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151: SaLUTaRy: Survey of Lung Transplant Rejection
Abstracts Methods and Materials: Whole blood Affymetrix microarrays and plasma iTRAQ proteomic analyses were carried out on 25 cardiac Tx patient samples. CAV diagnoses were made based on blinded quantitative analysis of angiograms, intravascular ultrasound images and clinical chart review. Genomics and proteomics data were assessed by moderated t-tests. Discriminant analyses identified genes and proteins for the combinatorial biomarker panel. Results: Ten genes and 10 proteins that were differentially expressed between the CAV and non-CAV subject samples were used to create the genomic and proteomic biomarker panels, respectively. Classification of test samples showed comparable sensitivities (83%) and specificities (83%) for both panels. Of the noted biomarkers, discriminant analyses identified 5 genes and 5 proteins which together constituted a combinatorial biomarker panel. Internal validation of the combinatorial panel demonstrated improved classification performance (100% sensitivity and 83% specificity) for identification of blood samples with or without CAV. Biomarkers belonging to the combinatorial panel were biologically diverse, but mainly associated with biologically plausible functions relevant to CAV. Conclusions: Whole blood-derived genomic and proteomic biomarkers may provide a minimally-invasive alternative for the diagnosis of CAV. Our results suggest that a multi-platform approach to biomarker panel development has the potential to provide additional classification power than one platform alone. All biomarker panels generated in this work are being evaluated in a prospective observational trial now in progress. 150 Flow Cytometric Parameters of CD8 Lavage Cells in Detection of Acute Cellular Rejection in Lung Transplant D.C. Neujahr,1 A. Mohammed,2 O. Ulukpo,1 A. Pelaez,1 A. Ramirez,1 E.C. Lawrence,1 S.D. Force,3 A.A. Gal,4 C.P. Larsen,3 A.D. Kirk.3 1 Emory University School of Medicine, Atlanta, GA; 2Emory University School of Medicine, Altanta, GA; 3Emory University School of Medicine, Atlanta, GA; 4Emory University School of Medicine, Atlanta, GA. Purpose: The standard for diagnosis of acute cellular rejection (ACR) in lung transplant patients is transbronchial biopsy (TBx). TBx is associated with morbidity and inter-observer reliability. Further, many patients cannot tolerate TBx due to bleeding risk. Methods to detect ACR without the need for TBx would allow for more frequent and widespread use of screening surveillance bronchoscopy with less risk. We performed flow cytometry (FC) of BAL CD8 cells from patients after lung transplant and correlated the ability of 3 T cell panels to predict ACR. Methods and Materials: All studies were approved by the IRB at Emory. We assessed BAL cells by FC in 24 patients who had at least 5 TBx in the first post transplant year. 141 BAL samples were analyzed of which 14 demonstrated ⱖ grade 2 ACR. We calculated the percentage of BAL CD8 cells according to 3 parameters: 1) GranzymeBhi 2) CD38hi HLA-DRhi 3) Bcl-2loKi67hi. We calculated the values for each patient for both normal biopsies (No ACR by ISHLT criteria) and for ⱖ grade 2 ACR (A or B score) evaluated pathologist in a blinded fashion. We then quantified the average frequency of CD8 BAL cells from all normal biopsies and all ACR biopsies. Results: BALs from all normal biopsy specimens showed lower CD8 GranzymeB, and Bcl-2loKi67hi frequencies versus ACR biopsies (16.9% vs. 39.1% for Granzyme B, p⬍.0001 and 5.29% vs. 11.9%, p⬍.01). There was a trend toward increased CD38hiHLA-DRhi cells in patients with ACR (16.2 vs. 23.3, p⫽0.13). GranzymeB had superior performance characteristics than Bcl2lo Ki67hi (Area under ROC curve .81 vs. .69). When episodes of viral pneunonitis were excluded from the analysis, the differences between normal and rejecting biopsies became more pronounced, but still did not reach statistical significance for CD38hi HLADRhi cells. No differences in the 3 panels were seen when analyzing peripheral blood lymphocytes. Conclusions: These data suggest that FC of BAL cells may have diagnostic utility for acute cellular rejection, particularly in patients in whom TBx is contraindicated.
S55 151 SaLUTaRy: Survey of Lung Transplant Rejection I.O. Gordon,1 S. Bhorade,2 W.T. Vigneswaran,3 P.J. McShane,2 E.R. Garrity,2 A.N. Husain.1 1The University of Chicago, Chicago, IL; 2 The University of Chicago, Chicago, IL; 3The University of Chicago, Chicago, IL. Purpose: The International Society for Heart and Lung Transplantation (ISHLT) guidelines on the interpretation of lung rejection in pulmonary allograft biopsies were revised most recently in 2007. The major changes from the 1996 guidelines were that four grades of airway inflammation (grade B) were collapsed to two grades, and that the distinction between active and inactive bronchiolitis obliterans (grade C) was no longer thought to be useful. The goal of our study was to determine how these revisions affect patient management. Methods and Materials: A web-based survey was emailed to pathologists and pulmonologists identified as being part of the lung transplant team at institutions in the United States with active lung transplant programs as determined from the United Network of Organ Sharing website (www. unos.org). Results were analyzed using Microsoft Excel. Results: Key findings were that while 25% and 70% of pulmonologists would give some treatment to asymptomatic patients with AXB1 and AXB2 (1996 classification), respectively, only 50% would for AXB1R (2007 classification). Interestingly, although the majority of pulmonologists would correctly interpret a pathologic diagnosis of lymphocytic bronchiolitis as grade B airway inflammation and treat accordingly, 12% of pulmonologists do not recognize this as grade B rejection. Finally, the 2007 classification does not encourage reporting of an active mononuclear inflammatory infiltrate in bronchiolitis obliterans, but 66% of pulmonologists would treat the patient differently if there were such an infiltrate, and 86% of pathologists would comment on the presence of such an infiltrate anyway. Results of other detailed questions will also be presented. Conclusions: The 2007 guideline of using two grades for airway inflammation may lead to overtreatment of patients with grade B1 rejection and undertreatment of those with grade B2 rejection, indicating that the issue of grading and treating airway inflammation continues to be problematic. Regarding bronchiolitis obliterans, the presence of an active inflammatory infiltrate is important for patient management.
