Session: 506. Bone Marrow Microenvironment: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research
METHODS: We conducted a functional study of BMSC obtained from MN-AIRD patients and carefully selected control populations to evaluate the damage induced by cytotoxic and DMARD, including non-cytotoxic biological and cytotoxic DMARD such as methotrexate, cyclophosphamide, and azathioprine in three cohorts: (1) MN-AIRD, patients who developed myeloid malignancy after long-term use of cytotoxic drugs and DMARD (n=33); (2) de novo MN without AIRD (de novo MN without AIRD; n=16); and (3) age-matched controls (n=17). We assessed morphology, proliferation by maximum cumulative population doublings (CPD), clonogenic potential by colony forming unit fibroblast (CFU-F), cellular senescence by the percentage of β-galactosidase-stained cells at passage 3, adipo- and osteogenic differentiation potential of BMSC, and cytokine levels (38-plex magnetic bead panel, Millipore).
RESULTS: BMSC isolated from MN-AIRD patients were significantly different from both age-matched healthy controls and MN without AIRD, with a decreased number of CFU-F (0.68±0.71 vs. 4.5±2.79 vs. 1.81±0.79 per 104 cells, respectively, P < 0.001) and maximum CPD (0.71±1.48 vs. 11.46±6.74 vs. 4.43±2.56 doublings respectively, P < 0.001).
Morphological indicators of senescence, as measured by β-galactosidase staining, were distinctly higher in MN-AIRD compared to de novo MN without AIRD and healthy controls (54±16% vs. 27±9% vs. 6±4%, respectively; P < 0.001). Remarkably, several inflammatory cytokines in the marrow interstitial fluid, including fractalkine, IFNα2, IL-1β, IFNγ and GM-CSF, were higher in MN-AIRD compared to de novo MN without AIRD.
The BMSC osteogenic differentiation potential, assessed by mineralization matrix formation, was significantly higher in MN-AIRD (134.4±65.76 Ca2+/µg DNA) compared to healthy controls (52.76±34.29 Ca2+/µg DNA) and de novo MN without AIRD (53.78±34.29 Ca2+/µg DNA; P = 0.0088). This was further confirmed by Alizarin red-stained mineralized deposits. In contrast, the quantification of lipid-laden Nile Red- marked adipocytes showed a significant reduction in MN-AIRD compared to de novo MN without AIRD and healthy controls (0.07±0.06 vs. 0.33±0.17 vs. 0.67±0.06; P<0.0001).
We further analyzed the association between the type of cytotoxic therapy or DMARD and differences in the BM stroma. Surprisingly, we did not observe a significant differences in clonogenic capacity (1.13±0.98, 0.41±0.33, 0.35±0.40, 0.83±0.85 per 104 cells; P=0.28), proliferative capacity (0.81±0.8, 0.46±0.76, 0±0, 0.67±1.6 doublings; P=0.83) or degrees of senescence (53±28.28%, 56±15.56%, 53±0%; P=0.99) in AIRD cases treated with azathioprine vs. methotrexate vs. other cytotoxics vs. biological DMARD.
To test the hypothesis that prior cytotoxic and DMARD therapy leads to long-term damage to the BM microenvironment, we assessed the proliferation, cellular senescence, and differentiation potential of BMSC samples before MN diagnosis in AIRD patients. Our preliminary results indicate a reduction in the proliferation capacity of BMSC years before MN diagnosis but subsequent to DMARD treatment.
CONCLUSION: Our study demonstrates that the marrow microenvironment in patients with MN-AIRD exhibits significant functional and morphological differences compared to healthy controls and patients with de novo MN without AIRD, indicating that long-term cytotoxic or DMARD therapy adversely affects BMSC. These findings suggest the need for more tailored and effective therapeutic strategies for MN-AIRD patients.
Disclosures: Hiwase: Astella Pharma: Honoraria; Otsuka: Honoraria; Abbvie: Honoraria.