ORIGINAL ARTICLE
Protein Expression Profiling Predicts Graft Performance in Clinical Ex Vivo Lung Perfusion Tiago N. Machuca, MD, PhD,∗ † Marcelo Cypel, MD, MSc,∗ † Jonathan C. Yeung, MD, PhD,∗ † Riccardo Bonato, MD,∗ † Ricardo Zamel, PhD,† Manyin Chen, MD,∗ † Sassan Azad, CRA,∗ Michael K. Hsin, MD,∗ † Tomohito Saito, MD,† Zehong Guan, MSc,† Thomas K. Waddell, MD, PhD,∗ † Mingyao Liu, MD, MSc,† and Shaf Keshavjee, MD, MSc∗ † Objectives: To study the impact of ex vivo lung perfusion (EVLP) on cytokines, chemokines, and growth factors and their correlation with graft performance either during perfusion or after transplantation. Background: EVLP is a modern technique that preserves lungs on normothermia in a metabolically active state. The identification of biomarkers during clinical EVLP can contribute to the safe expansion of the donor pool. Methods: High-risk brain death donors and donors after cardiac death underwent 4 to 6 hours EVLP. Using a multiplex magnetic bead array assay, we evaluated analytes in perfusate samples collected at 1 hour and 4 hours of EVLP. Donor lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcome [absence of primary graft dysfunction– (PGD) grade 3]; (II) PGD3: bilateral transplantation with PGD grade 3 anytime within 72 hours; (III) Declined: lungs unsuitable for transplantation after EVLP. Results: Of 50 cases included in this study, 27 were in Control group, 7 in PGD3, and 16 in Declined. From a total of 51 analytes, 34 were measurable in perfusates. The best marker to differentiate declined lungs from control lungs was stem cell growth factor -β [P < 0.001, AUC (area under the curve) = 0.86] at 1 hour. The best markers to differentiate PGD3 cases from controls were interleukin-8 (P < 0.001, AUC = 0.93) and growth-regulated oncogene-α (P = 0.001, AUC = 0.89) at 4 hours of EVLP. Conclusions: Perfusate protein expression during EVLP can differentiate lungs with good outcome from lungs PGD3 after transplantation. These perfusate biomarkers can be potentially used for more precise donor lung selection improving the outcomes of transplantation. Keywords: chemokines, cytokines, lung preservation, lung transplantation, primary graft dysfunction (Ann Surg 2015;261:591–597)
L
ung transplantation is a life-saving treatment for patients with end-stage lung disease. Despite the considerable amount of multiorgan donors available, only a small percentage of them present with lungs that fulfill donation criteria. In the majority of cases, lungs
From the ∗ Toronto Lung Transplant Program, University Health Network; and †Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, University of Toronto, Toronto, Ontario, Canada. Disclosure: This study was approved by the University Health Network Research Ethics Board. This study was funded by the Operating Grant FRN 93740 from Canadian Institutes for Health Research to Dr Keshavjee. Drs Keshavjee, Cypel, and Waddell are founding members of Perfusix Inc, a company that provides ex vivo organ perfusion services. But, the authors declare no conflicts of interest. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.annalsofsurgery.com). Reprints: Shaf Keshavjee, MD, MSc, Toronto Lung Transplant Program, Toronto General Hospital, 200 Elizabeth St, 9N946, Toronto, Ontario, M5G 2C4, Canada. E-mail: [email protected]. C 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright ISSN: 0003-4932/14/26103-0591 DOI: 10.1097/SLA.0000000000000974
Annals of Surgery r Volume 261, Number 3, March 2015
are not recovered during procurement due to poor function.1 Thus, the imbalance between a growing demand from lung transplant candidates versus the limited availability of donor lungs suitable for transplantation generates considerable waitlist mortality.2 Ex vivo lung perfusion (EVLP) is a modern preservation technique that provides a protective milieu (hyperosmolar extracellular solution, perfusion with 40% of donor-predicted cardiac output, protective volume control ventilation with low tidal volumes, gradual warming until normothermia, hourly recruitment maneuvers) for the donor lungs, keeping them functional and stable for periods as long as 12 hours. These advantages have set EVLP as the ideal platform to assess, diagnose, and improve function of injured donor lungs.3 This concept has been successfully translated to clinical practice: highrisk donor lungs subjected to EVLP yielded similar results as lungs that met conservative conventional criteria, thus safely expanding the donor pool.4 Currently, decision making during EVLP is based on physiologic criteria such as pulmonary hemodynamics, pulmonary mechanics, and gas exchange.5 Using these parameters, approximatelly 20% of donor lungs that are perfused for 4 to 6 hours end up being declined for clinical transplantation due to poor physiologic performance.6 Even more clinically relevant is the small percentage of cases that, in spite of presenting favorable physiology during EVLP, develop primary graft dysfunction (PGD) after transplantation. In these circumstances, we anticipate that biomarkers could refine diagnostic accuracy by objectively confirming good prognostic lungs or revealing suboptimal lungs early in the course of EVLP, either avoiding high-risk transplants or selecting those lungs that will require further ex vivo injury-specific treatment before transplantation. Cytokines and chemokines are involved in the pathogenesis of PGD. Even though previous studies have correlated specific proinflammatory cytokines with severe PGD, they were based on either donor samples taken immediately at the end of the cold ischemic time or recipient samples after reperfusion.7,8 Thus, although these markers can provide important information on mechanistic pathways and select potential therapeutic targets, several barriers to clinical translation still exist. The most important and obvious one is its retrospective nature: by the time the biomarker result is made available, the lungs have already been transplanted and the recipient is in the intensive care unit (ICU). In this study we report the cytokine, chemokine, and growth factor profiles in perfusates of lungs submitted to EVLP with intent to transplant. Analyte levels were studied according to different donor lung outcome phenotypes to define biomarkers that can improve the precision of donor lung selection during EVLP.
MATERIAL AND METHODS Study Design The cases included in this study were part of the Human Ex vivo Lung Perfusion Trial, conducted by the Toronto Lung Transplant Program to assess high-risk brain death donor lungs and donor lungs www.annalsofsurgery.com | 591
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from donation after cardiac death.4,6 Our EVLP technique has been described in detail elsewhere.3 Briefly, donor lungs were connected to the EVLP circuit, perfused and ventilated for 4 to 6 hours. Hourly functional assessment included partial pressure of oxygen (PaO2 ) in the perfusate, peak, mean, and plateau airway pressure; dynamic and static compliance; and pulmonary artery pressure. Lungs declined for transplantation demonstrated a PaO2 /FiO2 less than 400 mm Hg or greater than 15% worsening of any of the above parameters over the perfusion time. Donor lungs were divided into 3 groups: (1) Control: lungs accepted after EVLP, allocated for bilateral transplantation, and the recipient presented no PGD grade 3 within 72 hours; (2) PGD3: lungs accepted after EVLP, allocated for bilateral transplantation, and the recipient presented PGD grade 3 within 72 hours; (3) Declined: lungs declined for transplantation after EVLP due to failure to meet functional criteria. Single-lung transplants, lobar transplants, and recipients bridged to transplant with extracorporeal life support were not included in this study. PGD grade 3 was scored according to the International Society for Heart and Lung Transplantation PGD criteria, as PaO2 /FiO2 less than 200 within 72 hours in the presence of radiographic infiltrates.9
Sample Collection/Protein Analysis Perfusate samples were collected at 1 hour and 4 hours of EVLP and stored at −80o C. Perfusate levels of 51 analytes (cytokines, chemokines, and growth factors) were evaluated in 50 μL samples using 3 multiplex kits: Bio-plex Pro-Human Cytokine 27-plex Assay, Bio-plex Pro-Human Cytokine 21-plex Assay, and Bio-plex Pro TGF-β3-plex Assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) (Supplemental table, available online at http://links.lww.com/SLA/A745). Kits were used according to manufacturer’s instructions and read with a Luminex 100 analyzer (Luminex, Austin, CA). Data were extracted with Bio-plex Manager 6.0 (Bio-Rad Laboratories, Mississauga, Ontario, Canada), and the concentration of each analyte (pg/mL) was determined.
