ALBERT

Description: We characterized the mutational status of the FLT3 tyrosine kinase domain (FLT3-TLD) in 3082 patients with newly diagnosed AML. FLT3-TKD mutations were detected in 147 of 3082 (4.8%) patients. Similar to the FLT3 juxtamembrane domain mutations (FLT3-LM), there was a high correlation of FLT3-TKD mutations with normal karyotype (88 of 1472; 6.0%). FLT3-TKD mutations were most frequent in the AML FAB subtypes M5b (15 of 114; 13.2%), M3v (6 of 51; 11.8%), and M4 (39 of 484; 8.1%). Similar to FLT3-LM, the FLT3-TKD mutations show elevated peripheral leukocytes compared with FLT3wt AML. FLT3-TKD had a high incidence in cases with NPM1 mutations (23 of 262; 8.8%), CEBPA mutations (6 of 76; 7.9%), and NRAS mutations (6 of 78; 7.7%). FLT3-TKD in combination with FLT3-LM (17 of 594 patients; 2.9%) and KITD816 (1 of 44; 2.3%) was rare. Unlike the FLT3-LM, which are associated with inferior survival, prognosis was not influenced by FLT3-TKD in the total cohort of 1720 cases, where follow-up data were available (97 FLT3-TKD; 1623 FLT3-WT). In t(15;17)/PML-RARA with FLT3-TKD mutations, in FLT3-LM/TKD double-mutated, and in MLL-PTD/TKD double-mutated cases prognosis was unfavorably influenced by FLT3-TKD mutations. In contrast, we found an additional favorable impact of FLT3-TKD on EFS in prognostically favorable AML with NPM1- or CEBPA mutations.

Description: The mechanism that may cause progression of myelodysplastic syndrom (MDS) to acute myeloid leukemia (AML) is genetically poorly defined and several different scenarios might account for this phenomenon. Previously, RUNX1 mutations (RUNX1+) but also cytogenetic aberrations and mutations of the RAS signal transduction pathway have been implicated in this process. Therefore, we analyzed a cohort of MDS (n = 179) and s-AML following MDS (n = 93). The MDS cohort consisted of RARS (n=2), RCMD (n=15), 5q- syndrome (n=2), CMML-1/-2 (n=13), RAEB-1 (n=48), RAEB-2 (n=58) and MDS not further classified (n=41). The entire coding region was screened for RUNX1+ by denaturing high performance liquid chromatography (DHPLC) and mutations were called by direct DNA sequencing. Furthermore, FLT3 was screened for length mutations (FLT3- LM, FLT3-ITD), NRAS for mutations in codons 12/13 and 61, MLL for partial tandem duplications (MLL-PTD) and NPM1 for exon-12 gene mutations. Cytogenetic analysis was done by chromosome banding and if needed for further clarification with FISH analysis. RUNX1+ were detected in 26 (14.5%) of 179 MDS cases and in 32 (34.4%) of 93 sAML. Thus the incidence of RUNX1+ in the MDS cohort was significantly lower compared to the incidence of RUNX1+ in s-AML following MDS (p

Description: The genetic and cytogenetic events underlying the development of myelodysplastic syndrome (MDS) as well as the mechanisms leading to progression of MDS into acute myeloid leukemia (AML) are largely unknown. Activating mutations in receptor tyrosine kinases like FLT3 or members of the RAS family have been implicated in this process. Recently, mutations of RUNX1 (AML1) have been detected in MDS as well as in AML. As the development of AML is considered as a multistep process, in which mutations of genes with proliferative potential (class I) cooperate with gene mutations in hematopoietic transcription factors (class II), we hypothesized that similar mechanisms might be responsible for the progression of MDS into s-AML. Therefore, we investigated a cohort of 21 MDS patients (pts) (14 male, 7 female; median age at diagnosis