 (OR = 1.52, p = 0.02). The overall survival for patients with CC genotype of rs2853669 was significantly shorter compared to those with TT or TC genotypes (p = 0.036 and 0.029 (respectively). The influence of TERT rs2853669 CC on survival was confirmed in multivariable Cox regression analysis as an independent risk biomarker in addition to high risk group, age and treatment. No hot spot TERT promoter mutations at -228C>T or -250C>T or POT1 mutations could be identified in this AML cohort. We show for the first time that rs2853669 CC may be a risk factor for the development of AML that may also be used as a prognostic marker to identify relative high risk NK-AML patients, for treatment guidance. Note: This abstract was not presented at the meeting. Citation Format: Kourosh Lotfi, Mohamed Ali Mosrati, Kerstin Willander, Ingrid Jakobsen Falk, Monica Hermanson, Martin Höglund, Dick Stockelberg, Yuan Wei, Peter Söderkvist. Association of TERT polymorphism with acute myeloid leukemia risk and prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2015-5275

Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor–recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor–recipient pairs. Samples from nine of the donor–recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

The tumor-suppressor gene TP53, encoding the p53 protein, is vital for normal cellular processes including cell cycle regulation, apoptosis and senescence. Mutations in TP53 are frequent in many cancers, and have been associated with treatment resistance and inferior prognosis in acute myeloid leukemia (AML). Altered expression of MDM2 (mouse double minute 2), a negative regulator of p53, may also attenuate normal p53 signaling, thereby enhancing tumor transformation and resistance to apoptosis. The single nucleotide polymorphism (SNP) 309 (T>G, rs2279744) has been reported to increase MDM2 expression and impair normal p53 response. We investigated the frequency and impact of TP53 mutations (TP53mut) and MDM2SNP309 on treatment response and overall survival (OS) in 207 Swedish AML patients. To detect the TP53mut in exons 5-8, the single strand conformation analysis (SSCA) was performed. Samples with mobility shift were sequenced to determine the exact nucleotide sequence and compared to the corresponding TP53 reference sequence (NM_000546.4). MDM2SNP309 was analyzed using Pyrosequencing. We found a high frequency (22%) of TP53mut in patients with cytogenetic aberrations, with strong association to high risk cytogenetics (p Citation Format: Ingrid Jakobsen Falk, Kerstin Willander, Roza Chaireti, Johan Lund, Monica Hermanson, Henrik Greén, Peter Söderkvist, Kourosh Lotfi. TP53 mutations and MDM2 single nucleotide polymorphism 309T-G predicts outcome and treatment resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2014-3518

Background: De novo AML with normal karyotype (NK-AML) comprise a large group of patients without common cytogenetic alterations but with a large variation in treatment response. New tools such as the analysis of nucleophosmine 1 (NPM1), FMS-like tyrosine kinase 3 (FLT3) and CCAAT/enhancer binding protein (CEBPA) mutations has emerged as clinical markers of prognosis, but there is still a need for additional tools to explain the variation in outcome. Cytarabine (Ara-C), a cornerstone in AML chemotherapy, is activated intracellularly by deoxycytidine kinase (dCK), and deactivated by cytidine deaminase (CDA) and poor treatment outcome or development of chemotherapy resistance can be due to altered activity of these enzymes. Single nucleotide polymorphisms (SNPs) of CDA genes have previously been reported to result in lower CDA allelic expression and to affect the outcome of treatment. In addition, pharmacological inhibition of CDA has been shown to reduce the degree of DNA methylation. DNA methylation may be an important regulatory mechanism for the expression of genes related to drug effect, and we therefore aimed to investigate the relationship between CDA polymorphisms and outcome in NK-AML, DNA methylation, and in vitro drug cytotoxicity. Method: 207 Swedish NK-AML patients were genotyped for the CDA SNPs 79A>C (rs2072671) and -451C>T (rs532545) and the results were correlated to treatment response and overall survival (OS) in the material as a whole as well as stratified based on FLT3 and NPM1 status. Cells from a subset of the patients were also investigated for genotype association with in vitro drug cytotoxicity (n=56) and global DNA methylation (n=82). Results: CDA 79C/C or -451T/T genotype was associated to a shorter OS compared to other genotypes (0.44 vs. 1.86 years, p=0.004; and 0.45 vs. 1.79 years, p=0.051) for FLT3-ITD+ patients, and this was even more pronounced in the FLT3-ITD+/NPM1+ cases. There was significantly less DNA methylation in cells heterozygous for both polymorphisms (A/C+C/T) compared to homozygous A/A+C/C cells (p=0.018). There was also a lower degree of methylation in homozygous C/C+T/T cells, but due to the small number of samples in this group significance was not reached. An altered in vitro sensitivity towards topoisomerase inhibitory drugs, but not towards nucleoside analogues was seen for both CDA SNPs with homozygous C/C or T/T cells being more sensitive. Conclusions: We found a significant correlation between CDA genotype and OS in NK-AML, the degree of DNA methylation in AML cells, and in vitro drug sensitivity. The change in methylation related to CDA genotype may be associated with differences in drug activation and subsequent sensitivity, making these polymorphisms potential markers of use for future individual chemotherapy decisions such as dose adjustments. Citation Format: Ingrid Jakobsen Falk, Anna Fyrberg, Monica Hermanson, Martin Höglund, Hareth Nahi, Lars Palmqvist, Christer Paul, Esbjörn Paul, Richard Rosenquist, Dick Stockelberg, Yuan Wei, Henrik Green, Kourosh Lotfi. Correlation between cytidine deaminase single nucleotide polymorphisms and in vitro drug sensitivity, DNA methylation and outcome in normal karyotype acute myelogenous leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1170. doi:10.1158/1538-7445.AM2013-1170