of each biomarker to discriminate between groups was assessed with a receiver operating characteristics curve followed by calculation of the area under the curve (AUC).
RESULTS From September 2009 to November 2012, we performed 80 clinical EVLPs. In 22 EVLP cases, the inclusion criteria were not met. In addition, 8 of our earliest cases were excluded because there were no perfusate samples available. The remaining 50 EVLPs were included in this study with 27 Control, 7 PGD3, and 16 Declined cases (Fig. 1). Donor and Recipient variables are summarized in Table 1. The percentage of brain death donors was similar among groups, as well as donor age and P/F ratios. Recipient variables such as age, sex, and indication for transplant were similar between Control and PGD3 cases, although there was a trend of more interstitial pulmonary fibrosis patients in the PGD3 group. There was no case of primary pulmonary arterial hypertension. In the PGD3 group, all 7 cases presented PGD3 at ICU admission. In 2 cases, it persisted as PGD3 at 72 hours, with 1 case requiring veno-venous extracorporeal life support. One additional case persisted as PGD2 at 72 hours, and this patient died because of sepsis at day 7 posttransplant. PGD3 cases presented longer mechanical ventilation requirement (median 5 vs 2 days, P = 0.001) and longer ICU stay (median 7 vs 3 days, P = 0.01) in comparison to Controls.
EVLP Physiology EVLP physiology data are summarized in Figure 2. Briefly, pulmonary artery pressure was similar in all groups. As expected, peak airway pressure, plateau pressure, and PaO2 /FiO2 were significantly worse in Declined lungs than in Controls. Lungs in the PGD3 group presented similar PaO2 /FiO2 , peak airway pressure, and plateau pressure compared to Controls.
Statistical Analysis
Analysis of Cytokines, Chemokines, Growth Factors, and Interferons
Statistical analysis was performed using Graphpad Prism 5 (Graphpad Software Inc, La Jolla, CA). Comparisons for EVLP physiologic parameters among different groups were performed using 2way analysis of variance (time and phenotype). Continuous variables were compared using the nonparametric Mann-Whitney test, and categorical variables were compared with Fisher exact test. The ability
From 51 analytes, readable values were detected in at least 1 time point in 34. For the remaining 17, levels were below the detection limit (Supplemental table, available online at http://links.lww.com/SLA/A745). The highest median fold changes over time with respect to each donor lung phenotype are displayed in Figure 3.
FIGURE 1. Study design. ∗ Eight early cases did not have perfusates available for protein analysis. 592 | www.annalsofsurgery.com
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Annals of Surgery r Volume 261, Number 3, March 2015
Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
TABLE 1. Donor and Recipient Clinical Variables Donor variables Age (yr) BDD (%) Female (%) Best P/F ratio (mm Hg) Recipient variables Age (yr) Female (%) Diagnosis (% IPF) CPB
Control (n = 27)
PGD3 (n = 7)
P
Declined (n = 16)
P
46 56% 52% 335
48 57% 86% 301
0.983 1 0.197 0.582
40 56% 37% 318
0.289 1 0.358 0.370
53 52% 30% 22%
50 71% 71% 57%
0.808 0.426 0.078 0.157
BDD indicates brain death donor; CPB, cardiopulmonary bypass; IPF, interstitial pulmonary fibrosis.
FIGURE 2 . Physiologic assessment of high-risk donor lungs submitted to EVLP (∗ P < 0.05; †P < 0.01; ‡P < 0.001).
FIGURE 3. Highest median fold changes measurable analytes from 1 to 4 hours of EVLP.
The majority of cytokines were not detectable in the perfusate with the reported methodology. Among the detectable ones, interleukin (IL)-6 and IL-10 demonstrated the highest fold changes over time in all 3 groups. (Control, PGD3, and Declined). IL-1β absolute values were very low at 1 hour and remained low at 4 hours of EVLP in all groups. Tumor necrosis factor (TNF)-α and TNF-β were not detectable at either time point. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
Most of the chemokines were detectable in EVLP perfusate. Although IL-8, Eotaxin, and growth-regulated oncogene (GRO)-α presented high fold changes in all 3 groups, increases in macrophage inflammatory protein (MIP)-1α and MIP-1β were more striking in PGD3. Hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1, and stem cell growth factor (SCGF)-β had the highest www.annalsofsurgery.com | 593
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concentrations among growth factors at 1 hour of EVLP. At 4 hours, HGF remained as the highest in concentration. Granulocyte colonystimulating factor (G-CSF) was the growth factor with the highest fold change in all 3 groups. Both interferon (IFN)-γ and IFN-α2 were among the analytes with the lowest concentrations at 1 and 4 hours of EVLP, with minimal changes over time.
a sensitivity of 85.7% and specificity of 92.5% (AUC = 0.93), and with a cutoff of 892 pg/mL, GRO-α had a sensitivity of 85.7% and specificity of 85.1% (AUC = 0.89). G-CSF (7547 ± 3413 vs 3338
PGD3 vs Control
At 1 hour of EVLP, IL-8 (mean ± SE, 723.1 ± 543.6 vs 63.5 ± 11.2 pg/mL, P = 0.026) and macrophage colony-stimulating factor (M-CSF) (48.4 ± 12.8 vs 26.3 ± 2.1 pg/mL, P = 0.028) were significantly higher in PGD3 cases than in Controls (Fig. 4). For IL8, a cutoff of 84 pg/mL had a sensitivity of 71.4% and specificity of 85.1% (AUC = 0.77). For M-CSF, a cutoff of 37 pg/mL rendered a sensitivity of 57.1% and a specificity of 81.4% (AUC = 0.77) (Fig. 5). At 4 hours of EVLP, IL-8 (6195 ± 1456 vs 1746 ± 328.9 pg/mL, P < 0.001) and GRO-α (2031 ± 778.8 vs 519 ± 76.8 pg/mL, P = 0.001) were higher in PGD3 group. These 2 analytes had excellent discrimination with Control, with a cutoff of 3570 pg/mL, IL-8 had
FIGURE 5. Receiver operating characteristics curves for the potential biomarkers for PGD3 lungs in perfusates at 4 hours of EVLP. Numbers in brackets represent the AUC.