of MDS=67.1/range 49.1–76.5), with WHO categories: MDS RARS (n=1), MDS RCMD-RS (n=1), MDS RAEB-1 (n=8), MDS RAEB-2 (n=3), MDS/AML (n=1), CMML-1 (n=1), MDS/MPS overlap (n=1), MDS not further classifiable (n=4) and one patient with reactive condition in morphology only, but del(5q) in chromosome banding analysis. In all cases specimens at the time of progression into s-AML following MDS were available and all specimens were characterized for RUNX1 mutations (entire coding region), FLT3-ITD, MLL partial tandem duplications (MLL-PTD), NPM1-exon 12 mutations and NRAS (codons12/13 and 61) mutations. Furthermore, in all cases chromosome banding analyses (CBA) was performed. At diagnosis of MDS, a normal karyotype (NK) was present in 11/21 cases, whereas cytogenetic aberrations were detected in the remaining 10 cases (del(5q) (n=2), del(20q) (n=1), +8 (n=2), complex (n=2), −Y (n=1), t(1;3)(p36;q21) (n=1) and t(1;14) (p34;q32), −7 (n=1)). The following molecular aberrations were detected at diagnosis of MDS: RUNX1 (n=5), MLL-PTD (n=1), NRAS (n=1), NPM1 (n=1), but never FLT3-ITD. Only one pt had two of these molecular aberrations in combination at the MDS stage (RUNX1/NPM1). After progression to s-AML, 3 of the 11 pts with NK at the MDS stage had developed cytogenetic aberrations (+8 (n=1), +11 (n=1), del(5q), del(7q) (n=1)). The other 10 already at the MDS stage cytogenetically aberrant cases remained unchanged. Thus at the s-AML stage there were 8 pts with NK and 13 with cytogenetic aberrations. At this stage the cohort was again characterized for molecular aberrations and following gains were detected: RUNX1 (n=3), MLL-PTD (n=2), NRAS (n=1), NPM1 (n=1) and FLT3-LM (n=2). The median time between diagnosis of MDS and s-AML was 207 days (d) (range 27–960 d). However, no significant difference could be detected for pts with RUNX1 mutations at the MDS stage (n=5, mean=210 d) vs. the remaining cohort (n=16, mean=266 d) (p=0.536, t-test) and pts with NK at the MDS stage (n=11, mean=200 d) vs. pts with cytogenetic aberrations (n=10, mean=324 d) (p=0.190). The pattern of acquisition of the different aberrations was complex and therefore the cohort was divided in three different subgroups based on the cytogenetic profile: cytogenetically stable, NK: this group included pts without molecular aberration at all stages (n=5), pts with RUNX1 mutation at the MDS stage (n=2), where one acquired secondary a MLL-PTD and the other a FLT3-ITD and one pt, who acquired a RUNX1 mutation at progression. cytogenetically stable, AK: 3 pts without molecular aberrations, 4 pts with molecular aberrations (RUNX1, RUNX1/NPM1, MLL-PTD, NRAS), where the pt with RUNX1/NPM1 acquired FLT3-ITD at progression and 3 pts, who acquired molecular aberrations at progression (RUNX1, NPM1, NRAS). cytogenetically unstable: 2 pts without aberrations at MDS, who progressed with an AK with or without RUNX1 mutation and one pt with a RUNX1 mutation, who progressed with an additional AK plus MLL-PTD. In conclusion, RUNX1 mutations, when present at the MDS stage, seem to play a role in the progression to s-AML following MDS. Cooperating molecular events for progression to s-AML are the acquisition of FLT3-ITD (n=2) and MLL-PTD (n=2). FLT3-ITD was never detected at the MDS stage in our cohort and seems to be a strong indicator for the progression to s-AML, whereas MLL-PTD can be already detected at earlier stages. Finally, the combination of RUNX1 mutation with FLT3-ITD or MLL-PTD was always associated with progression.

