Thomas K. Waddell

Normothermic Ex Vivo Lung Perfusion in Clinical Lung Transplantation

BACKGROUND More than 80% of donor lungs are potentially injured and therefore not considered suitable for transplantation. With the use of normothermic ex vivo lung perfusion (EVLP), the retrieved donor lung can be perfused in an ex vivo circuit, providing an opportunity to reassess its function before transplantation. In this study, we examined the feasibility of transplanting high-risk donor lungs that have undergone EVLP. METHODS In this prospective, nonrandomized clinical trial, we subjected lungs considered to be high risk for transplantation to 4 hours of EVLP. High-risk donor lungs were defined by specific criteria, including pulmonary edema and a ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PO(2):FIO(2)) less than 300 mm Hg. Lungs with acceptable function were subsequently transplanted. Lungs that were transplanted without EVLP during the same period were used as controls. The primary end point was primary graft dysfunction 72 hours after transplantation. Secondary end points were 30-day mortality, bronchial complications, duration of mechanical ventilation, and length of stay in the intensive care unit and hospital. RESULTS During the study period, 136 lungs were transplanted. Lungs from 23 donors met the inclusion criteria for EVLP; in 20 of these lungs, physiological function remained stable during EVLP and the median PO(2):FIO(2) ratio increased from 335 mm Hg in the donor lung to 414 and 443 mm Hg at 1 hour and 4 hours of perfusion, respectively (P<0.001). These 20 lungs were transplanted; the other 116 lungs constituted the control group. The incidence of primary graft dysfunction 72 hours after transplantation was 15% in the EVLP group and 30% in the control group (P=0.11). No significant differences were observed for any secondary end points, and no severe adverse events were directly attributable to EVLP. CONCLUSIONS Transplantation of high-risk donor lungs that were physiologically stable during 4 hours of ex vivo perfusion led to results similar to those obtained with conventionally selected lungs. (Funded by Vitrolife; ClinicalTrials.gov number, NCT01190059.).

Technique for Prolonged Normothermic Ex Vivo Lung Perfusion

BACKGROUND The inhibition of cellular metabolism induced by hypothermia obviates the possibility of substantial reparative processes occurring during organ preservation. The aim of this study was to develop a technique of extended (12-hour) ex vivo lung perfusion (EVLP) at normothermia for assessment and protective maintenance of the donor lung. METHODS Six double-lung blocks from 35-kg pigs and 5 single human lungs were subjected to 12 hours of normothermic EVLP using acellular Steen Solution. In the animal studies, the left lung was transplanted into recipients at the end of EVLP and reperfused for 4 hours to evaluate the impact of prolonged EVLP on post-transplant lung function. A protective mode of mechanical ventilation with controlled perfusion flows and pressures in the pulmonary vasculature were employed during EVLP. Lung oxygenation capacity (DeltaPo(2)), pulmonary vascular resistance and airway pressures were evaluated in the system. Red blood cells were added to the perfusate to a hematocrit of 20% at the end of human lung EVLP to study lung functional assessment with and without cells. RESULTS Lung function was stable during 12 hours of EVLP. This stability during prolonged normothermic EVLP translated into excellent post-transplant lung function (Pao(2)/Fio(2): 527 +/- 22 mm Hg), low edema formation (wet/dry ratio: 5.24 +/- 0.38) and preserved lung histology after transplantation. The acellular perfusion assessment of lung function accurately correlated with post-transplant graft function. CONCLUSIONS Twelve hours of EVLP at physiologic temperatures using an acellular perfusate is achievable and maintains the donor lungs without inflicting significant added injury. This system can be used to assess, maintain and treat injured donor lungs.

Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction

BACKGROUND Bronchiolitis obliterans syndrome (BOS) with small-airway pathology and obstructive pulmonary physiology may not be the only form of chronic lung allograft dysfunction (CLAD) after lung transplantation. Characteristics of a form of CLAD consisting of restrictive functional changes involving peripheral lung pathology were investigated. METHODS Patients who received bilateral lung transplantation from 1996 to 2009 were retrospectively analyzed. Baseline pulmonary function was taken as the time of peak forced expiratory volume in 1 second (FEV(1)). CLAD was defined as irreversible decline in FEV(1) < 80% baseline. The most accurate threshold to predict irreversible decline in total lung capacity and thus restrictive functional change was at 90% baseline. Restrictive allograft syndrome (RAS) was defined as CLAD meeting this threshold. BOS was defined as CLAD without RAS. To estimate the effect on survival, Cox proportional hazards models and Kaplan-Meier analyses were used. RESULTS Among 468 patients, CLAD developed in 156; of those, 47 (30%) showed the RAS phenotype. Compared with the 109 BOS patients, RAS patients showed significant computed tomography findings of interstitial lung disease (p < 0.0001). Prevalence of RAS was approximately 25% to 35% of all CLAD over time. Patient survival of RAS was significantly worse than BOS after CLAD onset (median survival, 541 vs 1,421 days; p = 0.0003). The RAS phenotype was the most significant risk factor of death among other variables after CLAD onset (hazard ratio, 1.60; confidential interval, 1.23-2.07). CONCLUSIONS RAS is a novel form of CLAD that exhibits characteristics of peripheral lung fibrosis and significantly affects survival of lung transplant patients.

Three-gene prognostic classifier for early-stage non small-cell lung cancer.

PURPOSE Several microarray studies have reported gene expression signatures that classify non-small-cell lung carcinoma (NSCLC) patients into different prognostic groups. However, the prognostic gene lists reported to date overlap poorly across studies, and few have been validated independently using more quantitative assay methods. PATIENTS AND METHODS The expression of 158 putative prognostic genes identified in previous microarray studies was analyzed by reverse transcription quantitative polymerase chain reaction in the tumors of 147 NSCLC patients. Concordance indices and risk scores were used to identify a stage-independent set of genes that could classify patients with significantly different prognoses. RESULTS We have identified a three-gene classifier (STX1A, HIF1A, and CCR7) for overall survival (hazard ratio = 3.8; 95% CI, 1.7 to 8.2; P < .001). The classifier was also able to stratify stage I and II patients and further improved the predictive ability of clinical factors such as histology and tumor stage. The predictive value of this three-gene classifier was validated in two large independent microarray data sets from Harvard and Duke Universities. CONCLUSION We have identified a new three-gene classifier that is independent of and improves on stage to stratify early-stage NSCLC patients with significantly different prognoses. This classifier may be tested further for its potential value to improve the selection of resected NSCLC patients in adjuvant therapy.

Functional Repair of Human Donor Lungs by IL-10 Gene Therapy

Treatment of damaged donor lungs with the cytokine interleukin-10 improves their function, allowing previously unacceptable organs to be used for transplantation. Living Well After Lung Replacement Bumper stickers that counsel motorists to “just breathe” abound—easier said than done when it comes to patients with serious lung disorders. Lung transplantations are on the rise, from 203 in 1990 to more than 1200 in 2008 in the United States. Earlier this year, transplant surgeons at Johns Hopkins presented evidence that more is better—hospitals that perform 20 or more lung transplants per year have the best patient survival rates. However, successful surgeries require healthy donor lungs, a resource that remains in short supply. Now, Keshavjee and colleagues describe a gene therapy treatment protocol to repair lungs after removal from the donor and before transplantation into patients. Candidates for lung transplantation are patients suffering from end-stage lung diseases, such as emphysema, cystic fibrosis, pulmonary fibrosis, and pulmonary arterial hypertension. Organ donors are people who have undergone brain death, a process that is as violent as it sounds: Brain death is accompanied by the spewing of inflammation-inducing molecules called cytokines that damage more than 80% of donated lungs. These injured organs are highly inflamed, and their alveoli—the gas exchange machinery in lungs—are disrupted and only mildly functional. To avoid primary graft dysfunction—lung damage that occurs within the 72 hours after transplantation—transplant surgeons usually reject such injured organs. A method is needed to heal these fixer-upper organs so that they can be used to give patients a new lease on life. Using IL-10, an anti-inflammatory cytokine, Keshavjee’s team devised a treatment to quell inflammation in the injured donor lungs and refurbish the alveoli. Although the standard technique for the handling of organs is to keep them on ice in a sealed bag, this IL-10 gene therapy approach must be performed at body temperature so that the lung’s cellular machinery can express the gene efficiently. The researchers then carried out prolonged ex vivo lung perfusion (EVLP) and kept the lungs breathing outside the body in conditions that mimic physiological ones. Pig lungs that were subjected to IL-10 gene therapy and EVLP for 12 hours displayed reduced inflammation and enhanced function when transplanted into donor pigs, relative to control organs. The same treatment was applied to human lungs deemed unsuitable for transplantation, and these organs, relative to controls, displayed the presence of anti-inflammatory cytokines, repair of alveoli, and improved function, determined by measuring gas exchange and pulmonary vascular resistance. This procedure can yield a larger number of usable lungs and thus more successful transplantations so that patients can “just breathe.” More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10–treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10–treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar–blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients.