FIGURE 4. A, 1-hour analytes significantly higher in PGD 3 group than in Control; B, 4-hour analytes significantly higher in PGD 3 than in Control (∗ P < 0.05; †P < 0.01; ‡P < 0.001). Median and interquartile ranges are highlighted. The 2 cases with persistent PGD 3 are highlighted as orange triangle, whereas the PGD 3 case with early mortality is highlighted as a red triangle. 594 | www.annalsofsurgery.com
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Annals of Surgery r Volume 261, Number 3, March 2015
± 1122 pg/mL, P = 0.008, AUC = 0.83), MIP-1α (88.9 ± 31.2 vs 18.6 ± 5.4, P = 0.009, AUC = 0.82), and MIP-1β (4336 ± 1606 vs 737.1 ± 21.8 pg/mL, P = 0.008, AUC = 0.82) were also significantly higher in PGD3 cases, with a good discrimination versus Control. For IL-8, GRO-α, G-CSF, MIP-1α, and MIP-1β at 4 hours of EVLP, the 3 PGD3 cases previously detailed (2 persistent PGD3 and 1 persisting as PGD2 at 72 hours with mortality at day 7) were always above the proposed cutoff values.
Declined vs Control
In the Declined group, HGF (1935 ± 256.8 vs 1118 ± 88.4 pg/mL, P = 0.007), IL-1 receptor antagonist (ra) (77.6 ± 14.2 vs 40.5 ± 4 pg/mL, P = 0.017), stem cell factor (38.5 ± 6 vs 21.8 ± 2.3 pg/mL, P = 0.020), and SCGF-β (491.4 ± 58.1 vs 255 ± 19.1 pg/mL, P < 0.001) were significantly higher compared to Controls at 1 hour (Fig. 6). The last rendered the best discrimination: with a cutoff at 327 pg/mL, it had 81.2% sensitivity and 81.4% specificity (AUC = 0.86). Similarly to 1 hour of EVLP, HGF (3642 ± 639.9 vs 2242 ± 204.1 pg/mL, P = 0.038), IL-1ra (180.7 ± 35.8 vs 115 ± 17.7 pg/mL, P = 0.043), and SCGF-β (769.4 ± 133.5 vs 389.4 ± 32.5 pg/mL, P = 0.004) were higher in the Declined cases compared to Control at 4 hours of EVLP. Again, SCGF-β provided reasonable discrimination between groups (AUC = 0.77).
DISCUSSION In this study, we explored the temporal trends of cytokine, chemokine, and growth factor production in human donor lungs perfused ex vivo. Moreover, we correlated changes in the levels of these
Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
analytes with post-EVLP clinical outcomes as determined by PGD3 within 72 hours posttransplant or by nonutilization post-EVLP. The comparison of PGD3 cases to Controls is important because EVLP physiology alone was unable to predict the subsequent development of PGD3 in some cases. We found analytes (IL-8, GROα, MIP-1α, MIP-1β, and G-CSF) that were differentially accumulated between these groups. These compounds have biological plausibility as useful biomarkers. For example, IL-8 has been implicated in the pathogenesis of acute lung injury. In this study, IL-8 was the most powerful biomarker for predicting PGD3 cases. It was the only analyte significantly elevated at both 1 hour and 4 hours of EVLP in this group and rendered the best discrimination from Control cases. This corroborates the findings of de Perrot and coworkers, which reported increased levels of this cytokine in lung biopsies taken at the end of cold ischemic time, end of warm ischemic time, and at 1 hour after reperfusion.10 Indeed, they have also shown a correlation of IL-8 at 2 hours after reperfusion with graft function in the ICU. Others have similarly correlated increased levels of IL-8 in bronchoalveolar lavage (BAL) and donor lung biopsy before implantation with development of PGD3.11 In fact, it has been shown in a model of warm ischemia followed by reperfusion in rabbits that intravenous treatment with antibodies against IL-8 successfully reduced BAL neutrophil count and alveolar infiltration of neutrophils on histology, ultimately attenuating lung injury.12 The other analytes, GRO-α, MIP-1α, and MIP-1β, have been previously implicated in the early inflammatory response against skin allografts.13 Specific to the lung, the use of anti-GRO-α antibody was able to reduce myeloperoxidase activity and preserve alveolar-capillary membrane integrity in a model of acute kidney
FIGURE 6. A, 1-hour analytes significantly higher in Declined group than in Control; B, 4-hour analytes significantly higher in Declined group than in Control (∗ P < 0.05; †P < 0.01; ‡P < 0.001). Median and interquartile ranges are highlighted. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
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injury-induced lung injury.14 Moreover, in a model of pulmonary Klebsiella pneumoniae infection, GRO-α was shown to be involved not only in the recruitment of neutrophils but also to regulate its function (production of reactive oxygen species and reactive nitrogen species).15 MIP-1α knockout has been shown to decrease polymorphonuclear cell migration in lipopolysaccharide-induced and bleomycin-induced lung injury. In the first case, it was also capable of reducing lung edema formation.16 Intratracheal administration of MIP-1β was capable of inducing pulmonary capillary leak. Interestingly, in the same study, intratracheal anti-MIP-1β antibody reduced the pulmonary permeability index, the myeloperoxidase activity, and TNF-α in BAL of rats with immune complex-induced lung injury.17 G-CSF has been implicated in ischemia reperfusion lung injury. Kreisel et al18 have demonstrated that G-CSF blockade attenuated ischemia reperfusion in a lung transplantation model with Bcl-3 knockout mice. The same authors have further shown that G-CSF is increased early after reperfusion in grafts, which underwent prolonged (18 hours) cold ischemia and that anti-G-CSF antibody can abrogate its deleterious effects in the alveolar-capillary barrier, improving gas exchange.19 The next finding of this study was generated from comparisons between Control lungs and Declined lungs. Differences at the protein level between these 2 groups would reinforce the accuracy of the physiological assessment. Indeed, differential protein expression was observed and, interestingly, most of the differences noticed at 1 hour of EVLP were again observed at 4 hours. More importantly, SCGFβ was shown to have considerable discrimination power already at 1 hour of perfusion and thus could be used as an early biomarker for prediction of lungs that will perform poorly during EVLP. Once available in a timely fashion, this valuable information can point out which lungs will require additional therapeutic measures early in the EVLP process to rescue them for a safe transplantation. The fact that 4 declined lungs presented values below the proposed cutoff raises the question whether they could have been transplanted. Although this study is unable to provide this answer, these findings can certainly guide the design of future studies to improve diagnostic strategy for donor lung suitability. Analysis of the entire study cohort revealed interesting changes over time in cytokines, chemokines, and growth factors. The most significant fold changes (up to 60x increase from 1 to 4 hours of EVLP) were observed in IL-6, IL-8, and G-CSF. Several details should be taken into account when analyzing these results. First, this technique consists of a recirculated closed circuit, which lacks hepatic and renal clearance mechanisms. It is known from experimental models of bilateral nephrectomy that mediators such as IL-6, IL-1β, IL-10, and GRO-α accumulate after exogenous administration.20 Second, the acellular nature of the perfusate and the ex vivo status may have partly contributed to the homeostatic response of increased hematopoietic growth factors (M-CSF, G-CSF). Third, these are high-risk injured donor lungs and thus a proinflammatory profile is expected. Finally, even though under controlled conditions and ex vivo, there is a reperfusion process, albeit significantly modified. Previous clinical studies have shown that, regardless of the PGD score, IL-6, IL-8, and IL-10 increase in the plasma of patients early after reperfusion.7,8 These temporal trends in perfusate are similar to the ones in lung tissue described by Sadaria and coworkers.21 In a series of lungs procured with a research-only intent, they have shown significant increases in IL-6, IL-8, and G-CSF over time. Although proinflammatory cytokines increased locally, pulmonary physiology was stable for 12 hours, there was no edema formation (wet-to-dry ratio) and lung histology was unremarkable. Along with IL-6 and IL-8, elevated TNF-α and IL-1β mRNA expression before implantation have been previously shown to corre596 | www.annalsofsurgery.com
