Transforming Growth Factor-α Expression in Myeloid Disorders

Dr Simon Kavanagh PaLM, UWA Background

• Transforming Growth Factor-α (TGFα) • shares structural/functional similarities to epidermal growth factor (EGF) • exerts activity on binding to the EGF receptor (EGFR) • dimerisation • activation of downstream signalling • Ras/Raf/MAPK • PI3K/Akt

Okines. Nat. Rev. Clin. Oncol. 2011. • TGFα has several physiological roles • erythropoiesis (murine/chicken) • epidermal development • regulation of bone turnover through osteoclastogenesis

Expression in non-myeloid tissue Expression in myeloid tissue Epithelial cells (gut, kidney, liver, thyroid) Erythroblasts Ductal cells (pancreas, salivary gland) Megakaryocytes Schwann + ganglion cells Eosinophil precursors Smooth muscle Promyelocytes/myelocytes Macrophages (spleen, lung) Osteoclasts + precursors

Yasui, Virchows Archiv A Pathol Anat, 1992 Walz, Blood, 1995 • Abnormalities of TGFα may be pathological • congenital oro-facial clefts • induction of pulmonary fibrosis • expression in tumours (e.g. oesophageal, kidney, melanoma)

• Abnormalities in EGFR signalling are well recognised in malignancy • non-small cell lung cancer • glioblastoma multiforme

Study Aim

To assess TGFα expression in bone marrow biopsies of patients with acute and chronic myeloid malignancies Methods

• Bone marrow trephines were selected, sectioned and stained • TGFα antibody (clone 213-4.4, Thermo Scientific) on BOND RX immunostainer Diagnosis Number (Leica Biosystems, Australia) • haematoxylin counterstain Normal 69 Chronic myeloid leukaemia (CML) 45 • Evaluated by light microscopy Acute myeloid leukaemia (AML) 32 • Olympus BX53 research microscope Myelodysplastic syndromes (MDS) 13 (Olympus Imaging Australia Pty Ltd., Australia) Myeloproliferative neoplasms (MPN) 65 • photographed with Pixera Pro 600ES Systemic mastocytosis (SM) 4 microscope camera (Pixera Corporation, USA) Results

Normal, 600x magnification Normal, 600x magnification TGFα staining of normal bone marrow

Cell type Intensity of TGFα staining Myeloblasts Weak Promyelocytes Weak Mature granulocytes Negative Early erythroid precursors Moderate Late erythroid precursors Strong Megakaryocytes Weak Osteoblasts Moderate Osteoclasts Strong AML

AML, 600x magnification AML, 600x magnification CML

CML-AP, 600x magnification CML-BP, 600x magnification MDS

MDS, 400x magnification MDS, 400x magnification MPN

JAK2 negative ET, 600x magnification Myelofibrosis, 600x magnification SM

SM, 600x magnification SM, 400x magnification Osteoclasts/blasts

400x magnification 400x magnification Osteoclasts/blasts

400x magnification, TGFα stain 400x magnification, TRAP stain TGFα staining in myeloid malignancy

Cell type Intensity of TGFα staining Myeloblasts Weak - Moderate Malignant promyelocytes (APML) Weak Mature granulocytes Negative Early erythroid precursors Moderate Late erythroid precursors Strong Megakaryocytes Weak – Moderate Mast cells Weak Osteoblasts Moderate Osteoclasts Strong Summary

• We confirm TGFα is expressed in normal haematopoiesis • erythroid series • intensity increases with differentiation • megakaryocytes • intensity uniform throughout differentiation • granulocyte series • intensity diminishes with differentiation

• We confirm TGFα is similarly expressed in myeloid malignancies

• Myeloblasts • staining intensity overall more variable than in normal marrow • expression intensity is notably lower in APML than other AML subtypes

• Potential roles as • an immunohistochemical blast marker (e.g. CD34neg blasts) • a therapeutic target, potentially through targeted EGFR inhibition

• While TGFα appears involved in fibroblast growth, we have not identified consistent differences in staining between MPN subtypes • no differences in fibroblast/connective tissue staining to suggest a role in progression to myelofibrosis • no differences in megakaryocyte staining between mature/immature forms or between MPN subtypes • Osteoclasts, which are of myeloid (monocyte/macrophage) origin, are intensely TGFα positive • mononuclear (immature) and multinucleate (mature) forms are similarly stained

• Potential roles as • an immunohistochemical marker to identify immature osteoclasts in bone pathology • e.g. Paget’s disease of bone, renal osteodystrophy, myeloma • a therapeutic target – may allow for modulation of bone remodelling Acknowledgements

• Bob Mirzai • Kathy Heel • Wendy Erber