Clinical Molecular Imaging of Pulmonary CXCR4 Expression to Predict Outcome of Pirfenidone Treatment in Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive disease for which two antifibrotic drugs recently were approved. However, an unmet need exists to predict responses to antifibrotic treatment, such as pirfenidone. Recent data suggest that upregulated expression of CXCR4 is indicative of outcomes in IPF.Research QuestionCan quantitative, molecular imaging of pulmonary CXCR4 expression as a biomarker for disease activity predict response to the targeted treatment pirfenidone and prognosis in patients with IPF?Study Design and MethodsCXCR4 expression was analyzed by immunohistochemistry examination of lung tissues and reverse-transcriptase polymerase chain reaction analysis of BAL. PET-CT scanning with the specific CXCR4 ligand 68Ga-pentixafor was performed in 28 IPF patients and compared with baseline clinical characteristics. In 16 patients, a follow-up scan was obtained 6 to 12 weeks after initiation of treatment with pirfenidone. Patients were followed up in our outpatient clinic for ≥ 12 months.ResultsImmunohistochemistry analysis showed high CXCR4 staining of epithelial cells and macrophages in areas with vast fibrotic remodeling. Targeted PET scanning revealed CXCR4 upregulation in fibrotic areas of the lungs, particularly in zones with subpleural honeycombing. Baseline CXCR4 signal demonstrated a significant correlation with Gender Age Physiology stage (r = 0.44; P = .02) and with high-resolution CT scan score (r = 0.38; P = .04). Early changes in CXCR4 signal after initiation of pirfenidone treatment correlated with the long-term course of FVC after 12 months (r = −0.75; P = .0008). Moreover, patients with a high pulmonary CXCR4 signal on follow-up PET scan after 6 weeks into treatment demonstrated a statistically significant worse outcome at 12 months (P = .002). In multiple regression analysis, pulmonary CXCR4 signal on follow-up PET scan emerged as the only independent predictor of long-term outcome (P = .0226).InterpretationCXCR4-targeted PET imaging identified disease activity and predicted outcome of IPF patients treated with pirfenidone. It may serve as a future biomarker for personalized guidance of antifibrotic treatment. Idiopathic pulmonary fibrosis (IPF) is a progressive disease for which two antifibrotic drugs recently were approved. However, an unmet need exists to predict responses to antifibrotic treatment, such as pirfenidone. Recent data suggest that upregulated expression of CXCR4 is indicative of outcomes in IPF. Can quantitative, molecular imaging of pulmonary CXCR4 expression as a biomarker for disease activity predict response to the targeted treatment pirfenidone and prognosis in patients with IPF? CXCR4 expression was analyzed by immunohistochemistry examination of lung tissues and reverse-transcriptase polymerase chain reaction analysis of BAL. PET-CT scanning with the specific CXCR4 ligand 68Ga-pentixafor was performed in 28 IPF patients and compared with baseline clinical characteristics. In 16 patients, a follow-up scan was obtained 6 to 12 weeks after initiation of treatment with pirfenidone. Patients were followed up in our outpatient clinic for ≥ 12 months. Immunohistochemistry analysis showed high CXCR4 staining of epithelial cells and macrophages in areas with vast fibrotic remodeling. Targeted PET scanning revealed CXCR4 upregulation in fibrotic areas of the lungs, particularly in zones with subpleural honeycombing. Baseline CXCR4 signal demonstrated a significant correlation with Gender Age Physiology stage (r = 0.44; P = .02) and with high-resolution CT scan score (r = 0.38; P = .04). Early changes in CXCR4 signal after initiation of pirfenidone treatment correlated with the long-term course of FVC after 12 months (r = −0.75; P = .0008). Moreover, patients with a high pulmonary CXCR4 signal on follow-up PET scan after 6 weeks into treatment demonstrated a statistically significant worse outcome at 12 months (P = .002). In multiple regression analysis, pulmonary CXCR4 signal on follow-up PET scan emerged as the only independent predictor of long-term outcome (P = .0226). CXCR4-targeted PET imaging identified disease activity and predicted outcome of IPF patients treated with pirfenidone. It may serve as a future biomarker for personalized guidance of antifibrotic treatment. Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by progressive respiratory failure, resulting in a median survival of less than 3 years after diagnosis in untreated patients.1King Jr., T.E. Pardo A. Selman M. Idiopathic pulmonary fibrosis.Lancet. 2011; 378: 1949-1961Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar, 2Raghu G. Collard H.R. Egan J.J. et al.An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.Am J Respir Crit Care Med. 2011; 183: 788-824Crossref PubMed Scopus (4813) Google Scholar, 3Lederer D.J. Martinez F.J. Idiopathic pulmonary fibrosis.N Engl J Med. 2018; 379: 797-798PubMed Google Scholar However, interindividual outcome shows great variability. Recently, two antifibrotic drugs, pirfenidone and nintedanib, have been approved for treatment of IPF.4King Jr., T.E. Bradford W.Z. Castro-Bernardini S. et al.A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2083-2092Crossref PubMed Scopus (2087) Google Scholar,5Richeldi L. du Bois R.M. Raghu G. et al.Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2071-2082Crossref PubMed Scopus (2304) Google Scholar For both drugs, it has been demonstrated clearly that they attenuate the mean rate of FVC decline over a 12-month period in patients with IPF.4King Jr., T.E. Bradford W.Z. Castro-Bernardini S. et al.A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2083-2092Crossref PubMed Scopus (2087) Google Scholar,5Richeldi L. du Bois R.M. Raghu G. et al.Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2071-2082Crossref PubMed Scopus (2304) Google Scholar Some patients show stable or even improved FVC values after 12 months of treatment, whereas others deteriorate rapidly despite antifibrotic treatment.1King Jr., T.E. Pardo A. Selman M. Idiopathic pulmonary fibrosis.Lancet. 2011; 378: 1949-1961Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar,3Lederer D.J. Martinez F.J. Idiopathic pulmonary fibrosis.N Engl J Med. 2018; 379: 797-798PubMed Google Scholar Although FVC is a well-accepted primary end point in clinical trials, it needs to be monitored for a 12-month period to draw statistically significantly conclusions. Currently, no markers are available that allow the prediction of long-term response to any of the approved antifibrotic treatments as soon as treatment is started or as treatment progresses. Recently, the role of CXCR4 and its ligand CXCL12 (also known as SDF-1) in organ fibrosis has been highlighted.6Yuan A. Lee Y. Choi U. Moeckel G. Karihaloo A. Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors.Am J Physiol Renal Physiol. 2015; 308: F459-F472Crossref PubMed Scopus (34) Google Scholar The G-protein-coupled receptor CXCR4 has a crucial role in processes relying on cell migration, such as recruitment and homing of cells as well as metastatic spread.7Zlotnik A. Burkhardt A.M. Homey B. Homeostatic chemokine receptors and organ-specific metastasis.Nat Rev Immunol. 2011; 11: 597-606Crossref PubMed Scopus (372) Google Scholar,8Burger J.A. Kipps T.J. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment.Blood. 2006; 107: 1761-1767Crossref PubMed Scopus (951) Google Scholar CXCR4 is expressed in various immune, cancer, stem, and progenitor cells.8Burger J.A. Kipps T.J. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment.Blood. 2006; 107: 1761-1767Crossref PubMed Scopus (951) Google Scholar Several studies have reported CXCR4 expression of monocyte-derived M2 macrophages and have described the role of CXCR4 in homing of bone marrow-derived cells to injured organs.9Bertran E. Caja L. Navarro E. et al.Role of CXCR4/SDF-1 alpha in the migratory phenotype of hepatoma cells that have undergone epithelial-mesenchymal transition in response to the transforming growth factor-beta.Cell Signal. 