We investigated the expression of platelet-derived growth factor (PDGF) and its receptors in rat facial nuclei following axotomy by in situ hybridization and immunohistochemistry. Facial nuclei were examined on days 3, 6, 12, 19 and 26 postoperatively (p.o.). Strong immunoreactivity for PDGF was found in facial neurons and surrounding astrocytes on the ipsilateral side of the brainstem already after 3 days p.o. and persisted at a high level until day 26 p.o. in rats with a facial nerve cut injury. After crushing of the facial nerve, a similar increase was seen in PDGF immunoreactivity which, however, decreased after day 19 p.o., when reinnervation had occurred. Reactive gliosis appeared on the operated side and was confirmed by an increase in intensity of GFAP staining. The kinetics of PDGF A-chain mRNA expression corresponded to the PDGF immunoreactivity, whereas the B-chain mRNA was present only in the neurons. The PDGF alpha-receptor immunoreactivity as well as the mRNA were detected in scattered glial cells. The density of the PDGF alpha-receptor mRNA expressing glial cells was higher on the injured side, but the intensity of the expression per cell did not change after axotomy. An increase in PDGF beta-receptor immunoreactivity was seen in the ipsilateral facial nuclei after 3-6 days p.o., however, the increase in the mRNA could not be detected. The staining persisted until day 26 p.o., when transected facial neurons showed heavier staining than those that had been crushed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC(50)=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC(50) 1 μM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

Abstract 2535 Background: Minimal residual disease is well established as an important prognostic parameter in a number of hematologic diseases and is used for stratification and treatment decisions in adult and childhood acute lymphoblastic leukaemia (ALL). DNA- based PCR analysis of Ig-/TCR-rearrangements have been well standardised during the last decade, resulting in robust systems for MRD analysis with an intra- and inter-assay variability of less than half a log and low false positivity. In Ph+ALL, MRD analysis relies on RNA-based RT-PCR techniques that are highly sensitive but as yet lack standardisation between laboratories. Since laboratories differ substantially in both their methodology and analysis strategy, interpretation and meaningful comparison of results between laboratories and from different studies is difficult or impossible. Moreover, and in contrast to CML, there are no generally accepted definitions of molecular response that could identify thresholds on which to base therapeutic decisions. Aims: To assess i) the variability of BCR-ABL quantification between laboratories with recognized expertise in bcr-abl analysis, ii) identify optimal laboratory methods and iii) to standardize analysis and interpretation of RT-PCR results for Ph+ALL, the EWALL and ESG-MRD-ALL consortia initiated a multinational project for quality assurance involving more than 30 laboratories from 14 countries worldwide. We here report the results of the first six m-BCR-ABL laboratory control rounds, performed between march 2008 and august 2011, that focussed on inter-intraassay variability of rt-PR techniques, comparison of housekeeping genes, plasmids, platforms and reagents. Methods: Serial dilutions of the BCR-ABL positive cell line Sup B15 with the BCR-ABL negative cell line Nalm 6 covering 5 logs were produced. In the 6 lab rounds, 1355 aliquots, each generated from with a total amount of 5 × 10E+06 cells in a final volume of 1 ml were produced, stabilized in TRIZOL or buffer RLT and frozen at −20°C until shipment or centralized isolation of RNA and reverse transcribed. Participants were asked to process the material using predefined procedures. Results: In the first QC round a major finding was the high variability in RNA-yield between laboratories despite using the same extraction method, with an up to 3 log difference in ABL copy numbers. In addition, there was rare false-positivity and false negativity (specifity 94.8%). With centrally produced cDNA the variability improved and there was a lower frequency of false-positivity reaching 100% specifity in QC 2. By using standardized assays, in 12 of 33 laboratories no conversion factor is needed, 20 of 33 laboratories are at the moment within one log difference when comparing the BCR-ABL/ABL ratios. Comparison of housekeeping genes (ABL/GUS) revealed non-linearity at high transcript levels resulting in an underestimation of intraindividual log reductions and may be relevant when assessing disease prognosis. The quantitative range was generally above 10E-04. Conclusion: Initiation of laboratory control rounds and implementation of standardized methodology improved the variability and reduced the frequency of false-positive results. Sensitivity of current techniques is still unsatisfactory for diseases with aggressive growth kinetics such as Ph*ALL. Further standardisation of technical and analytical methodology will provide the basis for defining levels of molecular remission and relapse and help to ensure comparability between laboratories and clinical trials at the European level. Disclosures: Goekbuget: Micromet: Consultancy. Ottmann:Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