Description: Acquired resistance to imatinib is an important issue in CML. It was correlated to mutations in the tyrosine kinase domain (TKD) of the BCR-ABL fusion gene in 30–80% of cases. However, the mechanisms of resistance in cases without TKD mutations are still insufficiently understood. One potential further mechanism are additional chromosomal abnormalities (ACA) secondary to the Philadelphia translocation. We hypothezised that patients without TKD mutations might have a higher incidence of ACAs. Thus the aim of our analysis was to evaluate correlations between these two genetic mechanisms. In total 88 CML patients with acquired imatinib resistance were analyzed in parallel for additional chromosomal abnormalities by karyotyping and for resistance mutations in the TKD of the BCR-ABL fusion gene by sequence analysis. In 40 of the 88 pts (45.5%) at least one resistance mutation was detected. Of the 40 mutated cases 6 (15%) revealed two different resistance mutations whereas in 48 pts (54.5%) no mutation was detected with a sensitivity of 10–20% that is typical for conventional sequencing techniques. In the group not affected by TKD mutations 15 of 48 (31.3%) cases showed ACAs (−Y: n=2, t(3q26): n=4; +8: n=4; +Ph: n=5; del(17p): n=2; nonrecurrent reciprocal translocations: n=4; complex aberrant: n=1; others: n=2). 8 of these 15 cases had more than one ACA (median 3; range 2–7). In the group with resistance mutations a nearly equal number of 15/40 (37.5%) cases revealed ACAs and the spectrum of aberrations was very similar: (−Y: n=1, t(3q26): n=3; +8: n=5; +Ph: n=3; del(17p): n=2; complex aberrant: n=2; others: n=5). In this group 7 cases had more than one ACA (median:3; range: 2–3). Thus the total amount of pts with ACAs, the spectrum of ACA as well as the number of ACAs per patient is nearly equal in the mutated and the unmutated cohort. In the 40 TKD mutated cases 16 different mutations were detected (M244V: n=2 ; L248V: N=1; G250E: n=6; Y253H: n=3; E255K: n=2; D276G: n=1; E279K: n=2; L298V: n=1; L298V: n=1; F311I: n=1; F311L: n=1; T315I: n=11; F317L: n=1; F359C: n=3; F359I: n=1; M351T: n=6; H396R: n=4;). Most of the recurrent mutations are distributed equally in the groups with or without ACAs. Solely in cases with M244V, G250E or Y253H we never observed ACAs. Thus among 12 cases within the region of amino acid 244–253 there is no case with any ACA indicating that mutations within this region are strong enough to cause high resistance without further need of any ACA or alternatively are caused by different mechanisms that are not based on further genetic instability. In comparison 5 of 11 cases with T315I and 3 of 4 with H396R mutations are associated with multiple or complex chromosomal aberrations (p=0.014). Five pts were analyzed with both methods at 2–4 time points under dose escalation of imatinib. Two cases had stable G250E mutations without ACA for 2 and 10 months. One pt first revealed a T315I mutation and developed two reciprocal tranlocations t(5;10) (q31;q24) and t(6;17)(q26;q12) in addition to the T315I within 6 months. A second pt first had an E279K mutation and within one year developed an additional Y253H mutation and a +8. A third pt first had a t(3;21), after 2 months developed an additional T315I, and after further 5 months an additional F359C mutation. These few cases show that ACAs can preceed TDK mutations and vice versa. Although evolvement of resistance mainly is believed to be a process of selection some degree of genetic instability should be underlying to create some genetic diversity as a pool for any selection mechanism. Mutations within the TP53 gene have been associated with genetic instability and complex aberrant karyotypes in AML as well as in CLL. To analyze whether TP53 may also be involved in the genetic instability that leads to imatinib resistance we sequenced exons 4–9 of TP53 of 16 TKD mutated cases from this cohort (8 with complex aberrations and 8 without ACAs). No TP53 mutation was detected in all 16 cases indicating that TP53 is unlikely a major player in the development of resistance to imatinib. In conclusion, ACA are equally distributed between TKD mutated and unmutated cases. amino acid exchanges in the region 244–253 are not observed together with ACA whereas T315I and F359C are associated with multiple aberrations. TP53 mutations probably do not play a role in the development of genetic changes that are associated with imatinib resistance.