late with 30-day mortality in lung transplantation.22 Furthermore, our group has similarly reported higher TNF-α and IL-1β in lungs declined for transplantation after on-site assessment (a subset of lungs that currently would be considered for EVLP and thus overlaps with this study).23 Even though we measured perfusate protein levels, these proinflammatory cytokines were either not measurable during the entire EVLP (TNF-α) or had very low concentrations at both time points studied (IL-1β). This finding in metabolically active high-risk lungs is encouraging and may be explained by the protective nature of our current EVLP technique.3 The elevated levels of CXC and CC chemokines in PGD3 group in our study, along with previous experimental data, strongly highlight the involvement of neutrophils and macrophages in the modulation of ischemia reperfusion injury in lung transplantation. The benefits of alveolar macrophage depletion have been previously shown in models of both warm ischemia and cold ischemia reperfusion injury.24,25 Furthermore, in a study using in vivo 2-photon microscopy, Kreisel and coworkers26 explored the crucial interactions between monocytes and neutrophils, ultimately leading to ischemia reperfusion injury in a mouse lung transplantation model. On the basis of existing evidence, the potential advantages of manipulation of neutrophil and alveolar macrophage populations during EVLP certainly deserve further exploration. One of the main limitations of this study resides in the sample size. Although initially considered small, it includes the largest experience to date with perfusion of high-risk donor lungs for clinical transplantation. Another limitation is the inability to study recipient biologic factors related to PGD.7 The simultaneous assessment of biomarkers in recipient and donors has been reported to increase the predictive yield of biomarkers in lung transplantation and should be sought in future studies.27 In terms of clinical recipient and donor factors, we did not find significant differences between Control and PGD3 groups. Furthermore, recipient factors reportedly related to PGD3 such as diagnosis of primary pulmonary arterial hypertension or sarcoidosis and cases of single lung transplantation were not included in our study.28 Finally, the analysis of several analytes may potentially render misleading findings. However, most of the analytes predictive of PGD3 reported herein are well known, highly plausible biomarkers of lung injury. As EVLP gains widespread acceptance, multicenter collaborations will be instrumental for the validation of our findings. In summary, this study reveals changes in cytokines, chemokines, and growth factors at the protein level in high-risk donor lungs perfused ex vivo with clinical transplantation intent. Our findings suggest that adding biomarkers to EVLP assessment can improve the precision of donor lung selection. Besides adding more objectivity to decision making, these biomarkers can guide future studies on therapeutic strategies to safely increase the utilization of injured lungs for transplantation. Barriers to clinical translation can be overcome by the incorporation of already available advanced analytic tools able to provide quantitative results in a short period of time, ideally within minutes.29 Although previous studies reported candidate markers of primary graft dysfunction,8,10,22,30 this study has key advantages: (1) simplicity—it is based on readily available perfusate samples, avoiding potential caveats of lung biopsy including heterogeneity and time needed for sample preparation; (2) timing—at the moment of sample collection, the lungs are in a system capable of stably preserving them for hours3,31 ; and (3) reversibility— since the recipient is not on the operating table yet, the biomarker results can actually assist in the decision-making process. We hope that the combination of physiologic and biomarker assessment during EVLP will soon improve the precision of decision making in donor lung assessment and allow clinicians to safely expand the current donor pool improving outcomes after lung transplantation. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
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Annals of Surgery r Volume 261, Number 3, March 2015
ACKNOWLEDGMENTS For the preparation of this article, authors contributions were as follows: T.N.M.—designed and performed the study, analyzed data, and wrote manuscript; M.C.—designed the study, analyzed data, and wrote manuscript; J.C.Y.—performed the study, analyzed data, and reviewed manuscript; R.B.—performed data acquisition and reviewed the manuscript; R.Z.—analyzed data and reviewed manuscript; M.C.—performed data acquisition and reviewed the manuscript; M.K.H.—performed data acquisition, analyzed data, and reviewed manuscript; T.S.—analyzed and interpreted data, reviewed manuscript; Z.G.—performed the study and data acquisition, reviewed manuscript; T.K.W.—designed the study, analyzed data, and reviewed manuscript; M.L.—designed the study, analyzed data, and reviewed manuscript; S.K.—designed the study, analyzed data, supervised research, and reviewed manuscript. All authors approved the final version of the manuscript. We thank Paul Chartrand for assistance in the EVLP procedures and logistics.
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Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
13. Kondo T, Novick AC, Toma H, et al. Induction of chemokine gene expression during allogeneic skin graft rejection. Transplantation. 1996;61:1750–1757. 14. Ahuja N, Andres-Hernando A, Altmann C, et al. Circulating il-6 mediates lung injury via CXCL1 production after acute kidney injury in mice. Am J Physiol Renal Physiol. 2012;303:F864–F872. 15. Batra S, Cai S, Balamayooran G, et al. Intrapulmonary administration of leukotriene B(4) augments neutrophil accumulation and responses in the lung to Klebsiella infection in CXCL1 knockout mice. J Immunol. 2012;188:3458– 3468. 16. Smith RE, Strieter RM, Phan SH, et al. Production and function of murine macrophage inflammatory protein-1 alpha in bleomycin-induced lung injury. J Immunol. 1994;153:4704–4712. 17. Bless NM, Huber-Lang M, Guo RF, et al. Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats. J Immunol. 2000;164:2650–2659. 18. Kreisel D, Sugimoto S, Tietjens J, et al. Bcl3 prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis. J Clin Invest. 2011;121:265–276. 19. Kreisel D, Sugimoto S, Zhu J, et al. Emergency granulopoiesis promotes neutrophil-dendritic cell encounters that prevent mouse lung allograft acceptance. Blood. 2011;118:6172–6182. 20. Andres-Hernando A, Dursun B, Altmann C, et al. Cytokine production increases and cytokine clearance decreases in mice with bilateral nephrectomy. Nephrol Dial Transplant. 2012;27:4339–4347. 21. Sadaria MR, Smith PD, Fullerton DA, et al. Cytokine expression profile in human lungs undergoing normothermic ex-vivo lung perfusion. Ann Thorac Surg. 2011;92:478–484. 22. Kaneda H, Waddell TK, de Perrot M, et al. Pre-implantation multiple cytokine mRNA expression analysis of donor lung grafts predicts survival after lung transplantation in humans. Am J Transplant. 2006;6:544–551. 23. Cypel M, Kaneda H, Yeung JC, et al. Increased levels of interleukin-1β and tumor necrosis factor-α in donor lungs rejected for transplantation. J Heart Lung Transplant. 2011;30:452–459. 24. Naidu BV, Krishnadasan B, Farivar AS, et al. Early activation of the alveolar macrophage is critical to the development of lung ischemia-reperfusion injury. J Thorac Cardiovasc Surg. 2003;126:200–207. 25. Zhao M, Fernandez LG, Doctor A, et al. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2006;291:L1018–L1026. 26. Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocytedependent neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci U S A. 2010;107:18073–18078. 27. Salama M, Andrukhova O, Hoda MA, et al. Concomitant endothelin-1 overexpression in lung transplant donors and recipients predicts primary graft dysfunction. Am J Transplant. 2010;10:628–636. 28. Diamond JM, Lee JC, Kawut SM, et al. Clinical risk factors for primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med 2013;187:527–534. 29. Cederquist KB, Kelley SO. Nanostructured biomolecular detectors: pushing performance at the nanoscale. Curr Opin Chem Biol. 2012;16:415–421. 30. Shah RJ, Bellamy SL, Localio AR, et al. A panel of lung injury biomarkers enhances the definition of primary graft dysfunction (PGD) after lung transplantation. J Heart Lung Transplant 2012;31:942–949. 31. Cypel M, Rubacha M, Yeung J, et al. Normothermic ex vivo perfusion prevents lung injury compared to extended cold preservation for transplantation. Am J Transplant. 2009;9:2262–2269.
