Application of a Flow Cytometric Core Marker Set in the Diagnostic Workup of Patients with Suspected Myelodysplastic Syndromes

Background Flow cytometry (FC) performed according to ELN 2012 criteria (Westers et al., 2012) is recommended by ELN guidelines for diagnostic workup in suspected myelodysplastic syndromes (MDS, Malcovati et al., 2013). Recently, the ELN working group on FC in MDS identified 17 core markers independently related to MDS as well as >3% myeloid progenitor cells (MPC) to be strongly related to MDS. In parallel, during the last decade next generation sequencing (NGS) became increasingly relevant for MDS diagnostics.

Aim To validate the new ELN criteria (ELN 2022) in comparison to cytomorphology (CM) in an independent cohort of patients prospectively undergoing diagnostics for suspected MDS and their correlation with MDS specific variants as detected by NGS.

Methods Bone marrow samples of 4658 patients (female/male, 1833/2825; median age 72 years, range 18-96) with suspected MDS were analyzed in parallel by CM, FC and NGS (73 gene panel). Cases with non-MDS neoplasms were excluded.

Results CM and FC (ELN 2012) revealed concordant diagnostic results in 80%. 1342 (29%) cases were diagnosed MDS by both CM and FC (CM+FC+), 2384 (51%) were CM-FC-. Discordant cases were 329 (7%) CM+FC- and 603 (13%) CM-FC+, respectively.

Application of ELN 2022 criteria for the diagnostic of MDS by FC (either at least 3 out of 17 markers present or more than 3% MPC) resulted in a similar degree of concordance (78%). 1016 (22%) were CM+FC+ and 2626 (56%) were CM-FC-. Discordant cases were 655 (14%) CM+FC- and 361 (8%) CM-FC+, respectively.

Next, we determined the portion of cases with at least one MDS-related gene mutation as analyzed by NGS in cases discordant between CM and FC.

Regarding FC ELN 2012 criteria 248/329 (75%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 352/603 (58%). Considering ELN 2022 criteria 527/655 (81%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 236/361 (65%).

Focusing on mutations in detail revealed similar frequencies of CHIP mutations (ASXL1, DNMT3A, TET2) regardless of CM and FC results. Concordant CM and FC cases (ELN 2012) harbor at least one CHIP mutation in 38% (CM+FC+ 506/1341, 38%; CM-FC- 901/2384, 38%). In CM+FC- cases 137/329 (39%) had at least one CHIP mutation, while in CM-FC+ cases the rate was 220/603 (37%). Application of ELN 2022 criteria resulted in a similar distribution (CM+FC+ 379/1016, 37%; CM-FC- 983/2626, 37%; CM+FC- 254/655, 39%; CM-FC+ 138/361, 38%).

Analyzing cases discordant between CM and FC regarding non CHIP mutations showed a strong correlation between CM+FC- cases and SF3B1 mutations (ELN 2012: 102/324, 32%; ELN 2022: 190/651, 29%). In contrast, in CM-FC+ cases just 13/597 (2%, ELN 2012) and 11/357 (3%, ELN 2022), respectively, had an SF3B1 mutation. In line with this correlation, CM+FC- cases (ELN 2012) with mutated SF3B1 had ≥5-14% ring sideroblasts (RS) in 17/102 (17%) and ≥15% RS in 78/102 (77%). The respective figure for CM+FC- cases (ELN 2022) were ≥5-14% RS in 22/190 (12%) and ≥15% RS in 160/190 (84%).

On the other hand SRSF2 mutations were found in discordant cases slightly more frequently in CM-FC+ cases compared to CM+FC- cases (ELN 2012: 95/557 (17%) of CM-FC+ and 40/315 (13%) of CM+FC- cases). This correlation was even clearer with ELN 2022 criteria (75/334 (23%) of CM-FM+ and 101/628 (16%) of CM+FC- cases).

Evaluating remaining cases discordant between CM and FC after excluding those with CHIP mutations, SF3B1 mutations, and SRSF2 mutations revealed no major differences regarding presence of at least one of the remaining mutations (ELN 2012: 32/107 (30%) of CM+FC- cases and 62/275 (23%) of CM-FC+ cases; ELN 2022: 59/175 (34%) of CM+FC- cases and 43/146 (30%) of CM-FC+ cases).

Conclusion FC has already proven its power in MDS diagnostics. We here confirm the newly defined criteria of ELN for diagnosing MDS (ELN 2022) to reveal results comparable to ELN 2012 criteria. Thus, a reduced core set of markers is already sufficient for MDS diagnosis by FC without loss in accuracy. Furthermore, cases with results discordant between CM and FC are associated with MDS-related mutations with particular sensitivity of CM in SF3B1 mutated cases and of FC in SRSF2 mutated cases. The higher frequency of mutations in cases discordant between CM and FC applying ELN 2022 criteria as compared to ELN 2012 criteria argues in favor of a higher specificity of the ELN 2022 criteria. Follow-up analyses are indicated to reveal the significance of discordant findings.

Weiß:MLL Munich Leukemia Laboratory: Current Employment. Haferlach:Munich Leukemia Laboratory: Current Employment, Other: Part ownership. Baer:MLL Munich Leukemia Laboratory: Current Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Current Employment. Haferlach:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership. Kern:MLL Munich Leukemia Laboratory: Current Employment, Other: Ownership.