Bone marrow colony forming units-fibroblast in patients with chronic myelomonocytic leukemia: functions and clinical significance

doi:10.3969/j.issn.2095-4344.1725

Abstract

Abstract:

BACKGROUND: Studies have shown that abnormal bone marrow microenvironment is involved in the development of chronic myelomonocytic leukemia. Colony forming unit-fibroblast represents a group of multipotent mesenchymal stromal cells that can reflect the bone marrow stromal microenvironment in terms of cell function and number.
OBJECTIVE: To investigate the abilities of bone marrow mesenchymal stromal cells to form colony forming units-fibroblast and to differentiate towards osteoblasts in patients with chronic myelomonocytic leukemia and to study the relationship between colony forming unit-fibroblast counts and clinic characteristics (peripheral blood cell count and clinical phenotype).
METHODS: (1) Fifteen newly diagnosed chronic myelomonocytic leukemia patients admitted to the Department of Hematology, Guangdong Provincial People’s Hospital, and 10 volunteers (control) were enrolled. Functions and osteogenic potential of colony forming units-fibroblast were compared between two groups. (2) There was a correlation analysis between colony forming unit-fibroblast counts and clinical characteristics (peripheral blood cell count, clinical phenotype, percentage of bone marrow blasts and gene mutations). The study protocol was approved by the ethics committee of Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences) with the approval No. [2018]002.
RESULTS AND CONCLUSION: (1) The number of colony forming units-fibroblast formed in the patients with chronic myelomonocytic leukemia decreased as compared with the control group (P=0.04). The 10 of 15 (67%) patients showed remarkable reduction in the colony forming unit-fibroblast counts (P=0.00), while the rest 5 (33%) patients exhibited normal colony forming unit-fibroblast levels (P=0.14). (2) Expression levels of Osterix and Runx2 in the bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia were significantly lower than those in the control group. There was a limited osteogenic potential in the bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia (P=0.00). (3) Colony forming unit-fibroblast counts of patients with chronic myelomonocytic leukemia were negatively related to peripheral white blood cells, neutrocytes and monocytes counts, and were positively related to peripheral platelet counts. (4) Nine out of 10 (90%) patients with reduced colony forming unit-fibroblast counts exhibited a higher white blood cell level (≥ 13×10⁹/L) (P=0.01), and 5 out of 10 (50%) patients carried NRAS/KRAS mutations. Findings from this study show limited function of colony forming unit-fibroblast formation and osteogenic differentiation in bone marrow mesenchymal stem cells of patients with chronic myelomonocytic leukemia. Moreover, the number of colony forming units-fibroblast seems to be correlated to peripheral blood cell counts and clinical features, indicating underlying significances in the diagnosis and prognosis of chronic myelomonocytic leukemia.

Key words: colony forming unit-fibroblast, bone marrow, mesenchymal stem cells, chronic myelomonocytic leukemia, KRAS gene, NRAS gene, osteogenic differentiation, peripheral blood cell count, MPN-CMML type, MDS-CMML type, bone marrow microenvironment

CLC Number:

R457

Xu Ruohao, Li Chao, Wu Ping, Li Minming, Deng Chengxin, Geng Suxia, Lai Peilong, Lu Zesheng, Weng Jianyu, Du Xin. Bone marrow colony forming units-fibroblast in patients with chronic myelomonocytic leukemia: functions and clinical significance[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3398-3403.