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Protein Expression Profiling Predicts Graft Performance in Clinical Ex Vivo Lung Perfusion Tiago N. Machuca, MD, PhD,∗ † Marcelo Cypel, MD, MSc,∗ † Jonathan C. Yeung, MD, PhD,∗ † Riccardo Bonato, MD,∗ † Ricardo Zamel, PhD,† Manyin Chen, MD,∗ † Sassan Azad, CRA,∗ Michael K. Hsin, MD,∗ † Tomohito Saito, MD,† Zehong Guan, MSc,† Thomas K. Waddell, MD, PhD,∗ † Mingyao Liu, MD, MSc,† and Shaf Keshavjee, MD, MSc∗ † Objectives: To study the impact of ex vivo lung perfusion (EVLP) on cytokines, chemokines, and growth factors and their correlation with graft performance either during perfusion or after transplantation. Background: EVLP is a modern technique that preserves lungs on normothermia in a metabolically active state. The identification of biomarkers during clinical EVLP can contribute to the safe expansion of the donor pool. Methods: High-risk brain death donors and donors after cardiac death underwent 4 to 6 hours EVLP. Using a multiplex magnetic bead array assay, we evaluated analytes in perfusate samples collected at 1 hour and 4 hours of EVLP. Donor lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcome [absence of primary graft dysfunction– (PGD) grade 3]; (II) PGD3: bilateral transplantation with PGD grade 3 anytime within 72 hours; (III) Declined: lungs unsuitable for transplantation after EVLP. Results: Of 50 cases included in this study, 27 were in Control group, 7 in PGD3, and 16 in Declined. From a total of 51 analytes, 34 were measurable in perfusates. The best marker to differentiate declined lungs from control lungs was stem cell growth factor -β [P < 0.001, AUC (area under the curve) = 0.86] at 1 hour. The best markers to differentiate PGD3 cases from controls were interleukin-8 (P < 0.001, AUC = 0.93) and growth-regulated oncogene-α (P = 0.001, AUC = 0.89) at 4 hours of EVLP. Conclusions: Perfusate protein expression during EVLP can differentiate lungs with good outcome from lungs PGD3 after transplantation. These perfusate biomarkers can be potentially used for more precise donor lung selection improving the outcomes of transplantation. Keywords: chemokines, cytokines, lung preservation, lung transplantation, primary graft dysfunction (Ann Surg 2015;261:591–597)
L
ung transplantation is a life-saving treatment for patients with end-stage lung disease. Despite the considerable amount of multiorgan donors available, only a small percentage of them present with lungs that fulfill donation criteria. In the majority of cases, lungs
From the ∗ Toronto Lung Transplant Program, University Health Network; and †Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, University of Toronto, Toronto, Ontario, Canada. Disclosure: This study was approved by the University Health Network Research Ethics Board. This study was funded by the Operating Grant FRN 93740 from Canadian Institutes for Health Research to Dr Keshavjee. Drs Keshavjee, Cypel, and Waddell are founding members of Perfusix Inc, a company that provides ex vivo organ perfusion services. But, the authors declare no conflicts of interest. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.annalsofsurgery.com). Reprints: Shaf Keshavjee, MD, MSc, Toronto Lung Transplant Program, Toronto General Hospital, 200 Elizabeth St, 9N946, Toronto, Ontario, M5G 2C4, Canada. E-mail: [email protected]. C 2014 Wolters Kluwer Health, Inc. All rights reserved. Copyright ISSN: 0003-4932/14/26103-0591 DOI: 10.1097/SLA.0000000000000974
Annals of Surgery r Volume 261, Number 3, March 2015
are not recovered during procurement due to poor function.1 Thus, the imbalance between a growing demand from lung transplant candidates versus the limited availability of donor lungs suitable for transplantation generates considerable waitlist mortality.2 Ex vivo lung perfusion (EVLP) is a modern preservation technique that provides a protective milieu (hyperosmolar extracellular solution, perfusion with 40% of donor-predicted cardiac output, protective volume control ventilation with low tidal volumes, gradual warming until normothermia, hourly recruitment maneuvers) for the donor lungs, keeping them functional and stable for periods as long as 12 hours. These advantages have set EVLP as the ideal platform to assess, diagnose, and improve function of injured donor lungs.3 This concept has been successfully translated to clinical practice: highrisk donor lungs subjected to EVLP yielded similar results as lungs that met conservative conventional criteria, thus safely expanding the donor pool.4 Currently, decision making during EVLP is based on physiologic criteria such as pulmonary hemodynamics, pulmonary mechanics, and gas exchange.5 Using these parameters, approximatelly 20% of donor lungs that are perfused for 4 to 6 hours end up being declined for clinical transplantation due to poor physiologic performance.6 Even more clinically relevant is the small percentage of cases that, in spite of presenting favorable physiology during EVLP, develop primary graft dysfunction (PGD) after transplantation. In these circumstances, we anticipate that biomarkers could refine diagnostic accuracy by objectively confirming good prognostic lungs or revealing suboptimal lungs early in the course of EVLP, either avoiding high-risk transplants or selecting those lungs that will require further ex vivo injury-specific treatment before transplantation. Cytokines and chemokines are involved in the pathogenesis of PGD. Even though previous studies have correlated specific proinflammatory cytokines with severe PGD, they were based on either donor samples taken immediately at the end of the cold ischemic time or recipient samples after reperfusion.7,8 Thus, although these markers can provide important information on mechanistic pathways and select potential therapeutic targets, several barriers to clinical translation still exist. The most important and obvious one is its retrospective nature: by the time the biomarker result is made available, the lungs have already been transplanted and the recipient is in the intensive care unit (ICU). In this study we report the cytokine, chemokine, and growth factor profiles in perfusates of lungs submitted to EVLP with intent to transplant. Analyte levels were studied according to different donor lung outcome phenotypes to define biomarkers that can improve the precision of donor lung selection during EVLP.