2009; 21: 1595-1606Crossref PubMed Scopus (63) Google Scholar, 10Hughes R. Qian B.Z. Rowan C. et al.Perivascular M2 macrophages stimulate tumor relapse after chemotherapy.Cancer Res. 2015; 75: 3479-3491Crossref PubMed Scopus (234) Google Scholar, 11Chatterjee M. von Ungern-Sternberg S.N. 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Ozier A. et al.Blood fibrocytes are recruited during acute exacerbations of chronic obstructive pulmonary disease through a CXCR4-dependent pathway.J Allergy Clin Immunol. 2016; 137: 1036-1042 e1037Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Additional studies have reported reciprocal and amplifying interactions of the transforming growth factor β (TGF-β) and CXCR4 signaling pathways.6Yuan A. Lee Y. Choi U. Moeckel G. Karihaloo A. Chemokine receptor Cxcr4 contributes to kidney fibrosis via multiple effectors.Am J Physiol Renal Physiol. 2015; 308: F459-F472Crossref PubMed Scopus (34) Google Scholar,9Bertran E. Caja L. Navarro E. et al.Role of CXCR4/SDF-1 alpha in the migratory phenotype of hepatoma cells that have undergone epithelial-mesenchymal transition in response to the transforming growth factor-beta.Cell Signal. 2009; 21: 1595-1606Crossref PubMed Scopus (63) Google Scholar,13Feng Y.F. Yuan F. Guo H. Wu W.Z. TGF-beta1 enhances SDF-1-induced migration and tube formation of choroid-retinal endothelial cells by up-regulating CXCR4 and CXCR7 expression.Mol Cell Biochem. 2014; 397: 131-138Crossref PubMed Scopus (22) Google Scholar,14Bertran E. Crosas-Molist E. Sancho P. et al.Overactivation of the TGF-beta pathway confers a mesenchymal-like phenotype and CXCR4-dependent migratory properties to liver tumor cells.Hepatology. 2013; 58: 2032-2044Crossref PubMed Scopus (99) Google Scholar In various organs, TGF-β is considered to be the master cytokine driving fibrosis and to be related directly to disease activity.1King Jr., T.E. Pardo A. Selman M. Idiopathic pulmonary fibrosis.Lancet. 2011; 378: 1949-1961Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar,3Lederer D.J. Martinez F.J. Idiopathic pulmonary fibrosis.N Engl J Med. 2018; 379: 797-798PubMed Google Scholar,15Khalil N. Parekh T.V. O'Connor R. et al.Regulation of the effects of TGF-beta 1 by activation of latent TGF-beta 1 and differential expression of TGF-beta receptors (T beta R-I and T beta R-II) in idiopathic pulmonary fibrosis.Thorax. 2001; 56: 907-915Crossref PubMed Scopus (133) Google Scholar,16Selman M. King T.E. Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med. 2001; 134: 136-151Crossref PubMed Scopus (1480) Google Scholar Until now, it has been difficult to gauge levels of pulmonary TGF-β production by noninvasive biomarkers. Our own data indicate that CXCR4 expression of BAL cells is indicative of early mortality in IPF.17Prasse A. Binder H. Schupp J.C. et al.BAL cell gene expression is indicative of outcome and airway basal cell involvement in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2019; 199: 622-630Crossref PubMed Scopus (38) Google Scholar Hence, we investigated molecular imaging of CXCR4 expression in IPF as a noninvasive biomarker for disease activity and predictive value. For this purpose, we used a highly specific tracer, 68Ga-pentixafor, which recently was introduced for clinical PET imaging of CXCR4 in cardiovascular and oncologic diseases.18Philipp-Abbrederis K. Herrmann K. Knop S. et al.In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma.EMBO Mol Med. 2015; 7: 477-487Crossref PubMed Scopus (129) Google Scholar, 19Thackeray J.T. Derlin T. Haghikia A. et al.Molecular imaging of the chemokine receptor CXCR4 after acute myocardial infarction.JACC Cardiovasc Imaging. 2015; 8: 1417-1426Crossref PubMed Scopus (108) Google Scholar, 20Derlin T. Sedding D.G. Dutzmann J. et al.Imaging of chemokine receptor CXCR4 expression in culprit and nonculprit coronary atherosclerotic plaque using motion-corrected [(68)Ga]pentixafor PET/CT.Eur J Nucl Med Mol Imaging. 