Description: The aim of this study was to further evaluate the impact of minimal residual disease (MRD) in NPM1 mutated AML in comparison to other factors like FAB, cytogenetics, FLT3 mutations, NPM1 mutation type and age. In total 1002 samples of 219 NPM1 mutated (NPM1mut) patients (pts) were analysed at diagnosis, during, and after therapy. Pts were treated within different AML trials, and follow-up samples were referred to perform an NPM1 specific RQ-PCR for MRD. The cohort was comprised of 112 females and 107 males, median age was 58.8 years (range: 20–79 years). 207 had de novo AML (M0: n=5; M1: n=49; M2: n=55; M4=57; M5: n=28, M5: n=6; M7: n=1, nd: n=6), 4 s-AML and 5 t-AML. Cytogenetic data was available in 215 pts: 178 with normal (NK) and 37 with aberrant karyotypes (+4: n=4; +8: n=7; +21: n=2, two or more trisomies: n=4; -Y: n=4; del(7q): n=2; del(9q): n=3; del(20q): n=2; rare translocations: n=9). At diagnosis 87/219 pts (39.7%) had FLT3-ITDs in addition to the NPM1mut. FLT3-TKD status was available in 206 cases (14 mutated (6.7%) and 192 WT). The NPM1 mutation types were A (n=174), B (n=13), D (n=14), I: (n=4), L: (n=2), R: (n=4) and 8 with individual rare types. Univariate analysis for overall survival (OS) revealed unfavourable impact for age (p=0.049), and for FLT3-ITD (p=0.002), favourable impact for FLT3-TKD (p=0.046), and no impact for FAB, chromosomal aberrations or NPM1 mutation type. For MRD assessment for all 14 different NPM1 mutation types mRNA based RQ-PCR assays were established with sensitivities of 10,000–1,000,000. For each patient 2–17 samples (spls) were analyzed (median: 4) spanning a median follow up time of 252 days (range: 18–2347 days). Paired samples of diagnosis and relapse were available in 71 pts, in 8 pts also from second relapse. At relapse all cases had high NPM1 levels comparable to those at diagnosis. The FLT3-ITD status was mutated (+/+) at both time points in 25 pts and −/− in another 25 pts. 10 pts gained FLT3-ITD at relapse and 3 lost it. For 48 paired samples cytogenetics was available for both time points. A normal karyotype (NK) at both time points was detected in 36 pts, 7 cases showed a normal or aberrant karyotype (AK) at diagnosis and and AK at relapse (two of these gained additional aberrations at relapse), 2 different AK at both time points in were detected in 3 cases and a regression from AK to NK in 2 cases. These data show that NPM1 seems to be the primary genetic aberration in these cases and detection of NPM1 is more reliable to detect relapse than cytogenetics. To analyse the impact of NPM1 mutation levels on prognosis four different follow-up intervals were defined: interval 1: days 21–60 after start of therapy; interval 2: days 61–120; interval 3: days 121–365, 4: 〉365 days. First a set of 605 samples referred for analysis during first line treatment were analysed. Using Cox regression analysis a significant impact of MRD levels (as continuous variable) on EFS was detected for interval 2 (128 spls, p=0.008), interval 3 (214 spl; p

Description: Background: Bortezomib is a reversible proteasome inhibitor displaying significant clinical activity in the treatment of multiple myeloma. Apart from direct antitumor cell activity via inhibition of the nuclear factor kappa B pathway in myeloma cells additional anti-angiogenic effects have been reported. Aims: This study aimed to analyze the cellular effects and molecular targets of bortezomib in endothelial cells. In particular, we wanted to assess the its effects on proliferating as well as quiescent endothelial cells in vitro. Anti-angiogenic activity in vivo was studied in the chicken chorioallantoic membrane (CAM) model using tumor xenografts. Methods: Cell viablility of human umbilical vein endothelial cells (HUVEC) and endothelial colony forming cells (ECFC) was determined by flow cytometry; DNA synthesis was measured by BrDU-incorporation. All experiments were performed on mitotic and growth-arrested cells, respectively. Proteins involved in cell cycle arrest and apoptosis were analysed by Western Blots and antibody microarrays. Anti-angiogenic effects in vivo were studied in the chorioallantoic membrane (CAM) assay and the B16F10 xenograft model. Secretome analysis of