We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

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The recently evolved field of synthetic biology has revolutionized the way we think of biology as an “engineerable” discipline. The newly sprouted branch is constantly in need of simple, cost-effective and automatable DNA-assembly methods. We have developed a reliable DNA-assembly system, ZeBRα (Zero-Background Redα), for cloning multiple DNA-fragments seamlessly with very high efficiency. The hallmarks of ZeBRα are the greatly reduced hands-on time and costs and yet excellent efficiency and flexibility. ZeBRα combines a “zero-background vector” with a highly efficient in vitro recombination method. The suicide-gene in the vector acts as placeholder, and is replaced by the fragments-of-interest, ensuring the exclusive survival of the successful recombinants. Thereby the background from uncut or re-ligated vector is absent and screening for recombinant colonies is unnecessary. Multiple fragments-of-interest can be assembled into the empty vector by a recombinogenic E. coli -lysate (SLiCE) with a total time requirement of less than 48 h. We have significantly simplified the preparation of the high recombination-competent E. coli -lysate compared to the original protocol. ZeBRα is the least labor intensive among comparable state-of-the-art assembly/cloning methods without a trade-off in efficiency.

DNA databases are the most efficient tools in criminal investigations with unknown perpetrators. Due to a significant number of random matches in cross-border DNA profile exchanges, the European Network of Forensic Science Institutes (ENFSI) proposed the addition of further short tandem repeats (STRs) to European DNA databases. Therefore, the new Powerplex® ESX17 and Powerplex® ESI17 kits from Promega comprised the 11 established DNA database STRs and additionally the well-known loci D1S1656 and D12S391, as well as D2S441, D10S1248, and D22S1045. The latter three STRs are thereby established as so-called mini-STRs to fulfill the increasing requirements regarding sensitivity and reproducibility for analysis of minute amounts of DNA. Here, we provide allele frequencies for the five additional STRs from two populations from Germany. A test regarding suitability and robustness of the new kits for routine trace analysis showed that it is more likely to obtain a meaningful profile using Powerplex® ESX17 and Powerplex® ESI17 kits compared to the Powerplex® ES kit. However, for both new kits the range of template DNA amount is rather small, e.g., slightly more DNA than recommended resulted in DNA profiles which could not be reliably evaluated due to allelic drop-in or imbalances and overshoots. In our opinion, the new kits are very promising new tools in forensic trace analysis even though handling and evaluation should yet be carried out with great caution.

