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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

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

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

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.