2018; 45: 1934-1944Crossref PubMed Scopus (40) Google Scholar Immunohistochemistry analysis for CXCR4 was obtained from the lung tissue of 10 IPF lung explants and three healthy donors, as recently described, using an antibody against CXCR4 (no. ab124824; Abcam).17Prasse A. Binder H. Schupp J.C. et al.BAL cell gene expression is indicative of outcome and airway basal cell involvement in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2019; 199: 622-630Crossref PubMed Scopus (38) Google Scholar,21Demmer O. Dijkgraaf I. Schumacher U. et al.Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4.J Med Chem. 2011; 54: 7648-7662Crossref PubMed Scopus (69) Google Scholar Microarray data for CXCR4 expression of BAL cells were obtained from a recently published dataset consisting of 62 patients with IPF and 20 age-matched healthy volunteers.17Prasse A. Binder H. Schupp J.C. et al.BAL cell gene expression is indicative of outcome and airway basal cell involvement in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2019; 199: 622-630Crossref PubMed Scopus (38) Google Scholar For the purpose of the current study, we used only the normalized gene expression values for CXCR4 of the Freiburg cohort. BAL cells from 16 of the 28 IPF patients who were recruited into the study later were harvested at initial diagnosis during routine diagnostic workup. BAL cells were isolated as recently described,17Prasse A. Binder H. Schupp J.C. et al.BAL cell gene expression is indicative of outcome and airway basal cell involvement in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2019; 199: 622-630Crossref PubMed Scopus (38) Google Scholar,22Prasse A. Pechkovsky D.V. Toews G.B. et al.A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via CCL18.Am J Respir Crit Care Med. 2006; 173: 781-792Crossref PubMed Scopus (303) Google Scholar harvested in QIAzol lysis reagent (no. 79306; Qiagen), and archived at −80°C. RNA was isolated by RNeasy Mini Kit (no. 74104; Qiagen) in accordance with the standard protocol provided by the manufacturer. Total RNA were reverse transcribed to complementary DNA by using the High-Capacity cDNA Reverse Transcription Kit (no. 4368814; ThermoFisher Scientific). CXCR4 reverse-transcriptase polymerase chain reaction (RT-PCR) analyses were performed on a TaqMan platform with the following TaqMan primers: CXCR4 (assay identifier: Hs00976734_m1; no. 4331182; ThermoFisher Scientific) and glyceraldehyde 3-phosphate dehydrogenase (assay identifier: Hs99999905_m1; no. 4331182; ThermoFisher Scientific) as described recently.23Huppertz C. Jager B. Wieczorek G. et al.The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.Eur Respir J. 2020; 55: 1900119Crossref PubMed Scopus (19) Google Scholar A cycle threshold value was calculated and used to ascertain the relative level of CXCR4 messenger RNA by the following formula: relative expression = (2 (cycle threshold of glyceraldehyde 3-phosphate dehydrogenase - cycle threshold of CXCR4)) × 10,000 for each complementary DNA sample. For the analysis of in vitro modulation of CXCR4 expression, BAL cells from 10 additional patients with IPF were isolated as described recently22Prasse A. Pechkovsky D.V. Toews G.B. et al.A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via CCL18.Am J Respir Crit Care Med. 2006; 173: 781-792Crossref PubMed Scopus (303) Google Scholar: 1 × 106 BAL cells were cultured in Roswell Park Memorial Institute 1640 medium (no. 11879020; ThermoFisher Scientific) supplemented with 10% fetal bovine serum (no. TMS-013-B; Sigma-Aldrich) and 1% penicillin and streptomycin (no. A2212; Biochrom) for 24 h in an incubator (5% CO2, 37°C) with or without 2 mM pirfenidone (no. sc-203663; Santa Cruz Biotechnology) or 10 μM SB-431542 (a selective and potent inhibitor of the TGF-β/activin/nodal pathway; no. 72234; STEMCELL technologies) treatment. CXCR4 expression was tested by RT-PCR as described. In additional experiments, we tested in vitro stimulation of monocyte-derived macrophages and lung tissue slices by TGF-β. Using Ficoll gradient and MACS beads (no. 130-091-153; Miltenyi Biotec), we isolated monocytes from peripheral blood of five additional patients with IPF as indicated