152 Humoral Immune Responses in Acute Phase of Living Double Lobar Lung Transplantation: Donor-Specific IgM Can Predict Onset of Acute Rejection K. Miyoshi, Y. Sano, M. Yamane, S. Toyooka, T. Oto, S. Miyoshi. Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Purpose: Timely diagnosis of acute rejection is sometimes difficult in acute phase of lung transplantation because its clinical manifestation is usually complicated by other inflammatory pathologies. We review our clinical experience and evaluate the correlation between donor specific humoral response in the recipients and clinical onset of acute rejection for its early diagnosis. Methods and Materials: Ten patients who have received living lobar lung transplantation by donation from two individuals were retrospectively analyzed. Five of ten patients developed clinically-diagnosed acute rejection during the first 14 days after transplantation (Rejection group), and the others had no rejection episode during the period (Non-rejection group). The immunoreactivity against each side of donor T cells were daily monitored by detecting donor-specific IgM with a flow cytometric crossmatch technique. Recipient serum samples were incubated with donor lymphocytes and stained with anti-human IgM. T cells were sorted out and the titers of reacted donor-specific IgM were evaluated with flow cytometry. Results: In the Rejection group, the level of IgM for the donors of the affected side grafts started to be significantly higher than that for the donors
S55 151 SaLUTaRy: Survey of Lung Transplant Rejection I.O. Gordon,1 S. Bhorade,2 W.T. Vigneswaran,3 P.J. McShane,2 E.R. Garrity,2 A.N. Husain.1 1The University of Chicago, Chicago, IL; 2 The University of Chicago, Chicago, IL; 3The University of Chicago, Chicago, IL. Purpose: The International Society for Heart and Lung Transplantation (ISHLT) guidelines on the interpretation of lung rejection in pulmonary allograft biopsies were revised most recently in 2007. The major changes from the 1996 guidelines were that four grades of airway inflammation (grade B) were collapsed to two grades, and that the distinction between active and inactive bronchiolitis obliterans (grade C) was no longer thought to be useful. The goal of our study was to determine how these revisions affect patient management. Methods and Materials: A web-based survey was emailed to pathologists and pulmonologists identified as being part of the lung transplant team at institutions in the United States with active lung transplant programs as determined from the United Network of Organ Sharing website (www. unos.org). Results were analyzed using Microsoft Excel. Results: Key findings were that while 25% and 70% of pulmonologists would give some treatment to asymptomatic patients with AXB1 and AXB2 (1996 classification), respectively, only 50% would for AXB1R (2007 classification). Interestingly, although the majority of pulmonologists would correctly interpret a pathologic diagnosis of lymphocytic bronchiolitis as grade B airway inflammation and treat accordingly, 12% of pulmonologists do not recognize this as grade B rejection. Finally, the 2007 classification does not encourage reporting of an active mononuclear inflammatory infiltrate in bronchiolitis obliterans, but 66% of pulmonologists would treat the patient differently if there were such an infiltrate, and 86% of pathologists would comment on the presence of such an infiltrate anyway. Results of other detailed questions will also be presented. Conclusions: The 2007 guideline of using two grades for airway inflammation may lead to overtreatment of patients with grade B1 rejection and undertreatment of those with grade B2 rejection, indicating that the issue of grading and treating airway inflammation continues to be problematic. Regarding bronchiolitis obliterans, the presence of an active inflammatory infiltrate is important for patient management.
152 Humoral Immune Responses in Acute Phase of Living Double Lobar Lung Transplantation: Donor-Specific IgM Can Predict Onset of Acute Rejection K. Miyoshi, Y. Sano, M. Yamane, S. Toyooka, T. Oto, S. Miyoshi. Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. Purpose: Timely diagnosis of acute rejection is sometimes difficult in acute phase of lung transplantation because its clinical manifestation is usually complicated by other inflammatory pathologies. We review our clinical experience and evaluate the correlation between donor specific humoral response in the recipients and clinical onset of acute rejection for its early diagnosis. Methods and Materials: Ten patients who have received living lobar lung transplantation by donation from two individuals were retrospectively analyzed. Five of ten patients developed clinically-diagnosed acute rejection during the first 14 days after transplantation (Rejection group), and the others had no rejection episode during the period (Non-rejection group). The immunoreactivity against each side of donor T cells were daily monitored by detecting donor-specific IgM with a flow cytometric crossmatch technique. Recipient serum samples were incubated with donor lymphocytes and stained with anti-human IgM. T cells were sorted out and the titers of reacted donor-specific IgM were evaluated with flow cytometry. Results: In the Rejection group, the level of IgM for the donors of the affected side grafts started to be significantly higher than that for the donors