MF is a neoplastic stem cell disorder in which a multipotent hematopoietic stem cell acquires a clonal proliferative advantage, and its progeny inappropriately releases fibrogenic factors into the bone marrow microenvironment, leading to secondary bone marrow fibrosis. The cytokines implicated in the pathogenesis of MF include transforming growth factor β (TGF-β), basic fibroblastic growth factor (b-FGF), and platelet-derived growth factor (PDGF). Some of these cytokines, such as b-FGF and PDGF can act as angiogenic factors. Patients with MF have higher concentrations of circulating vascular endothelial growth factor (VEGF) and b-FGF than do control subjects. In addition, a higher degree of bone marrow angiogenesis has been reported in patients with MF.
Eph receptors form the largest subgroup of receptor tyrosine kinases (RTK). The Ephrins are the ligands of the Ephs and stimulate bi-directional signaling allowing cell movement and shape change. EphA3 is a member of the Eph family and is expressed mainly during fetal development. Aberrant expression of EphA3 is detected in some solid and hematologic tumors. Recently, the expression of EphA3 has been detected in bone samples from subjects with MF. Antibody targeting of EphA3 may therefore constitute a novel approach to treating MF and other hematologic malignancies.
Using well-characterized normal and diseased FFPE bone marrow biopsies, an IHC assay for EphA3 expression, which was developed and validated previously for use in AML and MDS patient screening, was evaluated further for use on MPNs, such as MF, polycythemia vera (PV) and essential thrombocythemia (ET).
This IHC assay for EphA3 expression was validated (intra-assay variability/precision and inter-assay variability/reproducibility established by variance of SHS for serial tissue sections) using MPNs with negative, low, medium and high intensity and/or percent positive expression of EphA3.
A numerical scoring scheme (semi-quantitative simplified H-Score [SHS]) was used by a-board certified anatomic & clinical pathologist to capture tumor & non-tumor (specifically, fibroblast) reactivity of EphA3-specific monoclonal antibody.
A survey of ten (10) normal bone marrow (NLBM), thirty five (35) cases of primary MF, five (5) post PV-MF cases, six (6) PV cases and four (4) cases with ET was made with the validated IHC assay.
For NLBM, there was low level reactivity in a subset of immature-blast cells that represent a small fraction (1-5% or less) of the total population of nucleated cells. There was no reactivity in fibroblasts, if present. Low EphA3 immunoreactivity of NLBM samples per se is consistent with published RT-PCR data.
For MPNs, significant EphA3 expression in both tumor and tumor-associated stromal fibroblasts was noted (not solely in areas of fibrosis, although not all MPN samples studied were in the fibrotic phase).
Interpretation of EphA3 status in these NLBM and MPNs was used to refine the semi-quantitative SHS scheme in anticipation of setting patient sample cut-off. Between 60-70% of MF samples were deemed EphA3+ using this IHC assay for EphA3 expression.
Targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating MF and other MPNs. The Phase 2 component of a clinical study is ongoing in which the activity of KB004, a non-fucosylated anti-EphA3 Humaneered® antibody, will be characterized in disease specific cohorts including AML, MDS, MF and other MPNs.
Locke:QualTek Molecular Labs: Employment. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership.
Asterisk with author names denotes non-ASH members.