7-ketocholesterol (7-KC) is a bioactive oxysterol generated under oxidative stress and may contribute to bone marrow niche reprogramming in acute myeloid leukemia (AML), thereby promoting stress tolerance and therapeutic resistance Bone marrow mesenchymal stromal cells (MSCs) from healthy donors and AML patients were exposed to subtoxic 7-KC concentrations for 24 h. We evaluated the ABC transporters involved in lipid transport, multidrug resistance and membrane microdomain remodeling; Hedgehog pathway proteins; stress–survival signaling; redox balance by glutathione measurements, and mitochondrial function and dynamics, including membrane potential and gene expression of mitochondrial fission and fusion regulators. Results were integrated using principal component analysis (PCA), heatmaps, and correlation-based networks. Multivariate analyses revealed an integrated, lineage-dependent response. Healthy donor MSCs showed greater plasticity of the efflux and microdomain axis and higher oxidative and mitochondrial vulnerability at high 7-KC doses. AML-MSCs exhibited a basal preconditioned state phenotype and preferentially routed the response toward Hedgehog and stress–survival modules, accompanied by glutathione expansion and adaptive mitochondrial remodeling. 7-KC acts as a broad modulator of several MSC functions, linking sterol homeostasis to Hedgehog signaling, stress–survival pathways, redox balance, and mitochondrial remodeling, potentially supporting a pro-survival, more therapy-tolerant leukemic niche.

Background/Objectives: T-cell receptor gamma (TCRγ) clonality testing by multiplex polymerase chain reaction (PCR) is an important ancillary method in the diagnostic evaluation of T-cell lymphoproliferative disorders. Interpretation is particularly challenging in HTLV-1 infection, in which persistent antigenic stimulation may produce clonal or oligoclonal T-cell expansions overlapping with non-neoplastic patterns. This study aimed to assess the diagnostic performance and interpretative contribution of two EuroClonality/BIOMED-2 assays (TCRγ-A and TCRγ-B), considered complementary components of the same analytical panel, and a single-tube multiplex PCR assay (TCRγ-O), and to develop an integrated workflow for TCRγ clonality interpretation. Methods: A total of 107 peripheral blood samples were analyzed, including 19 healthy donors, 75 HTLV-1 carriers, and 13 patients with adult T-cell leukemia/lymphoma (ATLL). TCRγ clonality was evaluated using three multiplex PCR assays followed by capillary electrophoresis. Profiles were classified as polyclonal, oligoclonal, or monoclonal. Diagnostic performance analysis was performed using healthy donors as the reference negative group and ATLL patients as the reference positive group. Results: The combined EuroClonality approach (TCRγ-A+B) showed the highest sensitivity (100%), whereas TCRγ-O showed the highest specificity (94.7%). TCRγ-A and TCRγ-B each showed a sensitivity of 76.9%, with specificities of 84.2% and 78.9%, respectively. Classification was highly concordant in healthy donors and ATLL patients employing both assays. However, among HTLV-1 carriers, classification varied across the assays. The integrated workflow was associated with a significant change in the distribution of molecular classification patterns in the HTLV-1 group compared with EuroClonality-based interpretation alone. (Stuart–Maxwell, p < 0.001). Conclusions: The EuroClonality/BIOMED-2 strategy provided greater sensitivity, whereas TCRγ-O showed greater specificity. Their integration supported a complementary and hierarchical approach to TCRγ clonality assessment, particularly in HTLV-1 infection. These findings support an exploratory integrated framework for TCRγ clonality interpretation in HTLV-1 infection and ATLL, requiring validation in larger independent cohorts.