66th American Society of Hematology (ASH)

Despite recent advances in the treatment of acute myeloid leukemia (AML), most patients with AML relapse and succumb to the disease. Achieving complete remissions (CRs) does not equate to cure, especially in TP53 mutated AML. Relapse after initial CRs is caused by measurable/minimal residual disease (MRD), which is resistant to prior therapies due to cell-intrinsic resistance mechanisms or is through protection by the bone marrow (BM) microenvironment. The mechanisms by which the BM microenvironment supports MRD cells are, however, largely unknown. We hypothesized that MRD AML cells possess unique characteristics distinct from leukemia cells prior to therapy and therefore may harbor targetable vulnerabilities. We performed multiplex protein analysis on AML BM biopsy specimens to dissect spatial characteristics of MRD cells and the BM microenvironment using spatial analytical techniques.

To comprehensively investigate the spatial architecture of TP53 mutated AML, we performed sequential multiplex immunofluorescence (seqIF) staining of formalin-fixed paraffin-embedded BM trephine biopsies using COMET (Lunaphore Technologies) with a 20-antibody panel including CD105 and CD90 for mesenchymal stromal cells (MSCs) and CD3, CD4, CD8, TIGIT, FoxP3, CD56 and CD20 for immune cell markers. We analyzed paired AML BM biopsies from 6 patients with AML with TP53 point mutations at diagnosis and at morphological CR with MRD detected by flow cytometry or PCRafter receiving Aza/Ven- or Ara-C/Idarubicin-based regimens, and 3 normal BM biopsies. Four patients had relapse after achieving CRs. TP53 mutated AML cells expressed high p53 protein levels at diagnosis and at MRD while no or extremely low levels of p53 positive cells were detected in BM biopsies from healthy donors and AML patients with TP53 truncating mutations, served as negative controls. seqIF stained slides were subjected to image analysis, where we segmented BM structures such as MSCs, lipid and bone, and classified hematopoietic cells and leukemia cells into 22 cell categories by combining deep-learning/AI-based methods and supervised classification using Visiopharm software. We determined the distances between any two cells (cell-cell relationships, e.g. AML cells and T-cells) or the distances from any one cell to each area class (cell-structure relationships, e.g. AML cells and MSCs), which allowed us to calculate spatial enrichment scores and evaluate proximity of each component in the BM.

On average, 25,000 (range 6,700-56,000) cells were analyzed per slide. seqIF detected 36.5 % (10 – 74%) of p53 positive (p53+) cells at diagnosis, and 0.23% (0.1 – 0.5%) at MRD. Clinical blast counts were 40% (22 – 74%) at diagnosis and MRD flow cytometry detected 1.1% (0 – 3.7%), suggesting that in some cases seqIF can detect malignant cells at MRD better than conventional multi-color flow cytometry in TP53 mutated AML. At both diagnosis and MRD, these p53+ cells tend to form clusters, not being randomly distributed throughout the BM. p53+ AML cells were classified using CD14, CD33, CD34, and CD71. p53+/CD71+ cells were more enriched in MRD BM samples compared to p53+/CD33+ cells (27% vs 6.8%). Intriguingly, BMs with p53+/CD71+ enrichment at MRD showed higher CD4/CD8 ratios compared to BMs at diagnosis collected from the same patients (0.68 vs 2.4). Moreover, cell-cell relationship analyses revealed that p53+/CD71+ AML cells are associated with a lower frequency of CD8+ T-cells compared to random distribution of all hematopoietic cells, suggesting that p53+/CD71+ cells are spatially sequestered from anti-AML immune cells. Cell-structure analyses revealed that the area of CD90+ MSCs is smaller than the CD105+ MSC area, consistent with previous reports (Cell 2024;187:1-21). In some cases, p53+ cells are not uniformly distributed in the BM but are proximal to specific BM niches such as CD90 or CD105 areas, suggesting that these specific MSCs support AML cell maintenance.

In conclusion, novel seqIF technique and spatial analytical approaches revealed novel MRD clusters and spatial characteristics of p53 mutated AML. Additional studies to characterize cell-cell and cell-structure interactions are ongoing.