MATERIAL AND METHODS Study Design The cases included in this study were part of the Human Ex vivo Lung Perfusion Trial, conducted by the Toronto Lung Transplant Program to assess high-risk brain death donor lungs and donor lungs www.annalsofsurgery.com | 591
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from donation after cardiac death.4,6 Our EVLP technique has been described in detail elsewhere.3 Briefly, donor lungs were connected to the EVLP circuit, perfused and ventilated for 4 to 6 hours. Hourly functional assessment included partial pressure of oxygen (PaO2 ) in the perfusate, peak, mean, and plateau airway pressure; dynamic and static compliance; and pulmonary artery pressure. Lungs declined for transplantation demonstrated a PaO2 /FiO2 less than 400 mm Hg or greater than 15% worsening of any of the above parameters over the perfusion time. Donor lungs were divided into 3 groups: (1) Control: lungs accepted after EVLP, allocated for bilateral transplantation, and the recipient presented no PGD grade 3 within 72 hours; (2) PGD3: lungs accepted after EVLP, allocated for bilateral transplantation, and the recipient presented PGD grade 3 within 72 hours; (3) Declined: lungs declined for transplantation after EVLP due to failure to meet functional criteria. Single-lung transplants, lobar transplants, and recipients bridged to transplant with extracorporeal life support were not included in this study. PGD grade 3 was scored according to the International Society for Heart and Lung Transplantation PGD criteria, as PaO2 /FiO2 less than 200 within 72 hours in the presence of radiographic infiltrates.9
Sample Collection/Protein Analysis Perfusate samples were collected at 1 hour and 4 hours of EVLP and stored at −80o C. Perfusate levels of 51 analytes (cytokines, chemokines, and growth factors) were evaluated in 50 μL samples using 3 multiplex kits: Bio-plex Pro-Human Cytokine 27-plex Assay, Bio-plex Pro-Human Cytokine 21-plex Assay, and Bio-plex Pro TGF-β3-plex Assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) (Supplemental table, available online at http://links.lww.com/SLA/A745). Kits were used according to manufacturer’s instructions and read with a Luminex 100 analyzer (Luminex, Austin, CA). Data were extracted with Bio-plex Manager 6.0 (Bio-Rad Laboratories, Mississauga, Ontario, Canada), and the concentration of each analyte (pg/mL) was determined.
of each biomarker to discriminate between groups was assessed with a receiver operating characteristics curve followed by calculation of the area under the curve (AUC).
RESULTS From September 2009 to November 2012, we performed 80 clinical EVLPs. In 22 EVLP cases, the inclusion criteria were not met. In addition, 8 of our earliest cases were excluded because there were no perfusate samples available. The remaining 50 EVLPs were included in this study with 27 Control, 7 PGD3, and 16 Declined cases (Fig. 1). Donor and Recipient variables are summarized in Table 1. The percentage of brain death donors was similar among groups, as well as donor age and P/F ratios. Recipient variables such as age, sex, and indication for transplant were similar between Control and PGD3 cases, although there was a trend of more interstitial pulmonary fibrosis patients in the PGD3 group. There was no case of primary pulmonary arterial hypertension. In the PGD3 group, all 7 cases presented PGD3 at ICU admission. In 2 cases, it persisted as PGD3 at 72 hours, with 1 case requiring veno-venous extracorporeal life support. One additional case persisted as PGD2 at 72 hours, and this patient died because of sepsis at day 7 posttransplant. PGD3 cases presented longer mechanical ventilation requirement (median 5 vs 2 days, P = 0.001) and longer ICU stay (median 7 vs 3 days, P = 0.01) in comparison to Controls.
EVLP Physiology EVLP physiology data are summarized in Figure 2. Briefly, pulmonary artery pressure was similar in all groups. As expected, peak airway pressure, plateau pressure, and PaO2 /FiO2 were significantly worse in Declined lungs than in Controls. Lungs in the PGD3 group presented similar PaO2 /FiO2 , peak airway pressure, and plateau pressure compared to Controls.
Statistical Analysis
Analysis of Cytokines, Chemokines, Growth Factors, and Interferons
Statistical analysis was performed using Graphpad Prism 5 (Graphpad Software Inc, La Jolla, CA). Comparisons for EVLP physiologic parameters among different groups were performed using 2way analysis of variance (time and phenotype). Continuous variables were compared using the nonparametric Mann-Whitney test, and categorical variables were compared with Fisher exact test. The ability
From 51 analytes, readable values were detected in at least 1 time point in 34. For the remaining 17, levels were below the detection limit (Supplemental table, available online at http://links.lww.com/SLA/A745). The highest median fold changes over time with respect to each donor lung phenotype are displayed in Figure 3.
FIGURE 1. Study design. ∗ Eight early cases did not have perfusates available for protein analysis. 592 | www.annalsofsurgery.com
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Annals of Surgery r Volume 261, Number 3, March 2015
Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
TABLE 1. Donor and Recipient Clinical Variables Donor variables Age (yr) BDD (%) Female (%) Best P/F ratio (mm Hg) Recipient variables Age (yr) Female (%) Diagnosis (% IPF) CPB
Control (n = 27)
PGD3 (n = 7)
P
Declined (n = 16)
P
46 56% 52% 335
48 57% 86% 301
0.983 1 0.197 0.582
40 56% 37% 318
0.289 1 0.358 0.370
53 52% 30% 22%
50 71% 71% 57%
0.808 0.426 0.078 0.157
BDD indicates brain death donor; CPB, cardiopulmonary bypass; IPF, interstitial pulmonary fibrosis.
FIGURE 2 . Physiologic assessment of high-risk donor lungs submitted to EVLP (∗ P < 0.05; †P < 0.01; ‡P < 0.001).
FIGURE 3. Highest median fold changes measurable analytes from 1 to 4 hours of EVLP.