by the manufacturer. Monocyte-derived macrophages were generated by stimulating isolated monocytes for five days with granulocyte-macrophage colony-stimulating factor (1 ng/mL; no. 11343123; Immunotools) in Roswell Park Memorial Institute 1640 medium supplemented with 10% human serum, 100,000 U/L penicillin and 100 mg/L streptomycin. Thereafter, cells were stimulated for 48 h with recombinant TGF-β (2 ng/mL; Immunotools) in Roswell Park Memorial Institute 1640 medium supplemented with 100,000 U/L penicillin and 100 mg/L streptomycin; after this, surface CXCR4 expression (CD184 PE; no. 130-098-354; Miltenyi Biotec) was analyzed by flow cytometry using protocols as described.22Prasse A. Pechkovsky D.V. Toews G.B. et al.A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via CCL18.Am J Respir Crit Care Med. 2006; 173: 781-792Crossref PubMed Scopus (303) Google Scholar,24Pechkovsky D.V. Prasse A. Kollert F. et al.Alternatively activated alveolar macrophages in pulmonary fibrosis-mediator production and intracellular signal transduction.Clin Immunol. 2010; 137: 89-101Crossref PubMed Scopus (207) Google Scholar Precision-cut lung slices were obtained from three lung explants of patients with fibrotic lung diseases, as recently described, and were cultured in vitro for 28 days in 1:1 bronchial epithelial cell growth medium (BEGM BulletKit medium [no. CC-3170;Lonza] and Gibco DMEM-Dulbecco's Modified Eagle Medium [no. 11965092; ThermoFisher Scientific]) and stimulated with or without TGF-β (2 ng/mL; Immunotools).25Hess A. Wang-Lauenstein L. Braun A. et al.Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.Toxicol In Vitro. 2016; 32: 347-361Crossref PubMed Scopus (23) Google Scholar Medium was replaced every other day. After 28 days, precision-cut lung slices were harvested, formalin fixed, and paraffin embedded. Immunohistochemistry analysis for CXCR4 expression of precision-cut lung slices materials was performed as described above. Twenty-eight patients (Table 1) were diagnosed with IPF according to the 2018 diagnostic criteria set forth together by the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Society2Raghu G. Collard H.R. Egan J.J. et al.An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.Am J Respir Crit Care Med. 2011; 183: 788-824Crossref PubMed Scopus (4813) Google Scholar,26Raghu G. Remy-Jardin M. Myers J.L. et al.Diagnosis of idiopathic pulmonary fibrosis. An official ATS/ERS/JRS/ALAT clinical practice guideline.Am J Respir Crit Care Med. 2018; 198: e44-e68Crossref PubMed Scopus (1262) Google Scholar,27American Thoracic SocietyIdiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS) and the European Respiratory Society (ERS).Am J Respir Crit Care Med. 2000; 161: 646-664PubMed Google Scholar and underwent baseline 68Ga-pentixafor PET. All patients were examined during their initial clinical workup, were treatment-naïve, and gave written consent to participate in studies of the German Center for Lung Research (DZL) and, in particular, in studies investigating biomarkers of disease progression and treatment outcomes in IPF. The local institutional review board approved the data analysis. None of the patients were being administered steroid or immunosuppressant therapy at the time of imaging. Pulmonary function tests, 6-min walk test, and capillary blood gas analysis were performed within four weeks before 68Ga-pentixafor PET scanning and during routine clinical follow-up every three months. Noninvasive PET imaging was obtained for clinical purposes, and 68Ga-pentixafor was used according to §13.2b of the German Pharmaceuticals Act. In 16 patients, follow-up imaging was obtained 6 to 12 weeks after initiation of treatment with pirfenidone. The 12 remaining patients were treated with nintedanib. Preliminary imaging and in vitro data in five patients suggested that nintedanib induced CXCR4 upregulation in the context of TGF-β signaling. Because of this observation, we did not perform further PET scans in these patients. For pirfenidone treatment, we applied a dose-escalating regimen and