bortezomib-resistant cells was performed by size exclusion chromatography (SEC), 2D gel electrophoresis (2D-PAGE) and mass spectroscopy (MS, Maldi-TOF). Results: In proliferating endothelial cells, bortezomib significantly reduced cell viability and proliferation in a dose-dependent fashion with an EC50 of 5 ng/mL. This is about ten times lower than the EC50 determined for established multiple myeloma cell lines (OPM-2, LP1 and RPMI 8226). A dose-dependent increase of cell cycle arrest was accompanied by upregulation of the cycline-dependent kinase inhibitors p21CIP1, p27 KIP1 and the tumor suppressor p53. Bortezomib at higher concentrations induced apoptosis only in proliferating endothelial cells by induction of the pro-apoptotic Bok and Noxa proteins. Vessel formation in the CAM-assay was strongly inhibited by bortezomib. In contrast,, growth and vascularization of melanoma (B16F10) xenografts in the CAM assay was not inhibited by bortezomib. A protein secreted spontaneously by melanoma cells neutralized the antiangiogenic action of bortezomib. By size exclusion chromatography (SEC) and proteasome activity assays a 50–60 KDa protein could be isolated and identified to cause this activity. Besides melanoma cells a variety of other cell lines derived from soldi tumors, such as the colorectal carcinoma cells HRT18 and prostate carcinoma cells PC3 were found to release this protein constitutively at high concentrations. Mass spectrometry data on this protein will be presented at the meeting. Conclusions: Besides its direct anti-myeloma activity by suppressing the nuclear factor kappa B pathway in myeloma cells bortezomib induces apoptosis in proliferating endothelial cells via the p53 pathway and its downstream targets Noxa and Bok, and inhibits angiogenesis in vivo.. Unexpetedly, we observed many solid tumor cell lines to release a soluble protein that can effectively block the antiangiogenic activity of bortezomib in vivo..

Description: Human cytomegalovirus (CMV) reactivation and disease is still a frequent complication after allogeneic stem cell transplantation (allo SCT). It is well accepted that T-cell immunity is mandatory to control CMV infection and disease and much effort has been put into the development of cell-based monitoring assays. Nevertheless, no reliable marker for protective immunity has been established to date. Most studies use one CMV model antigen (pp65) to compare the frequencies of cytokine producers (mainly IFNg) or multimer-specific T-cells. Methods: In total, we recruited 16 patients after allo SCT, (7 high risk, 9 standard risk pts.). We used 8-colour flow cytometry to detect degranulation (mobilized CD107a/b), intracellular IFNg, TNFa, IL-2 production and CD28-expression in peptide pool stimulated pp65 and IE-1 specific CD8 T-cells. Results were compared to 7 healthy CMV exposed donors. Results: Degranulation identifies the highest percentage of CMV-specific T-cells in allo-transplanted patients (pp65: 0,94% degranulation and 0,31% IFNg; IE-1: 1,44% degranulation and 0,87% IFNg, mean frequency). These T-cells are relatively cytokine deficient compared to those in healthy donors (cytokine-production/degranulation ratio: SCT=0,42, healthy=0,72 for pp65, p=0,048; SCT=0,61, healthy= 1,00 for IE-1, p=0,133, U-test). The cytokine expression pattern differs between antigens used for stimulation, for example more IL-2-producers could be detected in the pp65 specific compartment (12,5% for pp65 and 4,5% for IE-1 of all activated CD8 T-cells, p=0,015). Conclusion: This study demonstrates that degranulation is the most prominent marker of CMV-specific T-cells (pp65 and IE-1) in allo SCT patients. Looking at IFN-g producers only may underestimate the frequencies of CMV specific T-cells in this setting. Furthermore, these subsets have a divergent functionality in transplant recipients compared to healthy individuals. Our data challenge the concept of enumerating CMV specific T-cells to estimate immunity. We rather propose measuring functional differences in the T-cell response may help to identify patients with a high risk of CMV reactivation. A careful dissection of these differences is a prerequisite for the development of monitoring tools and adoptive T-cell transfer.