The majority of cytokines were not detectable in the perfusate with the reported methodology. Among the detectable ones, interleukin (IL)-6 and IL-10 demonstrated the highest fold changes over time in all 3 groups. (Control, PGD3, and Declined). IL-1β absolute values were very low at 1 hour and remained low at 4 hours of EVLP in all groups. Tumor necrosis factor (TNF)-α and TNF-β were not detectable at either time point. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
Most of the chemokines were detectable in EVLP perfusate. Although IL-8, Eotaxin, and growth-regulated oncogene (GRO)-α presented high fold changes in all 3 groups, increases in macrophage inflammatory protein (MIP)-1α and MIP-1β were more striking in PGD3. Hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1, and stem cell growth factor (SCGF)-β had the highest www.annalsofsurgery.com | 593
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concentrations among growth factors at 1 hour of EVLP. At 4 hours, HGF remained as the highest in concentration. Granulocyte colonystimulating factor (G-CSF) was the growth factor with the highest fold change in all 3 groups. Both interferon (IFN)-γ and IFN-α2 were among the analytes with the lowest concentrations at 1 and 4 hours of EVLP, with minimal changes over time.
a sensitivity of 85.7% and specificity of 92.5% (AUC = 0.93), and with a cutoff of 892 pg/mL, GRO-α had a sensitivity of 85.7% and specificity of 85.1% (AUC = 0.89). G-CSF (7547 ± 3413 vs 3338
PGD3 vs Control
At 1 hour of EVLP, IL-8 (mean ± SE, 723.1 ± 543.6 vs 63.5 ± 11.2 pg/mL, P = 0.026) and macrophage colony-stimulating factor (M-CSF) (48.4 ± 12.8 vs 26.3 ± 2.1 pg/mL, P = 0.028) were significantly higher in PGD3 cases than in Controls (Fig. 4). For IL8, a cutoff of 84 pg/mL had a sensitivity of 71.4% and specificity of 85.1% (AUC = 0.77). For M-CSF, a cutoff of 37 pg/mL rendered a sensitivity of 57.1% and a specificity of 81.4% (AUC = 0.77) (Fig. 5). At 4 hours of EVLP, IL-8 (6195 ± 1456 vs 1746 ± 328.9 pg/mL, P < 0.001) and GRO-α (2031 ± 778.8 vs 519 ± 76.8 pg/mL, P = 0.001) were higher in PGD3 group. These 2 analytes had excellent discrimination with Control, with a cutoff of 3570 pg/mL, IL-8 had
FIGURE 5. Receiver operating characteristics curves for the potential biomarkers for PGD3 lungs in perfusates at 4 hours of EVLP. Numbers in brackets represent the AUC.
FIGURE 4. A, 1-hour analytes significantly higher in PGD 3 group than in Control; B, 4-hour analytes significantly higher in PGD 3 than in Control (∗ P < 0.05; †P < 0.01; ‡P < 0.001). Median and interquartile ranges are highlighted. The 2 cases with persistent PGD 3 are highlighted as orange triangle, whereas the PGD 3 case with early mortality is highlighted as a red triangle. 594 | www.annalsofsurgery.com
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Annals of Surgery r Volume 261, Number 3, March 2015
± 1122 pg/mL, P = 0.008, AUC = 0.83), MIP-1α (88.9 ± 31.2 vs 18.6 ± 5.4, P = 0.009, AUC = 0.82), and MIP-1β (4336 ± 1606 vs 737.1 ± 21.8 pg/mL, P = 0.008, AUC = 0.82) were also significantly higher in PGD3 cases, with a good discrimination versus Control. For IL-8, GRO-α, G-CSF, MIP-1α, and MIP-1β at 4 hours of EVLP, the 3 PGD3 cases previously detailed (2 persistent PGD3 and 1 persisting as PGD2 at 72 hours with mortality at day 7) were always above the proposed cutoff values.
Declined vs Control
In the Declined group, HGF (1935 ± 256.8 vs 1118 ± 88.4 pg/mL, P = 0.007), IL-1 receptor antagonist (ra) (77.6 ± 14.2 vs 40.5 ± 4 pg/mL, P = 0.017), stem cell factor (38.5 ± 6 vs 21.8 ± 2.3 pg/mL, P = 0.020), and SCGF-β (491.4 ± 58.1 vs 255 ± 19.1 pg/mL, P < 0.001) were significantly higher compared to Controls at 1 hour (Fig. 6). The last rendered the best discrimination: with a cutoff at 327 pg/mL, it had 81.2% sensitivity and 81.4% specificity (AUC = 0.86). Similarly to 1 hour of EVLP, HGF (3642 ± 639.9 vs 2242 ± 204.1 pg/mL, P = 0.038), IL-1ra (180.7 ± 35.8 vs 115 ± 17.7 pg/mL, P = 0.043), and SCGF-β (769.4 ± 133.5 vs 389.4 ± 32.5 pg/mL, P = 0.004) were higher in the Declined cases compared to Control at 4 hours of EVLP. Again, SCGF-β provided reasonable discrimination between groups (AUC = 0.77).
DISCUSSION In this study, we explored the temporal trends of cytokine, chemokine, and growth factor production in human donor lungs perfused ex vivo. Moreover, we correlated changes in the levels of these
Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
analytes with post-EVLP clinical outcomes as determined by PGD3 within 72 hours posttransplant or by nonutilization post-EVLP. The comparison of PGD3 cases to Controls is important because EVLP physiology alone was unable to predict the subsequent development of PGD3 in some cases. We found analytes (IL-8, GROα, MIP-1α, MIP-1β, and G-CSF) that were differentially accumulated between these groups. These compounds have biological plausibility as useful biomarkers. For example, IL-8 has been implicated in the pathogenesis of acute lung injury. In this study, IL-8 was the most powerful biomarker for predicting PGD3 cases. It was the only analyte significantly elevated at both 1 hour and 4 hours of EVLP in this group and rendered the best discrimination from Control cases. This corroborates the findings of de Perrot and coworkers, which reported increased levels of this cytokine in lung biopsies taken at the end of cold ischemic time, end of warm ischemic time, and at 1 hour after reperfusion.10 Indeed, they have also shown a correlation of IL-8 at 2 hours after reperfusion with graft function in the ICU. Others have similarly correlated increased levels of IL-8 in bronchoalveolar lavage (BAL) and donor lung biopsy before implantation with development of PGD3.11 In fact, it has been shown in a model of warm ischemia followed by reperfusion in rabbits that intravenous treatment with antibodies against IL-8 successfully reduced BAL neutrophil count and alveolar infiltration of neutrophils on histology, ultimately attenuating lung injury.12 The other analytes, GRO-α, MIP-1α, and MIP-1β, have been previously implicated in the early inflammatory response against skin allografts.13 Specific to the lung, the use of anti-GRO-α antibody was able to reduce myeloperoxidase activity and preserve alveolar-capillary membrane integrity in a model of acute kidney
FIGURE 6. A, 1-hour analytes significantly higher in Declined group than in Control; B, 4-hour analytes significantly higher in Declined group than in Control (∗ P < 0.05; †P < 0.01; ‡P < 0.001). Median and interquartile ranges are highlighted. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
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injury-induced lung injury.14 Moreover, in a model of pulmonary Klebsiella pneumoniae infection, GRO-α was shown to be involved not only in the recruitment of neutrophils but also to regulate its function (production of reactive oxygen species and reactive nitrogen species).15 MIP-1α knockout has been shown to decrease polymorphonuclear cell migration in lipopolysaccharide-induced and bleomycin-induced lung injury. In the first case, it was also capable of reducing lung edema formation.16 Intratracheal administration of MIP-1β was capable of inducing pulmonary capillary leak. Interestingly, in the same study, intratracheal anti-MIP-1β antibody reduced the pulmonary permeability index, the myeloperoxidase activity, and TNF-α in BAL of rats with immune complex-induced lung injury.17 G-CSF has been implicated in ischemia reperfusion lung injury. Kreisel et al18 have demonstrated that G-CSF blockade attenuated ischemia reperfusion in a lung transplantation model with Bcl-3 knockout mice. The same authors have further shown that G-CSF is increased early after reperfusion in grafts, which underwent prolonged (18 hours) cold ischemia and that anti-G-CSF antibody can abrogate its deleterious effects in the alveolar-capillary barrier, improving gas exchange.19 The next finding of this study was generated from comparisons between Control lungs and Declined lungs. Differences at the protein level between these 2 groups would reinforce the