Niche-Targeted Clinical Grade Immunohistochemistry Reveals Heterogeneous Bone Marrow Neuropathy and Novel Stromal Patterns in Primary Myelofibrosis Patients

Regulation of the hematopoietic stem cell (HSC) niche includes elements of the nervous system, immune system as well as the vasculature and associated stroma. Evidence in murine models demonstrate microenvironmental influence on HSC and hematopoiesis however correlative clinical and translational data remain scarce. The sympathetic nervous system may contribute to the pathogenesis of myeloproliferative neoplasms (MPNs) mechanistically through effects on nestin+ mesenchymal stromal/stem cells. Temporally, bone marrow neuropathy may even precede overt MPN by promoting aberrant premalignant HSC expansion, at least in genetically engineered MPN mouse models. We hypothesize that clinical characterization of the bone marrow niche via conventional immunohistochemistry (IHC) on routine diagnostic trephine biopsies is feasible and may yield novel stromal architectural data supplementing classification and/or prognostic stratification of BCR-ABL1 negative MPN. Herein, we describe our initial results on normal bone marrows and patients with primary myelofibrosis (PMF) with a focus on the pan-neuronal marker (PGP9.5) and the stromal marker smooth muscle actin (SMA). Results: Bone marrow neuropathy, as assessed by loss of punctate or linear perivascular PGP9.5 reactivity around arterioles is highly variable at initial PMF diagnosis. This suggests that in PMF, bone marrow neuropathy may not be universal and if present may only occur in a subset of patients or affect only focal regions of the marrow. SMA staining reproducibly yielded 3 patterns of reactivity in PMF: paratrabecular, perivascular and interstitial. While perivascular SMA staining was observed in all cases of PMF, the interstitial SMA pattern exhibited both inter-patient and intra-patient variability. Intriguingly, SMA was capable of highlighting "hot" and "cold" spots in PMF marrows otherwise appearing homogeneous by conventional reticulin staining. These SMA patterns are presently being investigated in the context of clinical information and molecular profiling to further assess their prognostic relevance. From longitudinal biopsies, we show resolution of the PMF interstitial SMA pattern as early as 2 months post allo-hematopoietic stem cell transplant, far earlier than any reduction in reticulin fibrosis which suggests that SMA signal may have important biological implications - including as a biomarker of early response to JAK inhibition, superior to standard reticulin/collagen assessment. In summary, visualization of the bone marrow niche by routine clinical grade IHC is feasible and reveals stromal heterogeneity within WHO-defined PMF of potential interest for classification refinement, prognostication and therapeutic monitoring.