Description: It has previously been shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human Organic Cation Transporter 1 (hOCT1; SLC22A1). In more recent work on clinical samples it was further shown that low hOCT1 expression of this influx transporter may be an important mechanism of imatinib resistance. To further evaluate this issue we have retrospectively quantified pretreatment hOCT1 mRNA expression in 92 CML patients (pts) that responded with major molecular remission within the first year of treatment and compared these results to 19 pts with primary resistance to imatinib. We found that all 19 resistant pts had low hOCT1 expression (median: 2.032 (expressed as %hOCT1/ABL); range 0.18–4.24). Although the median hOCT1 expression at diagnosis in the responders was higher (median 8.417) the range was very heterogeneous (0.45–188.2) with only 30% of all responders having a significantly higher expression than the resistant pts. As in vitro studies have shown that genetic variants of the SLC22A1 gene that codes for hOCT1 can have a negative effect on the transport of some substrates we hypothesized that not only certain hOTC1 expression levels but also different genetic variants within the SLC22A1 gene may be associated with different efficiencies of imatinib uptake. Using high resoluting melting and subsequent sequencing we have genotyped exons 1, 2, 5, 6, 7, 9, 10, and 11 in 109 responders as well as in 55 resistant pts, thus each 326 alleles were evaluated. We detected 12 different exonic polymorphisms. Two of these, a G38D and a Y404C were so far undescribed variants. Both nonsynonymous variants were detected in heterozygeous forms, the G38D in one responder and the Y404C variant in one resistant pt. All other variants were detected in frequencies similar to those that have already been described (R61C: 0.07, L160F: 0.76, P341L: 0.01, G401S:

Description: Leukemia specific fusion genes such as CBFB-MYH11 play a major role in the pathogenesis of distinct AML entities. However, additional genetic aberrations seem necessary for the development of full blown leukemia. This study was performed to decipher CBFB-MYH11 rearrangements and their accompanying genetic lesions at the molecular level. Therefore, Affymetrix SNP Array 6.0 analyses, featuring 〉1.8 million markers for genetic variation (〉906,600 SNPs and 〉946,000 probes for the detection of copy number variations), were performed in 35 newly diagnosed AML with inv(16) (p13q22) or t(16;16)(p13;q22) and CBFB-MYH11-rearrangement. First, as a proof of principle, additional gains and losses of chromosomal material as observed by cytogenetics were also detected by the SNP technology. This included gains of whole chromosome 8 (n=7) and 22 (n=8). In addition, a partial trisomy 13 and a partial trisomy 6 resulting from an unbalanced translocation were confirmed. In two cases a 7q deletion was observed by chromosome banding analysis. One of these was missed by SNP array as the 7q deletion occurred in a subclone only (11% of cells with 7q deletion as determined by interphase FISH). However, SNP array analyses detected loss of 7q in two additional cases which was missed by cytogenetics. Based on SNP array data the commonly deleted region was identified to range from 7q36.1 to 7q36.3 (size: 8.5 MB; physical map position 147,549,804–156,038,680). In addition to a gain of the whole chromosome 8, frequently observed as an additional aberration, in one case SNP array analyses revealed only a partial gain on 8q ranging from 