Arginase and YKL-40, Effectors of Immunosuppressive Myeloid Cells, Are Over-Expressed In the Bone Marrow of Most Chronic Myelomonocytic Leukemia Patients, and Are Potential Prognostic Biomarkers In Myelodysplastic Syndrome

Solid tumors commonly recruit and condition immature myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMΦs), which impair immune-surveillance and facilitate growth. Whether they play a pathogenic role in hematological malignancies is not known. Our previous studies in an MDS/CMML model (SHIP-/- mice) revealed MΦs that are skewed to an alternatively activated (M2) phenotype (reminiscent of TAMΦs), characterized by arginase 1 (Arg1)-mediated redirection of L-arginine metabolism away from cytotoxic NO and towards the production of healing intermediates, as well as over-expression of the Th2-skewing and anti-apoptotic chitinase-like protein, Ym1. Moreover, MDSCs are also increased in SHIP-/- mice, leading to T-cell suppression. We therefore hypothesized that MDSCs/M2-MΦs may be expanded in the BM of MDS and CMML patients and/or their properties hijacked by the malignant clones.

With informed consent, BM mononuclear cells (MNCs) and plasma were obtained from 40 specimens in the Sunnybrook MDS Biobank (6 normals, 11 CMML-1, 1 CMML-2, 1 RARS, 2 del(5q), 6 RCMD, 2 RCMD-RS, 8 RAEB-1, and 3 RAEB-2) (20 at presentation; 20 at follow-up). BM plasma was subjected to YKL-40 ELISA and Quantibody (RayBiotech) cytokine array (bFGF, BMP6, IL-1α, IL-1β, IL-6, PDGF-BB, TGFβ1, TNFα, VEGF, and VEGF-R2). BM-MNC protein lysates were prepared and subjected to arginase enzymatic assay, and Arg1/YKL-40 Westerns. Micro-vessel density (MVD) was recorded as the concordant counts of two observers per mm² of H&E BM biopsies. Surrogate MVD analysis was performed by automated CD31 immunohistochemistry (IHC) analysis (Vias, Ventana). Linear regression and unpaired, two-tailed t tests were performed with Prism (GraphPad) software.

Mean BM-MNC arginase activity (μg urea/h/μg protein, recorded as % of control mean) and BM plasma YKL-40 levels were significantly higher in CMML patients, as compared to controls (350 ± 69 % vs 100 ± 5 %, p = 0.022, and 300 ± 32 μg/L vs 150 ± 22 μg/L, p = 0.0033, respectively). Westerns confirmed intracellular Arg1/YKL-40 over-expression in CMML BM-MNCs. Heterogeneous Arg1/YKL-40 over-expression was observed in MDS patients, and was strongly associated with the lowest risk IPSS and WPSS MDS scoring categories (Figure 1) (CMML not included). Moreover, high Arg1, but not YKL-40, was able to further discriminate IPSS Low/Int1-risk patients with poor prognosis using the low-risk MD Anderson score (Figure 1). This low-risk MDS/high-Arg1/YKL-40 profile was confirmed by negative correlations with (poor-prognostic) MVD (Arg1: p = 0.049, YKL-40: p = 0.089) (similar trends with CD31), albeit in a seemingly paradoxical pro-angiogenic BM plasma milieu (trends to increased bFGF, PDGF-BB, VEGF, and VEGF-R2). These results were unexpected, given that Arg1/YKL-40 are normally thought to be pro-angiogenic, and were reminiscent of recent studies suggesting VEGF-mediated recruitment of suppressive myeloid cells may inhibit angiogenesis and tumor progression in some situations (Stockmann et al., Nature 456:814-8). Accordingly, MDS BM arginase activity was associated with a larger BM myeloid compartment (using > 60% as a cutoff; p = 0.042) and a striking linear correlation with peripheral blood (PB) neutrophil counts on the day of BM collection (p = 0.0003), likely reflecting BM and systemic expansion of suppressive myeloid populations. MDS BM-MNC Arg1/YKL-40 Westerns, stratified by PB neutrophil counts, confirmed these associations.

1) Arg1/YKL-40, effectors of MDSCs/M2-MΦs, are over-expressed in BM MNCs and plasma in most CMML patients, as predicted from SHIP-/- mouse studies. Perturbations in SHIP and associated pathways may warrant investigation in CMML. 2) Arg1/YKL-40 are over-expressed in low-risk MDS patients, as assessed using 3 prognostic scoring systems. Their immune-suppressive nature and pro-proliferative/anti-apoptotic effects may create a permissive environment for low-risk MDS clones. 3) The unexpected association of high Arg1/YKL-40 with a pro-angiogenic BM milieu and paradoxically low MVD, may be in keeping with the ability of MDSCs/M2-MΦs to inhibit angiogenesis under certain conditions. Understanding what regulates the “immune/angiogenic switch” to low suppressive myeloid/high MVD/high-risk MDS may have important future treatment implications.

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