Clinical Significance of Myelofibrotic (MF) Changes in Myelodysplastic Syndromes (MDS): A Prospective Evaluation Including Mutational Analysis By Next-Generation Sequencing (NGS)

MDS with MF is not recognized as a distinct entity in 2008 WHO classification, and the prognostic role of MF remains controversial after several retrospective evaluations (Della Porta, 2009; Greenberg, 2012). In order to minimize inter-observer variability, a European Consensus (EC) has been reached for MF grade assignation (Thiele, 2005). IHC analysis of p53 protein BM accumulation has been proposed as prognosticator in MDS (Ramos, 2002), and more specifically in MDS with isolated 5q- (Jädersten, 2011; Saft, 2014) where it correlates with TP53 mutations. Several gene mutations have a prognostic role in MDS (Bejar,2011; Papaemmanuil, 2013; Haferlach, 2014), mutTP53 ranking as the most potent molecular prognosticator. By contrast, CALRmut, common in MPN, seems rare in MDS (Klampfl, 2013; Nangalia, 2013) albeit a focused evaluation taking into consideration EC grading has not been performed so far.

The aim of this study was to evaluate the events that underlie MF changes in MDS by studying 77 MDS patients (52M/ 25F; median age 76, range 31-88; WHO-2008: RCUD 2, RARS 4, RCMD 40, RAEB-1 15, RAEB-2 7, 5q- 7, MDS-U 2), that were recruited prospectively since June 2006 and followed up to June 2104 in 7 GESMD centers. Diagnosis and cytogenetic evaluation followed GESMD SOP’s (Schanz’s cytogenetic score: Very good 6, Good 51, Intermediate 12, Poor 5, Very Poor 5). The study was approved by the IRB at each study site and all patients gave written informed consent. Median follow-up was 3.0 years (0.1-7.7). During this time, 49 patients (63.6%) died and 2 (2.6%) were lost to follow-up, while 20 (21.6%) progressed into acute leukemia.

MF was evaluated independently by 2 pathologists following EC guidelines (MF-0 29, MF-1 31, MF-2 15, MF-3 2). IHC accumulation of p53 protein was evaluated as described previously and soluble p53 protein was analyzed in plasma by ELISA. Serum levels of 9 cytokines and chemokines were analyzed using cytometric bead arrays and a FACScanto flow cytometer (Becton-Dickinson). WT1 gene expression was evaluated by RT-PCR in PB and calculated as the WT1/GUSratio. The presence of mutations was evaluated in 67 patients. Sequencing was performed on a MISEQ NGS (Illumina) system using a panel of 111 genes previously related to MDS or MF. Sequencing results were analyzed by using the VariantStudio software package (Illumina), the threshold for mutation calling was set to 5%. Descriptive statistics and nonparametric statistical procedures were performed as appropriate, and univariate and multivariate analysis were performed for OS prediction.

MF grade showed a statistically significant nonparametric correlation with the proportion of bone marrow erythroid precursors in BMA (-0.408), Hb levels (-0.271), number of PRBC transfused in the first 16 wks (0.302), ferritin levels (0.294), EPO levels (0.331) and p53 IHC score (0.231), as well as the presence of ALIPs (0.358), WT1/GUSratio (0.264) and serum levels of MIG (-0.251), but not with other covariates.

Mutation incidence was highest for genes involved in splicing machinery (38.8%) as well as ASXL1 (18%). Mutated SF3B1 was associated with the absence of fibrosis (MF-0 23.1%, MF-1 7.4%, MF-2/3 0%; p=0.024) while the opposite was true for mutated ETV6 (MF-0 and MF-1 no mutations, MF-2/3 14.3%; p=0.041). Only 1 patient harbored a mutation in CALR. In our series, p53 protein accumulation and mutTP53 were not statistically linked. Mutations in GATA1 and SMC3 were more frequent in patients with IPSS-R High/Very High (25.0% vs. 3.6%, p=0.037 and 25.0% vs. 0%, p=0.005, respectively), but no single mutation was linked to Schanz’s cytogenetic score (Very Good/Good vs. Intermediate vs. Poor/Very Poor) with this sample size.

Survival analysis showed MF-2 or higher as a relevant predictor for OS (median OS for MF-0/1 4.0 years [CI95% 1.4-6.6] vs. 1.2 [0-3.3] for MF-2/3; HR 2.3, CI95% 1.2-4.3, p=0.008), that was independent of IPSS-R in multivariate analysis (IPSS-R HR 2.47 [1.3-4.7], p=0.006; MF HR 2.18 [1.2-4.1], p=0.013).

In conclusion, MF changes observed in MDS patients are not linked to mutations in CALR, cytogenetic score or IPSS-R, but to erythroid failure. Mutations in SF3B1 and ETV6 seem to diverge on their influence over MF. MF grade 2 or higher seems relevant for OS and independent of IPSS-R.