Pathology of the idiopathic pulmonary fibrosis-facts and controversies Pathologists, radiologists and pulmonologists together for the patients… Anca Maria Cimpean, Marius Raica Department of Microscopic Morphology/Histology, Angiogenesis Research Center “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

Idiopathic pulmonary fibrosis

Lung interstitium

Lung interstitium histology…brief, neglected but important for IPF Known Neglected Cellularity - fibroblasts, fibrocytes and macrophages Fibers: reticulin, elastic, collagen ECM components Blood and lymphatic networks MAST CELLS? RETICULIN? GROWTH FACTORS? HOW LYMPHATIC VESSELS ARE INVOLVED

Blood and lymphatic vessels One shot , multiple targets! Lung alveoli Interalveolar CN Blood and lymphatic vessels Cellular milieu

Using ‘guidelines’ for microscopic diagnosis of IPF Where do we are?

How often SLB is performed? Diagnostic approach Italy, Belgium, Greece, Spain, United States and Finland: 28– 38% of the cases Therapy

IPF classification HRCT vs histopathology 2011 IPF classification HRCT vs histopathology 2013

HISTOPATHOLOGICAL CRITERIA FOR UIP MORE “ABSENCE” THAN “PRESENCE” FOR UIP

Histopathologic Pattern Diagnosis of IPF by Lung Biopsy Histopathologic Pattern UIP Probable UIP Possible UIP Not UIP Not performed IPF Not IPF +/- IPF Inconsistent with UIP Radiologic Pattern Raghu G, et al. Am J Respir Crit Care Med. 2011;183:788-824. Wells: The revised ATS/ERS/JRS/ALAT diagnostic criteria for idiopathic pulmonary fibrosis (IPF) - practical implications. Respiratory Research 2013 14(Suppl 1):S2.

Weaknesses General considerations only… Incomplete criteria for cellular assessement Fibroblastic foci- present but incomplete… “Marked fibrosis”-does this notion could help the patient? Angiogenesis and lymphangiogenesis No molecular data stated in the current classification

KNOWN UNKNOWN FACES OF IPF PATHOLOGY

Usual Interstitial Pneumonia Normal Lung Usual Interstitial Pneumonia

2015 Heterogeneous phenotype of fibroblasts Myofibroblasts behaviour and function Macrophages –same but…different Mast cells 2015 Microscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis., DePianti et al, Thorax 2015 Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression. Wygrecka et al Am J Pathol. 2013 

Reticulin fibers The MMP-3 enzyme degrades collagen types II, III, IV, IX, and X,proteoglycans, fibronectin, laminin, and elastin

I II III IV

Immunohistochemistry Morphologic and immunohistochemical algorithm of IPF assessement Immunohistochemistry IPF III IPF IV Keratin Bronchiolar epithelium, isolated alveolar cells Vimentin +++ in fibrotic area +, fibrotic area with heterogeneous distribution SMAct +++ Myofibroblasts +++ Desmin +++ in nodular fibrotic areas focal in septal regions highly proliferative myofibroblasts VAD Vim-Act-Des) Negative Ki 67 <1 % alveolar epithelium 5% in fibrotic area <1% fibrotic areas FGF + ++ FGFR 1 FGFR 3 PDGF + in nodular areas , vessels, bronchiolar epithelium and myofibroblasts Negativ PDGFR VEGF-A + bronchiolar epithelium +macrophages VEGFR 2 + fibrotic area, vessels + macrophages, negative in fibrotic areas D2-40 IPF III IPF IV Morphology Col.HE Trichrome stain Silver staining Severe alteration of lung histology Massive distorsion of lung architecture Homogeneous and/or heterogeneous collagen fibers Nodular arrangements of collagen bundles Reticulin fibers. Fragmented or absent on extensive areas Completely absent

Fibroblastic foci Controversial regarding their impact on prognosis and therapy Their simple microscopic identification-not enough The quantitative %FF score was a significant predictor of survival in IPF patients Enomoto et al, Chest, 2006

Fibroblastic foci and HRCT

A minute lesion of alveolar damage A minute lesion of alveolar damage. Alveolar epithelial cells have disappeared from the surface of the alveoli (arrows), leaving cytokeratin‐positive cell debris. B, Mild extravasation and mild intra‐alveolar oedema, probably resulting from lung injury. C1, Some of the alveolar macrophages are positive for hypoxia‐inducible factor‐2α. C2, Some of the alveolar macrophages are positive for tumour necrosis factor‐α. D, Accumulation of CD15‐positive cells in alveolar spaces. Most of them are neutrophils. Scale bars: 30 μm (A–C); 50 μm (D). A,C1,C2,D, Immunohistochemistry for AE1/AE3 (A), hypoxia‐inducible factor‐2α (C1), tumour necrosis factor‐α (C2), and CD15 (D). 23

Angiogenesis and lymphangiogenesis in IPF Angiogenesis: pros and cons Lymphangiogenesis?

IPF concept: “lung scar” or never healing wound…. Similarities with defective scars or tumors Same growth factors involved Epithelial to mesenchymal transition… Reactive changes of blood and lymphatic vessels

How should we improve? Use of SLB for any suspicious HRCT image of IPF Reevaluation of microscopic criteria Therapy based on multimodal approach of the disease