started with a dose of 267 mg 3 times daily for 7 days, then 574 mg 3 times daily for 7 days, and then escalated to the final dose of 801 mg 3 times daily. All of the 16 patients treated with pirfenidone tolerated the dose of 3 × 801 mg daily until the last follow-up visit. Patients were followed up in our outpatient clinic for ≥ 12 months. None of the patients were lost to follow-up or died. Data are presented as No. (%) or median (interquartile range). Dlco = diffusing lung capacity for carbon monoxide; HRCT = high-resolution CT; Spo2 = oxygen saturation as measured by pulse oximetry; TLC = total lung capacity. Material for synthesis of 68Ga-pentixafor was obtained by Scintomics. Synthesis was performed as described previously28Gourni E. Demmer O. Schottelius M. et al.PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent.J Nucl Med. 2011; 52: 1803-1810Crossref PubMed Scopus (143) Google Scholar,29Martin R. Juttler S. Muller M. Wester H.J. Cationic eluate pretreatment for automated synthesis of [(6)(8)Ga]CPCR4.2.Nucl Med Biol. 2014; 41: 84-89Crossref PubMed Scopus (68) Google Scholar at Hannover Medical School's Department of Nuclear Medicine (Hannover, Germany) using a 68Ge/68Ga generator (ITG Garching) connected to a Scintomics good radiopharmaceutical practice synthesis module. All study data were obtained using a dedicated PET/CT system (Biograph mCT Flow; Siemens) equipped with an extended field-of-view lutetium oxyorthosilicate PET component, a 128-slice spiral CT component, and a magnetically driven table optimized for continuous scanning. The patients received an IV injection of 132 MBq (interquartile range [IQR], 91-158 MBq) of 68Ga-pentixafor. Imaging started with a low-dose nonenhanced helical CT (120 kV; mA modulated; pitch, 1.2; reconstructed axial slice thickness, 5.0 mm) performed for attenuation correction of PET acquisitions. PET images of the entire body then were acquired using continuous bed motion at a speed of 2.0 mm/s for head and neck and 0.5 mm/s for chest and abdomen at 60 min after injection. All studies were reconstructed using time-of-flight and point-spread function information combined with an iterative algorithm (Ultra HD; Siemens Healthcare; settings: 2 iterations; 21 subsets; matrix, 200; zoom, 1.0; Gaussian filter, 5.0). Standardized uptake values of normalized by body weight were calculated from Ultra HD PET images. In addition, noncontrast breath-hold high-resolution CT (HRCT) scanning (120 kV, mA modulated) of the chest was performed to obtain contiguous 1.0-mm cross-sectional slices throughout the thorax. Raw data were reconstructed using the reconstruction kernels B31f and B70f and were displayed at window settings suitable for viewing the lung parenchyma. Transaxial PET, CT, and fused PET/CT images were analyzed in a random order and blinded for further clinical information on a dedicated workstation equipped with a commercial software package (syngo.via VB30A; Siemens Healthcare) by an expert in PET/CT reading (13 years of experience). PET images were evaluated visually for the presence of elevated radiotracer uptake in lung parenchyma in areas of usual interstitial pneumonia pattern on CT scans. Then, 68Ga-pentixafor uptake was quantified using a 3-D volume-of-interest (VOI) technique with isocontour thresholding at a maximum standardized uptake value (SUVmax) threshold of 45%, yielding a mean standardized uptake value (SUVmean) and SUVmax of usual interstitial pneumonia areas. Separate measurements were performed in three subpleural regions in each lung lobe, and values for each region then were averaged for the subsequent statistical analysis. Similar analyses were performed for assessment of tracer uptake in unaffected lung parenchyma without fibrotic changes. Tracer uptake in mediastinal and hilar lymph nodes was assessed in three thoracic lymph node stations using 3-D VOIs with isocontour thresholding and were averaged for the subsequent statistical analysis. In addition, tracer uptake (SUVmean) in the spleen and bone marrow was recorded to evaluate