Assessment of ASC specks as a putative biomarker of pyroptosis in myelodysplastic syndromes: an observational cohort study

Abstract

Background: NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes.

Methods: This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis.

Findings: Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log₁₀-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97).

Interpretation: Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes.

Funding: T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).

Figure 1:. Visualisation of ASC specks in patients with myelodysplastic syndromes

Representative confocal microscopy micrographs of ASC specks in bone marrow mononuclear cells from three patients with myelodysplastic syndromes from the discovery cohort. (A) DAPI nuclear staining. (B) Anti-ASC antibody staining. (C) Merged DAPI and anti-ASC image (A–C × 2100 magnification). (D and E) Digitally enlarged images of parts of C to highlight specks (white arrows). (F) Surface rendering of E. (G) Purified GFP-ASC specks from lipopolysaccharide-stimulated or nigericin-stimulated THP-1-GFP-ASC cell lines. (H) Transmission electron microscopy image of GFP-ASC specks isolated from stimulated THP-1 cells (× 15 000 magnification). ASC=apoptosis-associated speck-like protein containing a CARD. GFP=green fluorescent protein

Figure 2:. Quantification of ASC specks in bone marrow and peripheral blood from patients with myelodysplastic syndromes relative to patients with other haematological cancers, type 2 diabetes, or healthy donors

ASC=apoptosis-associated speck-like protein containing a CARD. MDS=myelodysplastic syndromes. del(5q)=chromosome 5 deletion. ALL=acute lymphocytic leukaemia. CLL=chronic lymphocytic leukaemia. CML=chronic myelogenous leukaemia. CMML=chronic myelomonocytic leukaemia. AML=acute myeloid leukaemia. LGL=large granular lymphocytic leukaemia. SLD=single-lineage dysplasia. MLD=multilineage dysplasia. EB=excess blasts. Boxes show median (centre line) and IQR (outer lines) and whiskers show SD. (A) Glucose-adjusted percentage of ASC specks in bone marrow and peripheral blood plasma, and difference between peripheral blood and bone marrow in samples from patients with myelodysplastic syndromes in the discovery cohort, quantified by flow cytometry. (B) Glucose-adjusted log₁₀-transformed percentage of ASC specks in peripheral blood plasma from healthy controls and patients with myelodysplastic syndromes in the discovery cohort, as well as patients with non-myelodysplastic syndrome haematological cancers and type 2 diabetes, quantified by flow cytometry. (C) Glucose-adjusted log₁₀-transformed percentage of ASC specks quantified by flow cytometry in patients with different subtypes of myelodysplastic syndromes from the discovery cohort and patients with del(5q) mutation, as categorised by 2016 WHO guidelines. (D) Glucose-adjusted log₁₀-transformed percentage of ASC specks quantified by flow cytometry in peripheral blood plasma samples from patients with myelodysplastic syndromes versus healthy controls in the validation cohort.

Figure 3:. Sensitivity and specificity of ASC specks in peripheral blood plasma from patients with myelodysplastic syndromes

Receiver operating characteristic AUC analysis of glucose-adjusted log₁₀-normalised ASC speck percentage in patients with lower-risk, higher-risk, or any myelodysplastic syndromes from the discovery and validation cohorts. ASC=apoptosis-associated speck-like protein containing a CARD. MDS=myelodysplastic syndromes. AUC=area under the curve.

Figure 4:. Percentage of peripheral blood plasma ASC specks from patients with myelodysplastic syndromes according to number of somatic gene mutations

Boxes show median (centre line) and IQR (outer lines) and whiskers show SD. Data are from the discovery and validation cohorts (n=96). ASC=apoptosis-associated speck-like protein containing a CARD.