Karyotype evolution and acquisition of FLT3 or RAS pathway alterations drive progression of myelodysplastic syndrome to acute myeloid leukemia Karyotype evolution and acquisition of FLT3 or RAS pathway alterations drive progression of myelodysplastic synd

Haematologica 2015 [Epub ahead of print] Citation: Meggendorfer M, De Albuquerque A, Nadarajah N, Alpermann T, Kern W, Steuer K, Perglerová K, Haferlach C, Schnittger S, and Haferlach T. Karyotype evolution and acquisition of FLT3 or RAS pathway alterations drive progression of myelodysplastic syndrome to acute myeloid leukemia. Haematologica. 2015; 100:xxx doi:10.3324/haematol.2015.127985 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. to s-AML. We identified mutations in genes of the cellular signal transduction to drive transformation, and several MDS typical mutations to predispose to s-AML transformation. In addition, karyotype evolution was detected in more than one third of patients underlining its important impact on s-AML transformation. We identified 38 patients (11 female, 27 male) that were analyzed in our laboratory by cytomorphology and cytogenetics both at diagnosis of MDS and later at progression to s- Cytogenetically, a total of 14/38 (37%) MDS and 22/38 (58%) s-AML samples presented an aberrant karyotype and 13 patients (34%) gained chromosome abnormalities during progression to s-AML. One of these patients showed in parallel clonal evolution in one cytogenetic clone but lost another cytogenetic clone completely. These findings again confirm that cytogenetic evolution in MDS has been associated with progression to AML. 2;3 The most frequently occurring recurrent abnormality at MDS stage was trisomy 8 (+8; n=5), followed by del(5q) and del(20q) both in two patients each. While in s-AML patients a +8 occurred in four additional patients amounting to nine cases in total, del(5q) and del(20q) 3 were rarely gained at s-AML (one case with del(5q)). Although a normal karyotype was more often found at the MDS stage compared to the s-AML transformed samples, this difference was not statistically significant. Illustration of the cytogenetic patterns at both MDS and s-AML states is shown in We next analyzed the distribution of molecular mutations in patients at MDS and s-AML stages. All samples presented at least one mutation in both MDS and s-AML disease states. The most frequently mutated genes were largely matching for MDS and s-AML and showed the following mutation frequencies, respectively: SRSF2 (47% MDS; 47% s-AML), RUNX1 (37%; 40%), ASXL1 (37%; 42%), TET2 (34%; 34%), and SF3B1 (24%; 24%). On the other hand, mutations in GATA1, MPL and RAD21 genes were found at neither MDS nor s-AML stages. Furthermore, one KIT mutation was detected in a patient only at MDS stage and was lost at s-AML stage, while FLT3-ITD and FLT3-TKD mutations were identified in s-AML samples only (16% and 8%, respectively). A total of 20 (53%) patients gained mutations in at least one gene of the analyzed 33 genes during progression to s-AML, four (11%) patients lost at least one mutation and six (16%) patients had simultaneous losses and gains of mutations (figure 2). But there were no recurrently lost gene mutations at transformation. Looking at the distribution of molecular mutations revealed that FLT3-ITD was significantly associated with s-AML compared to the corresponding MDS cases (16% vs 0%, p=0.025). Although not significant, FLT3-TKD (8% vs 0%), NRAS (26% vs 11%), and KRAS (16% vs 3%) were more often mutated in s-AML compared to MDS cases, while mutations in all other genes were equally distributed between both disease states (figure 2). Summing up the cases with at least one of this four gene mutations resulted in significant association of mutations in these genes to s-AML compared to MDS state (47% vs 13%, p=0.002). In total, 15/20 patients (75%) that acquired new mutations, showed mutations in the latter mentioned signal transduction proteins (FLT3 or RAS pathway), indicating that these might be mutations driving s-AML transformation. FLT3 and NRAS mutations are thought to be important genetic events contributing to the pathogenesis of AML 4-6 and as expected, the increase in mutation frequencies in s-AML cases was observed, confirming previously reported data. Comparing the frequencies of mutational acquisition in cases with and without karyotype alteration at leukemia transformation revealed that only eight of the 26 cases that acquired mutations showed also gains of cytogenetic aberrations, while 18 cases remained cytogenetically stable. Interestingly, none of the patients that acquired a mutation in NRAS or FLT3 (n=12) presented with karyotype transformation, and only three patients acquiring a KRAS mutation showed also cytogenetic transformation (figure 2). We further compared the MDS data set of the presented study to our results of an independent previously published MDS cohort. 11 WHO diagnosis categories were matched between cohorts, and cases showing transformation to s-AML were excluded. Cases with follow-up less than 18 months (the median time to transformation of the MDS/s-AML cohort) were also excluded, resulting in a final cohort of 494 patients. A comparable frequency of patients showed aberrant karyotypes at their MDS stage (37% vs 29% in the published control cohort, respectively). However, trisomy 8 appeared more frequently in MDS patients with progression to s-AML than in the control MDS cohort (13% vs 3%; p=0.015) (figure 3). We further compared the mutation frequencies between these two cohorts, and found that in MDS cases transforming to s-AML the following mutations were more frequent than in the MDS control cohort: ASXL1 (37% vs 19%; p=0.019), CBL (11% vs 3; p=0.027), GATA2 (5% vs 0; p=0.027), IDH2 (18% vs 2%; p<0.001), NRAS (11% vs 2; p=0.010), RUNX1 (39% vs 5; p<0.001), and SRSF2 (47% vs 14%; p<0.001). Mutations in these genes might thus predispose to s-AML progression (figure 3). To further validate our findings we then focused on the patients previously excluded from the control cohort that also transformed to s-AML (n=78) and analyzed the differences in cytogenetics and mutation frequencies to the MDS cases of the present MDS/s-AML cohort. Both MDS to s-AML cohorts showed comparable frequencies of aberrations and mutations, again supporting that the previous findings are specific for MDS cases transforming to s-AML (supplemental