Immune cell profiling in CML bone marrow by multiplex IHC

Aim/Background: We aimed to characterize the cellular and molecular immunological profiles of chronic myeloid leukemia (CML) patients’ bone marrow (BM) samples and investigate their clinical relevance. Methods: Tissue microarrays (TMA) were constructed from BM biopsy samples of CML patients (n = 59). Using a novel multiplex immunohistochemistry (IHC) method, we stained TMA slides with up to six markers simultaneously from a total of 25 different lymphoid, dendritic, macrophage, leukemic, immune checkpoint and immune cell activation markers. Results were correlated with therapy responses (complete cytogenetic remission, CCyR; major molecular remission, MMR; complete molecular remission, CMR) using Kaplan-Meier analysis. Results: Patients with high vs. low CD3+ T cell levels achieved faster CCyR (median 97 vs. 189 days, p < 0.001), MMR (210 vs. 543 days, p = 0.008) and CMR (462 vs. 1272 days, p = 0.03). Similarly, high CD4+ T helper cell levels associated with faster CCyR (92 vs. 182 days, p < 0.001), MMR (182 vs. 518 days, p < 0.001) and CMR (336 vs. 927 days, p < 0.001). High CD8+ cytotoxic T cell levels correlated only with faster CMR (548 vs. 927 days, p = 0.03). More detailed immunophenotypic analysis of putative exhaustion markers revealed that high levels of CD4+PD1+ and CD4+PD1+TIM3+ T cells correlated with slower CCyR (171 vs. 96 days, p = 0.02 and 239 vs. 98 days, p = 0.01, respectively), MMR (362 vs. 240 days, p = 0.02 and 742 vs. 252 days, p = 0.002, respectively) and CMR (903 vs. 672 days, p = 0.04 and 239 vs. 98 days, p = 0.01, respectively). Similarly, high levels of CD8+PD1+ and CD8+PD1+TIM3+ T cells associated with slower MMR (455 vs. 199 days, p = 0.04 and 909 vs. 277 days, p = 0.001) and CMR (927 vs. 672 days, p = 0.02 and 1969 vs. 741 days, p = 0.01). Conclusions: Multiplex IHC allows detailed characterization of immune cell subtypes and their phenotypes. High levels of CD3+ T cells, including both CD4+ T helper and CD8+ cytotoxic T cells, associated with better therapy responses. However, dysfunctional PD1+ and PD1+TIM3+ T cells correlated with slower remission rates. The analysis of other immune cell subclasses with comprehensive survival and laboratory data is ongoing. Together, the results will clarify the clinical relevance of immunologic profiles in CML patients. Legal entity responsible for the study: Helsinki University Hospital Funding: Academy of Finland Disclosure: All authors have declared no conflicts of interest.