Transcriptional Characterization of Myelofibrotic Bone Marrow Microenvironment Reveals Distinct Tumor Microenvironment in JAK2⁺ and Calr⁺ PMF Marrows

Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status.

Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84).

Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F⁺ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR⁺ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation.

Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2⁺ or CALR⁺). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis.

Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339.