Abstract 894 Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia (CML) resistant to imatinib or second generation tyrosine kinase inhibitors. This variation may at least partially explain the different frequencies of mutations that have been reported. Furthermore, the pattern of individual mutations reported seems to depend on the specific method used for mutation detection. Standardized techniques and protocols for the detection of BCR-ABL mutations will be necessary to obtain comparable mutation results within clinical studies. The first objective of this study conducted within the EUTOS (European Treatment and Outcome Study) for CML program was to record the mutation analysis techniques and protocols that are used for routine diagnostics by 28 national reference laboratories in 23 European countries, 9 of whom perform regular mutation analyses as a central laboratory for national or international clinical trials. The second objective was to evaluate the techniques by analysis of blinded samples containing various BCR-ABL kinase domain mutations. Initially a web-based survey was conducted with a total of 39 technical and PCR-specific questions. Most laboratories (n=17; 61%) perform 1–10 mutation tests per month (range 100) and use published protocols for nested RT-PCR from peripheral blood or bone marrow leukocytes. Failure to amplify BCR-ABL mRNA at low BCR-ABL transcript levels was reported to be the most common PCR related difficulty in 16 (57%) laboratories. Sanger sequencing is applied for routine BCR-ABL mutation analysis in 27 (96%) laboratories. Additional screening techniques (e.g. denaturing high-performance liquid chromatography, high-resolution melting) or more sensitive detection methods (e.g. allele-specific oligonucleotide PCR, pyrosequencing) are used routinely by eight laboratories each, respectively. The application of positive or negative mutation control samples was reported by 13 (46%) or 17 (61%) laboratories, respectively. Quantitative mutation analysis is performed routinely in nine (32%) laboratories. To evaluate laboratory performance we prepared a series of control samples that were distributed in a blinded fashion to testing laboratories. Seventeen Ba/F3BCR-ABL cell lines harboring various BCR-ABL kinase domain mutations were mixed with non-mutated Ba/F3BCR-ABL to produce dilutions ranging from 1% to 100% of mutant alleles. Three samples were non-mutated Ba/F3BCR-ABL only and were used as negative controls. Mutated and non-mutated Ba/F3BCR-ABL cell lines were diluted into HL60 cells to simulate a BCR-ABL level of 10% on the International Scale. Twenty blinded cDNA samples were sent out on dry ice to each participating laboratory (total of 560 samples). Twenty-three labs have reported their results so far (460 samples). The three non-mutated samples were identified correctly in 68/69 (99%) tests. For the 20 mutated samples we distinguished those with high (≥20%; n=11) and low (≤10%; n=9) levels of mutation. For the high level samples 218/253 (86%) tests were reported correctly. Of the incorrect results, 22 were scored as negative and 13 were false positives (T315I, n=7; D276G, K357E, D363Y, N374I, S386S, E505K, n=1 each). For the low level samples only 15/138 (11%) tests correctly identified the mutation. Most (122/138; 88%) were reported as undetectable and a false positive result (L323H) was reported in one case. We conclude that Sanger sequencing is the most frequently applied technique for routine analysis of BCR-ABL kinase domain mutations in CML in Europe. In general it reliably identifies mutations when the proportion of mutant alleles comprise 20% or more. Nevertheless, false negative and false positive results were reported in a substantial proportion of samples with ≥20% mutation level (35/253, 14%). For mutations that are present at 10% or less mutant alleles, routine methods mainly failed to identify mutations. This study provides a basis for further comparisons and standardization efforts comparable with the introduction of the international scale for quantification of BCR-ABL transcripts. Disclosures: No relevant conflicts of interest to declare.

Previous studies have indicated that a candidate tumor suppressor gene resides telomeric of the RB1 gene at 13q14, a region that is commonly deleted in B-cell chronic lymphocytic leukemia (B-CLL). In this study, we have evaluated the frequency and minimal region of overlap for 13q deletions in malignant cells from various lymphoid neoplasms. We observed losses at 13q14 in 33/75 (44%) B-CLL cases, four of 16 (25%) non-Hodgkin's lymphoma (NHL) cases, eight of 29 (28%) patients with acute lymphocytic leukemia (ALL), and one of 15 T-cell lines. In some ALL cases, inactivation of the RB1 gene is suggested as the important event in the 13q deletions. The most commonly deleted marker in CLL and NHL was D13S319, between RBkpt and the D13S25 loci. Homozygous deletions of this marker were observed in 10 of 75 B-CLL cases, in six of which the homozygous deletions included only the D13S319 locus. Our data suggest that 13q deletions are common in lymphoid neoplasms, and that deletion of a candidate tumor suppressor gene(s) in the vicinity of the D13S319 marker is a tumorigenic event in these diseases.