Figures/Tables 3

2.1 纳入患者一般信息见表2。 2.2 骨髓间充质干细胞功能与纯度鉴定结果见图1。图中可见典型间充质干细胞形态，具有成脂、成骨分化能力。对5例慢性粒单核细胞白血病患者及4例对照组志愿者体外培养的间充质干细胞进行流式表面抗原检测：CD90阳性率为(96.2±1.9)%，CD73阳性率为(95.7±1.7)%，CD105阳性率为(96.0±2.1)%，CD45阴性率为(98.0±1.2)%，CD34阴性率(99.5±0.9)%。 2.3 慢性粒单核细胞白血病患者CFU-F与成骨分化功能受损见图2。大体观察，慢性粒单核细胞白血病患者形成的CFU-F密度降低，数量减少(图2A，D)。低倍镜下间充质干细胞失去了特征性的梭形外观，细胞间距增大，伴有较多有核细胞吸附，CFU-F集落数量显著减少(图2B，表2)。慢性粒单核细胞白血病患者的骨髓间充质干细胞经成骨诱导后茜素红染色降低(图2C)，A560降低(图2E，P=0.00)，提示钙盐沉积减少，成骨分化能力受损。 2.4 慢性粒单核细胞白血病患者骨髓间充质干细胞成骨相关基因表达水平见图3。qPCR结果显示，与对照组比较，慢性粒单核细胞白血病患者骨髓间充质干细胞中成骨分化关键基因Osterix和RUNX2的表达水平降低(Osterix ??Ct值：3.85±0.57 vs. 5.12±1.11，P=0.01；RUNX2 ??Ct值：2.33±0.57 vs. 3.15±1.22，P=0.00)，而早期成骨分化指标ALP的表达量无明显改变(??Ct值：3.21±1.02 vs. 3.67±0.66，P=0.21)。 2.5 CFU-F数量与患者外周血细胞计数相关性见图4。10例CFU-F降低组患者中，9例(90%)外周血白细胞计数≥13×109 L-1，其余5例CFU-F正常患者(100%)的外周血白细胞计数均<13×109 L-1(P=0.01)；慢性粒单核细胞白血病患者CFU-F数量与外周血白细胞、中性粒细胞计数显著负相关；与外周血小板计数显著正相关；与外周血单核细胞具有负相关趋势；CFU-F数量与外周血红蛋白水平以及骨髓原始细胞比例无明显相关性。 2.6 CFU-F数量与基因突变见表3。10例CFU-F降低组患者中，5例(50%)伴有NRAS/KRAS位点突变，5例CFU-F正常组患者中，仅1例(20%)伴有NRAS/KRAS位点突变(P=0.58)。CFU-F降低组患者中70%(7/10)与CFU-F正常组患者中40%(2/5)携带有≥3个基因位点的突变(P=0.33)。"

References

[1] Tefferi A, Vardiman JW.Myelodysplastic syndromes.N Engl J Med. 2009;361(19):1872-1885.

[2] Arber DA, Orazi A, Hasserjian R, et al.The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.Blood. 2016;127(20): 2391-2405.

[3] Kuznetsov SA, Friedenstein AJ, Robey PG. Factors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol. 1997; 97(3):561-570.

[4] Nifontova I, Svinareva D, Petrova T, et al. Sensitivity of mesenchymal stem cells and their progeny to medicines used for the treatment of hematoproliferative diseases. Acta Haematol. 2008;119(2):98-103.

[5] Caplan AI. Mesenchymal stem cells. J Orthop Res.1991; 9: 641-650.

[6] Balaian E, Wobus M, Weidner H, et al.Erythropoietin inhibits osteoblast function in myelodysplastic syndromes via the canonical Wnt pathway. Haematologica. 2018;103(1):61-68.

[7] Tauro S, Hepburn MD, Bowen DT, et al. Assessment of stromal function, and its potential contribution to deregulation of hematopoiesis in the myelodysplastic syndromes. Haematologica. 2001; 86: 1038-1045.

[8] Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell. 2008; 2:141-150.

[9] Geyh S, Oz S, Cadeddu RP, et al. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells. Leukemia.2013;27:1841-1851.

[10] Chen S, Zambetti NA, Bindels EM, et al. Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs. Leukemia.2016; 30: 1938-1942.

[11] Ricci C, Fermo E, Corti S, et al. RAS mutations contribute to evolution of chronic myelomonocytic leukemia to the proliferative variant. Clin Cancer Res.2010; 16: 2246-2256.

[12] Patnaik MM, Tefferi A. Chronic myelomonocytic leukemia: 2018 update on diagnosis, risk stratification and management. Am J Hematol. 2018; 93(6):824-840.

[13] Niyongere S, Lucas N, Zhou JM, et al. Heterogeneous expression of cytokines accounts for clinical diversity and refines prognostication in CMML. Leukemia. 2019;33(1):205-216.

[14] Fedders H, Alsadeq A, Schmah J, et al. The role of constitutive activation of FMS-related tyrosine kinase-3 and NRas/KRas mutational status in infants with KMT2A-rearranged acute lymphoblastic leukemia. Haematologica.2017; 102: e438-e442.

[15] Liang DC, Chen SH. Mutational status of NRAS, KRAS, and PTPN11 genes is associated with genetic/cytogenetic features in children with B-precursor acute lymphoblastic leukemia. Pediatr Blood Cancer. 2018; 65(2). doi: 10.1002/pbc.26786.