Abstract
Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow. Lower-grade MDS (bone marrow blasts <5%) is challenging to diagnose, as the morphologic dysplasia may be subtle and many patients lack characteristic cytogenetic abnormalities.
Multiple studies of the bone marrow in MDS have demonstrated excessive hematopoietic cell death, and this increased cell death is thought to play an important role in the pathogenesis of the disease. Early studies of MDS revealed increased in situ end labeling and increased TUNEL staining which led to the conclusion of apoptotic cell death, but these methods cannot properly distinguish between necroptosis and apoptosis. Apoptosis is driven by caspases that break down the cytoskeleton to cause collapse of the cell in a predominantly immune silent process. Recently, necroptosis has been identified as a new pathway of programmed cell death, which is driven by Rip kinases and is characterized by premature rupture of the plasma membrane, resulting in an immune response. Necroptosis has been shown to play a role in inflammatory diseases as well as host response to infection, and tumors.
Methods: To assess cell death status in normal bone marrow as compared to lower-grade MDS (RCUD, RA, RARS, RCMD) and higher-grade MDS (RAEBI/RAEBII, RAEB-t), we evaluated a total of 37 de novo MDS cases and 36 normal control cases. The MDS specimens included 20 cases of lower-grade MDS and 17 cases of higher-grade MDS. All cases were reviewed and the diagnosis confirmed by board-certified hematopathologists. Necroptosis was assessed by measuring Rip1 and phosphorylation of Mixed Lineage Kinase domain-like protein (MLKL) and apoptosis was measured by cleaved-caspase 3.
Formalin fixed paraffin embedded bone marrow core biopsies were immunostained with the apoptosis marker (caspase 3) and necroptosis markers (Rip1, MLKL) and visualized by immunofluorescence. Whole slide images were obtained using Aperio Versa 200 (Leica Microsystems). We developed a pipeline for statistical analysis to evaluate the immunofluorescence of stained and scanned images using CellProfiler (Broad Institute) image analysis software. For each slide over 10,000 cells were analyzed.
Results: We found that the proportion of RIP1 positive cells was significantly different between normal controls (6.91±1.32%) and lower-grade MDS (32.80±6.19%, p value 0.0042). Likewise, RIP1 staining in higher-grade MDS was less common than in lower-grade MDS (15.99±4.76% vs 32.90±6.19%, p value 0.0506). There was no significant difference between normal controls and high-grade MDS. In lower-grade MDS samples, RIP1 positive cells were mononuclear cells arranged in clusters, morphologically consistent with erythroid progenitors. Similar trends were seen with MLKL staining.
To evaluate apoptosis, we performed cleaved caspase 3 staining on the same patient samples. There was no difference in the proportion of caspase 3 positive cells among the groups: normal controls (2.02±1.08%), lower-grade MDS (2.77±0.86%) or higher-grade MDS (1.99±0.71%). As apoptotic and necroptotic cells have markedly different cellular ultrastructural morphologies, we performed electron microscopy on fresh bone marrow cells to define cell death. After scoring cells with a nucleus as either being live, apoptotic (shrinkage of cell membrane and pyknotic nucleus), or necrotic (swelling of cell membrane and nucleus), we found that there was a significant increase in the number of necrotic cells in the bone marrow of the lower-grade MDS patients relative to the normal control or higher-grade MDS patients.
We then performed over-representation analysis of the publicly available MDS microarray data (GEO GSE15061) from the international multi-institutional Microarray Innovations in Leukemia (MILE) research program. Although the study was underpowered to identify differences between normal controls and early MDS, we found that RIP1, RIP3 and MLKL had significantly increased expression in the MDS group vs the de novo AML group, suggesting that genes in the necroptotic pathway may be upregulated in MDS as compared to AML.
In summary, our findings reveal that the necroptosis cell death pathway is upregulated in early MDS relative to normal controls and warrants further study to define its role in the pathogenesis of MDS and as a potential biomarker for the diagnosis of lower-grade MDS.
Savona: Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Incyte Corporation: Consultancy, Research Funding; Karyopharm: Consultancy, Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.
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