406 Introduction: Mutations in the spliceosome gene SF3B1 (splicing factor 3b, subunit 1; SF3B1 mut) are frequent in patients with myelodysplastic syndromes (MDS) and ring sideroblasts (RS). In contrast, in AML occurrence of SF3B1mut has been published to be comparatively low (2–5%). However, analysis of SF3B1 mut in AML with RS is lacking. We aimed to determine the frequency of SF3B1 mut in AML patients according to the percentage of RS and the association of SF3B1 mut with other genetic markers. Patients and Methods: 275 AML patients (115 f/160 m; median age: 71.2 years, range: 20.8 – 89.9 years) were analyzed for SF3B1 mut by Sanger sequencing of the coding region (exon 11 to 16). RS were detectable in 176/275 cases (RS in ≥15% of erythroid precursors: n=106; RS in 1–14%: n=70), 99 cases showed no RS. The cohort comprised 202 de novo AML (FAB: M0 n=27, M1 n=36, M2 n=55, M4 n=32, M5 n=1, M6 n=51), 65 s-AML and 8 t-AML patients. Data on other mutations were available as follows: FLT3-ITD n=242, FLT3-TKD n=173, MLL-PTD n=235, NPM1 n=230, RUNX1 n=199, CEBPA n=152, NRAS n=141, KRAS n=56, ASXL1 n=90, IDH1 n=78, IDH2 n=58, TP53 n=74, and DNMT3A n=52. Chromosome banding analysis (combined with FISH if needed) was performed in 262 cases. According to MRC criteria, favorable karyotypes were found in 6, intermediate in 169, and adverse in 87 cases. 120/262 (45.8%) cases had a normal karyotype (NK-AML). Results: Overall, in 44/275 (16.0%) patients SF3B1 mut were detected with a median mutation/wildtype ratio of 45% (range: 10 – 50%). The most frequent mutation was Lys700Glu (19/44, 43.2%) followed by Lys666Asn/Arg/Thr (15/44, 34.1%) and Arg625Cys/Leu (3/44, 6.8%) and other mutations found in single cases only. Patients without detectable RS had almost no SF3B1 mut in contrast to cases with RS (3/99, 3.0% vs 41/176, 23.3%, p<0.001). Of note, all three patients with SF3B1 mut without RS were s-AML. SF3B1 mut were significantly more frequent in AML with RS ≥15% as compared to RS 1–14% (33/106, 31.1% vs 8/70, 11.4% p=0.003). The frequency of SF3B1 mut was significantly increasing by higher RS categories: group 1, RS 0–14%: SF3B1 mut in 6.5%; group 2, RS 15–34%: 17.9%; group 3, RS 35–54%: 45.4%, group 4, RS 55–74%: 41.2%, group 5, RS 75–100%: 54.5% (group 1 vs 2: p=0.017; 2 vs 3: p=0.020; comparison of the frequency of SF3B1 mut for all groups: p<0.001). In line, SF3B1 mut had higher percentages of RS vs SF3B1 wt (mean: 37% vs 13%, p<0.001), higher age (mean: 72 vs 69 years, p=0.044) and higher platelet counts (mean: 108 vs 71 x109/L, p=0.014). SF3B1 mut was not detected in any of the analyzed t-AMLs. Within cases with RS ≥15% the frequency of SF3B1 mut was only slightly higher in s-AML vs de novo AML (12/34, 35.3% vs 21/69, 30.4%, p=0.657), and likewise was the percentage of RS (mean: 45% vs 37%, p=0.137). In de novo AML, SF3B1 mut occurred more often in FAB M2 and M4 (M2: 13/55, 23.6%; M4 10/32, 31.3%; M2 and M4 combined vs others 23/87, 26.4% vs 4/115, 3.5%, p<0.001). Frequency was lower in FAB M6 vs others (3/51, 5.9% vs 24/151, 15.9%, p=0.094) and M0 (1/27, 3.7% vs 26/175, 14.9%, p=0.138), whereas SF3B1 mut were mutually exclusive of FAB M1 (0/36, 0% vs 27/166, 16.3%, p=0.005). In intermediate MRC karyotypes frequency of SF3B1 mut was much higher vs all others (38/169, 22.5% vs 4/93, 4.3%, p<0.001). In detail, SF3B1 mut showed high occurrence in NK patients (NK vs aberrant: 28/120, 23.3% vs 14/142, 9.9%, p=0.004) and within this subgroup a very high frequency of 48.9% (23/47) in cases with RS ≥15%. In contrast, SF3B1 mut were nearly mutually exclusive of complex karyotype (complex vs all others: 1/57, 1.8% vs 41/205, 20.0%, p<0.001). Furthermore, SF3B1 mut were associated with FLT3-ITD (7/21, 33.3% vs 30/221, 13.6%, p=0.025) and RUNX1 mutations (19/65, 29.2% vs 12/134, 9.0%, p=0.001). Conclusions: So far, SF3B1 mut were considered to be mainly relevant for MDS. In this study, SF3B1 mut were found in 31.1% of AML with RS ≥15% and even more striking in 48.9% of AML-NK with RS ≥15%. SF3B1 mut were associated with higher age, AML FAB M2 and M4 subtypes, normal karyotype, FLT3-ITD and RUNX1 mutations. Our study adds both de novo and s-AML with RS ≥15% to the myeloid entities with frequent occurrence of SF3B1 mut and suggests analysis of a possible prognostic impact of SF3B1 mut in AML with increased RS. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Bayer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.