Detection of Prognostically Relevant Mutations and Translocations in Myeloid Sarcoma by Next Generation Sequencing

Myeloid sarcoma (MS) is a subgroup of acute myeloid leukemia (AML) where myeloid blasts form a tumoral mass in extramedullary tissues [1]. MS may occur at any point during the disease course and almost every site of the body can be affected. Although MS is often diagnosed as an isolated event without concomitant bone marrow involvement, virtually all of these patients will develop overt hematologic disease if left untreated [2,3], which further highlights the systemic nature of this disease. Beside isolated MS, extramedullary manifestations might also occur simultaneously with leukemic bone marrow (BM) infiltration. The incidence rate of this situation in newly diagnosed AML patients is estimated to be around 9%, however, some authors report incidence rates of up to 20–40% [2,4]. MS is usually treated with standard AML induction regimens, even if it occurs as isolated event. However, little is known about optimal consolidation strategies after achievement of complete remission [5,6]. This is particularly true for cases with isolated MS, which is due to the fact that material from MS biopsies is often sparse and usually Formalin-Fixed-ParaffinEmbedded (FFPE), thereby often precluding comprehensive AML risk stratification. In this study analyzing 18 cases of MS, we developed a next-generation sequencing (NGS) based approach, which enables the detection of both mutations and translocations with prognostic relevance from FFPE tissues of MS. Additionally, we analyzed the effects of this MS tissue based NGS profiling on AML risk stratification in cases where MS coincides with systemic AML and where cytogenetic/molecular analyses have already been performed from leukemic BM. All patient specimens of MS and corresponding BM biopsies were collected at the Division of Hematology, Medical University of Graz (MUG), between June 2003 and December 2016 and stored as FFPE samples. Patient characteristics are presented in Table 1. Briefly, MS coincided with systemic AML in 11 patients, whereas isolated MS was present in seven cases. The study was approved by the MUG-ethical committee (vote number 24-036 ex 11/12) and performed in accordance with the Declaration of Helsinki. In a first step, we focused on the detection of translocations with relevance for AML risk stratification [5]. This is of relevance, as complete cytogenetic analysis from MS specimens is usually not possible, which is due to the frequent unavailability of fresh material. Additionally, although fluorescence in situ hybridization (FISH) was successfully applied to MS specimens previously [7], this approach is limited by the fact that the amount of MS material is often sparse and that every abnormality tested requires at least one FFPE-section slide. Significant progress in this area came from Mirza et al., who recently analyzed six cases of MS by chromosomal microarray analysis (CMA) [8]. Without the need to target their analyses to selected abnormalities, they successfully detected unbalanced chromosomal aberrations and complex karyotypes. However, a limitation of this approach was the fact that prognostically relevant balanced chromosomal rearrangements could not be detected. To circumvent this limitation, we now performed NGS-based translocation analysis using only 100 ng of RNA extracted from FFPE-MS specimens discovering CBFB-MYH11, DEK/CANNUP214, DEK-NUP214, MLL-MLLT3, PML-RARA, RBM15MKL1, RPN1-MECOM, and RUNX1-RUNX1T1 (for details see also the Supplementary information). By studying 18MS specimens, we were able to unambiguously detect the presence of CBFB-MYH11 in three of them (Figure 1). In two of these cases, MS presented as an isolated event without any evidence of systemic disease. While the follow up of these patients was too short to allow any conclusions about a prognostic relevance of this lesion in MS, previous reports suggested a potential correlation of