8q24.13 to 8q24.3 (size: 25.3 MB; physical map position 120,986,982–146,268,936). Furthermore, a recurrent deletion (n=2) on chromosome 18 was detected by SNP array but not detected by cytogenetics. The commonly deleted region was localized in 18q23 (size: 3.1 MB; physical map position 72,481,657–75,604,994). In two cases the CBFB-MYH11 rearrangement was cryptic and could not be detected by chromosome banding analysis or FISH using two probes flanking the breakpoints within the CBFB gene, however, a CBFB-MYH11 transcript was amplified by RT-PCR. In one of these cases SNP array data revealed a small gain on 16p13 including 3′ part of the MYH11 gene (size: 71 kb; physical map position 15,654,558–15,725,636) suggesting the insertion of additional 3′ MYH11 sequences into the CBFB rearrangement leading to a CBFB-MYH11 fusion gene. Interestingly, four cases showed a deletion on 16p13 (sizes: 176 kb, 461 kb, 464 kb, 468 kb; physical map positions 15,729,932–15,906,308, 15,726,920–16,188,116, 15,725,663–16,189,984, 15,721,133–16,189,807). All included the 5′ part of the MYH11 gene, and in 3 cases, the ABCC1 gene (multidrug resistance-associated protein 1) was included in the deleted region, which could have an impact on prognosis. The patient with the smallest deletion in 16p13 also showed a deletion on 16q22 including the ′ part of CBFB (size: 35 kb, physical map position 65,672,864–65,707,954). This would be in line with findings in chronic myeloid leukemia where comparable small deletions in the breakpoint region of BCR and ABL have been described. Furthermore, large regions of copy-neutral loss of heterozygosity were observed for the whole short arm of chromosome 1 in two cases, for 17q12 to 17qter and 19q in one case each. In conclusion, a novel mechanism leading to a CBFB-MYH11 fusion gene was identified: A cytogenetically cryptic insertion of additional MYH11 sequences into the CBFB locus. A distinct pattern of additional aberrations was confirmed showing gains of whole chromosomes 8 and 22. Small copy number changes not observable in chromosome banding analysis were detected on 7q, 8q and 18q. A recurrent region of loss of heterozygosity without copy number change was found for the whole short arm of chromosome 1 suggesting that candidate genes in this region are mutated and potentially play a pathogenetic role in AML with CBFB-MYH11-rearrangement.

Description: Mutations in the DNA-binding domain, the Runt homology domain of the AML1/RUNX1 gene have been described mostly in therapy-related myelodysplastic syndrome, in therapy-related AML as well as in AML after MDS (s-AML). Recently we have shown that RUNX1 mutations also can be found in de novo AML with normal karyotype and single or simple chromosomal imbalances. To further address the importance of RUNX1 in these kind of AML we analyzed the RUNX1 mutational status in a selected cohort of 389 de novo AML with: normal karyotype (NK): n=221, +8 (n=40), +11 (n=9), +13 (n=26), +21 (n=14), rare trisomies (n=11), −7/7q- (n=22), 5q- (n=3), 9q- (n=6), 20q- (n=4), or any combinations of these (n=33). Median age was 67.5 years (range: 20.4–88.2), male:female ratio was 216:173. In this selected cohort 134/389 mutations (34.4%) were detected, showing that RUNX1 is one of the most frequently mutated genes in certain de novo AML. The mutations were not randomly distributed according to FAB subtypes: AML M0 (58.3%, n=36), M1 (32.1%, n=84), M2 (34%, n=126), M4 (20.5%, n=39), but only 10.5% in M6 (n=19) and never detected in M5 (n=12). Also within the single different cytogenetic groups the RUNX1 mutations (RUNX1mut) revealed different frequencies: NK: 28.5%; +8: 35%, +11: 44 %, +13: 96%, +21: 56%, −7/7q-: 27%, 20q-: 75% and 23.5% in the combination group. The patients (pts) were also analyzed for CEBPA, FLT3ITD, FLT3TKD, JAK2, MLLPTD, NPM1 and NRAS. 49/134 RUNX1mut cases (36.6%) revealed at least one of these additional mutations. CEBPA and JAK2 mutations were never detected in combination with RUNX1. An NPM1 mutation was observed in one RUNX1mut pts with +21. The mutation found most frequently together with RUNX1 was MLLPTD that was detected in 28/134 pts (20.9%) followed by FLT3-ITD that was detected in 24/134 cases (17.9%). The distribution of additional mutations in the different cytogenetic groups was heterogeneous. Of the 63 pts with RUNX1mut NK 20 had MLLPTD (31.7%) and 13 had FLT3ITD (20.6%). In 14 pts with +8 and RUNX1mut no MLLPTD but 3 (21.4%) FLT3ITD were detected, in addition to one case with NRAS. Two of the 4 RUNX1mut +11 pts had MLL-PTD. The 8 cases with RUNX1 mut and +21 had a very high additional mutational rate with 2 MLLPTD and 6 FLT3ITD and 1 FLT3TKD, 1 NPM1 and 1 NRAS (3 double mutated cases). In contrast only 3 of 25 (12%) RUNX1mut pts with +13 had additional mutations (2 MLLPTD and 1 FLT3ITD). Similarily the −7/7q- group with RUNX1mut (n=6) revealed no additional markers. This finding suggest that some chromosomal aberrations are biologically “ more potent” than others and require less additional molecular events to cause overt leukemia. Clinical follow up data were available for 213 pts (74 RUNX1mut and 139 RUNXunmut). A direct comparison of these two groups showed a trend to shorter overall survival (OS) in the RUNX1mut pts (p=0.094) and a significantly shorter event free survival (EFS) (p=0.008). A subanalysis for RUNX1 and MLLPTD showed worse EFS for all three groups (RUNX1+/MLLPTD+ (n=21), RUNX1+/MLLPTD− (n=53), RUNX1−/MLLPTD+ (n=18) compared to the unmutated group (n=121) (p=0.064, 0.009, 0.081). Similar results were obtained for the combination of RUNX1/FLT3ITD with (RUNX1+/FLT3ITD+ (n=15), RUNX1+/FLT3ITD− (n=59), RUNX1−/FLT3ITD+ (n=24)) compared to the unmutated group (n=114) (p=0.006, 0.033, 0.223). Subsequently an analysis in the NK group was performed (25 RUNX1mut, 82 RUNX1wt). Surprisingly, RUNX1mut AML were not worse than the RUNX1unmut AML. In a subanalysis taking MLL-PTD and FLT3-ITD into account there were only 12 sole RUNX1mut pts and all revealed no event for OS and EFS (median follow up time: 196 days). In a further analysis taking cytogenetic as well as molecular aberrations into account it could be shown that RUNX1mut without any of these further aberrations have no event and are favourable compared to pts with single aberrations (p=0.063) and even more favourable compared to those with two or more cytogenetic or molecular aberrations (p=0.031). A multivariate analysis including age, cytogenetics, molecular genetics, and additional further genetic events shows that only age (p=0.027) and additional genetic events (p=0.005) are independent prognostic markers in this analysis. In conclusion, RUNX1 mutations are frequent in AML with NK or single chromosomal imbalaces, tend to go together with additional mutations which differ dependent on underlying cytogenetics. cooperate most frequently with MLL-PTD 4) per se are prognostically favourable as single genetic aberration but deteriorate by acquisition of other cytogenetic and/or molecular aberrations.