accuracy of the physiological assessment. Indeed, differential protein expression was observed and, interestingly, most of the differences noticed at 1 hour of EVLP were again observed at 4 hours. More importantly, SCGFβ was shown to have considerable discrimination power already at 1 hour of perfusion and thus could be used as an early biomarker for prediction of lungs that will perform poorly during EVLP. Once available in a timely fashion, this valuable information can point out which lungs will require additional therapeutic measures early in the EVLP process to rescue them for a safe transplantation. The fact that 4 declined lungs presented values below the proposed cutoff raises the question whether they could have been transplanted. Although this study is unable to provide this answer, these findings can certainly guide the design of future studies to improve diagnostic strategy for donor lung suitability. Analysis of the entire study cohort revealed interesting changes over time in cytokines, chemokines, and growth factors. The most significant fold changes (up to 60x increase from 1 to 4 hours of EVLP) were observed in IL-6, IL-8, and G-CSF. Several details should be taken into account when analyzing these results. First, this technique consists of a recirculated closed circuit, which lacks hepatic and renal clearance mechanisms. It is known from experimental models of bilateral nephrectomy that mediators such as IL-6, IL-1β, IL-10, and GRO-α accumulate after exogenous administration.20 Second, the acellular nature of the perfusate and the ex vivo status may have partly contributed to the homeostatic response of increased hematopoietic growth factors (M-CSF, G-CSF). Third, these are high-risk injured donor lungs and thus a proinflammatory profile is expected. Finally, even though under controlled conditions and ex vivo, there is a reperfusion process, albeit significantly modified. Previous clinical studies have shown that, regardless of the PGD score, IL-6, IL-8, and IL-10 increase in the plasma of patients early after reperfusion.7,8 These temporal trends in perfusate are similar to the ones in lung tissue described by Sadaria and coworkers.21 In a series of lungs procured with a research-only intent, they have shown significant increases in IL-6, IL-8, and G-CSF over time. Although proinflammatory cytokines increased locally, pulmonary physiology was stable for 12 hours, there was no edema formation (wet-to-dry ratio) and lung histology was unremarkable. Along with IL-6 and IL-8, elevated TNF-α and IL-1β mRNA expression before implantation have been previously shown to corre596 | www.annalsofsurgery.com
late with 30-day mortality in lung transplantation.22 Furthermore, our group has similarly reported higher TNF-α and IL-1β in lungs declined for transplantation after on-site assessment (a subset of lungs that currently would be considered for EVLP and thus overlaps with this study).23 Even though we measured perfusate protein levels, these proinflammatory cytokines were either not measurable during the entire EVLP (TNF-α) or had very low concentrations at both time points studied (IL-1β). This finding in metabolically active high-risk lungs is encouraging and may be explained by the protective nature of our current EVLP technique.3 The elevated levels of CXC and CC chemokines in PGD3 group in our study, along with previous experimental data, strongly highlight the involvement of neutrophils and macrophages in the modulation of ischemia reperfusion injury in lung transplantation. The benefits of alveolar macrophage depletion have been previously shown in models of both warm ischemia and cold ischemia reperfusion injury.24,25 Furthermore, in a study using in vivo 2-photon microscopy, Kreisel and coworkers26 explored the crucial interactions between monocytes and neutrophils, ultimately leading to ischemia reperfusion injury in a mouse lung transplantation model. On the basis of existing evidence, the potential advantages of manipulation of neutrophil and alveolar macrophage populations during EVLP certainly deserve further exploration. One of the main limitations of this study resides in the sample size. Although initially considered small, it includes the largest experience to date with perfusion of high-risk donor lungs for clinical transplantation. Another limitation is the inability to study recipient biologic factors related to PGD.7 The simultaneous assessment of biomarkers in recipient and donors has been reported to increase the predictive yield of biomarkers in lung transplantation and should be sought in future studies.27 In terms of clinical recipient and donor factors, we did not find significant differences between Control and PGD3 groups. Furthermore, recipient factors reportedly related to PGD3 such as diagnosis of primary pulmonary arterial hypertension or sarcoidosis and cases of single lung transplantation were not included in our study.28 Finally, the analysis of several analytes may potentially render misleading findings. However, most of the analytes predictive of PGD3 reported herein are well known, highly plausible biomarkers of lung injury. As EVLP gains widespread acceptance, multicenter collaborations will be instrumental for the validation of our findings. In summary, this study reveals changes in cytokines, chemokines, and growth factors at the protein level in high-risk donor lungs perfused ex vivo with clinical transplantation intent. Our findings suggest that adding biomarkers to EVLP assessment can improve the precision of donor lung selection. Besides adding more objectivity to decision making, these biomarkers can guide future studies on therapeutic strategies to safely increase the utilization of injured lungs for transplantation. Barriers to clinical translation can be overcome by the incorporation of already available advanced analytic tools able to provide quantitative results in a short period of time, ideally within minutes.29 Although previous studies reported candidate markers of primary graft dysfunction,8,10,22,30 this study has key advantages: (1) simplicity—it is based on readily available perfusate samples, avoiding potential caveats of lung biopsy including heterogeneity and time needed for sample preparation; (2) timing—at the moment of sample collection, the lungs are in a system capable of stably preserving them for hours3,31 ; and (3) reversibility— since the recipient is not on the operating table yet, the biomarker results can actually assist in the decision-making process. We hope that the combination of physiologic and biomarker assessment during EVLP will soon improve the precision of decision making in donor lung assessment and allow clinicians to safely expand the current donor pool improving outcomes after lung transplantation. C 2014 Wolters Kluwer Health, Inc. All rights reserved.
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Annals of Surgery r Volume 261, Number 3, March 2015
ACKNOWLEDGMENTS For the preparation of this article, authors contributions were as follows: T.N.M.—designed and performed the study, analyzed data, and wrote manuscript; M.C.—designed the study, analyzed data, and wrote manuscript; J.C.Y.—performed the study, analyzed data, and reviewed manuscript; R.B.—performed data acquisition and reviewed the manuscript; R.Z.—analyzed data and reviewed manuscript; M.C.—performed data acquisition and reviewed the manuscript; M.K.H.—performed data acquisition, analyzed data, and reviewed manuscript; T.S.—analyzed and interpreted data, reviewed manuscript; Z.G.—performed the study and data acquisition, reviewed manuscript; T.K.W.—designed the study, analyzed data, and reviewed manuscript; M.L.—designed the study, analyzed data, and reviewed manuscript; S.K.—designed the study, analyzed data, supervised research, and reviewed manuscript. All authors approved the final version of the manuscript. We thank Paul Chartrand for assistance in the EVLP procedures and logistics.
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Perfusate Proteins in Clinical Ex Vivo Lung Perfusion
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