systemic interactions. As a control measurement to address variability between scans, SUVmean of liver (average of three VOIs) and blood pool (average of three VOIs placed inside the superior vena cava) was determined. The end points were not corrected for air volumes or tissue density because we used serial measurements in the same patient and assumed no major changes in air volumes or tissue density in the short observation time. This assumption was supported by stable CT findings. To assess the reproducibility of the PET analysis, the analysis of pulmonary tracer uptake was repeated by a second reader (4 years of experience). Interrater agreement was good to excellent (intraclass correlation coefficient, 0.95 for analysis of SUVmax and 0.88 for analysis of SUVmean), underlining high reproducibility of the image analysis. Baseline and follow-up HRCT findings were scored using a standardized form, as previously described30Lynch D.A. Godwin J.D. Safrin S. et al.High-resolution computed tomography in idiopathic pulmonary fibrosis: diagnosis and prognosis.Am J Respir Crit Care Med. 2005; 172: 488-493Crossref PubMed Scopus (381) Google Scholar: the overall extent of fibrosis (ie, extent of reticulation and honeycombing) was determined for each entire lung using a 5-point scale (0, no involvement; 1, 1%-25% involvement; 2, 26%-50% involvement; 3, 51%-75% involvement; and 4, 76%-100% involvement). Pulmonary function tests were performed routinely with a standard methodology according to the European Respiratory Society and American Thoracic Society recommendations.31Graham B.L. Brusasco V. Burgos F. et al.Executive summary: 2017 ERS/ATS standards for single-breath carbon monoxide uptake in the lung.Eur Respir J. 2017; 49: 16E0016Crossref PubMed Scopus (56) Google Scholar After treatment, decreasing FVC was defined as any reduction in FVC, whereas all other outcomes were considered to be stable. The Gender Age Physiology (GAP) index, a staging system for patients with IPF, was calculated using gender, age, FVC, and diffusing lung capacity for carbon monoxide (Dlco).32Ley B. Ryerson C.J. Vittinghoff E. et al.A multidimensional index and staging system for idiopathic pulmonary fibrosis.Ann Intern Med. 2012; 156: 684-691Crossref PubMed Scopus (660) Google Scholar Continuous variables were expressed as median with IQR. Categorical variables were presented with absolute and relative frequencies. For between-group comparisons of continuous data, P values were calculated by the Student t test or Wilcoxon test, as appropriate. The Pearson correlation coefficient r was used to correlate imaging findings. Tracer uptake in lung lobes was compared using a one-way analysis of variance with Tukey's multiple comparisons test. Multiplicity-adjusted P values were reported. Ex vivo validation data among study groups were analyzed using the paired Student t test. The Fisher exact test was used to test the association between changes in FVC and changes in imaging parameters. Receiver operating characteristic curve analysis was performed to obtain standardized uptake value cutoffs for FVC outcomes. Simple linear regression analysis and multivariate linear regression analysis were performed to identify predictors of long-term outcome in terms of FVC. Survival by CXCR4 expression was compared by Kaplan-Meier method univariate Cox regression. A P value of less than .05 was regarded as statistically significant. Statistical analysis and graph generation were performed using GraphPad Prism version 7 and 8 software (GraphPad Software) and RStudio version 1.2.5033 software (RStudio PBC). In IPF tissue specimens, immunohistochemistry revealed high expression of CXCR4 by macrophages, lymphocytes, and epithelial cells compared with normal tissues (Fig 1A-D). Notably, airway epithelial cells lining honeycomb cysts and epithelial cells covering fibroblast foci showed positive staining results for CXCR4; however, neither the fibroblasts nor endothelial cells showed positive results for CXCR4 expression. In normal lung tissue, a weak CXCR4 expression