Normothermic Ex Vivo Perfusion Prevents Lung Injury Compared to Extended Cold Preservation for Transplantation

Treatment of injured donor lungs ex vivo to accelerate organ recovery and ameliorate reperfusion injury could have a major impact in lung transplantation. We have recently demonstrated a feasible technique for prolonged (12 h) normothermic ex vivo lung perfusion (EVLP). This study was performed to examine the impact of prolonged EVLP on ischemic injury. Pig donor lungs were cold preserved in Perfadex® for 12 h and subsequently divided into two groups: cold static preservation (CSP) or EVLP at 37°C with Steen™ solution for a further 12 h (total 24 h preservation). Lungs were then transplanted and reperfused for 4 h. EVLP preservation resulted in significantly better lung oxygenation (PaO2 531 ± 43 vs. 244 ± 49 mmHg, p < 0.01) and lower edema formation rates after transplantation. Alveolar epithelial cell tight junction integrity, evaluated by zona occludens‐1 protein staining, was disrupted in the cell membranes after prolonged CSP but not after EVLP. The maintenance of integrity of barrier function during EVLP translates into significant attenuation of reperfusion injury and improved graft performance after transplantation. Integrity of functional metabolic pathways during normothermic perfusion was confirmed by effective gene transfer and GFP protein synthesis by lung alveolar cells. In conclusion, EVLP prevents ongoing injury associated with prolonged ischemia and accelerates lung recovery.

Tumor-initiating cells are rare in many human tumors

Tumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions, raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic, non-small cell lung, and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice, it remained low (<1 in 2500 cells) in all cases. Moreover, aldehyde dehydrogenase-positive (ALDH(+)) and CD44(+)CD24(+) cells, phenotypically distinct cells enriched in TICs, were equally tumorigenic in NOD/SCID and NSG mice. Our findings demonstrate that TICs are rare in these cancers and that the identification of TICs and their frequency in other human malignancies should be validated via primary tumors and highly permissive xenotransplantation conditions.

Experience with the first 50 ex vivo lung perfusions in clinical transplantation.

OBJECTIVE Normothermic ex vivo lung perfusion is a novel method to evaluate and improve the function of injured donor lungs. We reviewed our experience with 50 consecutive transplants after ex vivo lung perfusion. METHODS A retrospective study using prospectively collected data was performed. High-risk brain death donor lungs (defined as Pao(2)/Fio(2) <300 mm Hg or lungs with radiographic or clinical findings of pulmonary edema) and lungs from cardiac death donors were subjected to 4 to 6 hours of ex vivo lung perfusion. Lungs that achieved stable airway and vascular pressures and Pao(2)/Fio(2) greater than 400 mm Hg during ex vivo lung perfusion were transplanted. The primary end point was the incidence of primary graft dysfunction grade 3 at 72 hours after transplantation. End points were compared with lung transplants not treated with ex vivo lung perfusion (controls). RESULTS A total of 317 lung transplants were performed during the study period (39 months). Fifty-eight ex vivo lung perfusion procedures were performed, resulting in 50 transplants (86% use). Of these, 22 were from cardiac death donors and 28 were from brain death donors. The mean donor Pao(2)/Fio(2) was 334 mm Hg in the ex vivo lung perfusion group and 452 mm Hg in the control group (P = .0001). The incidence of primary graft dysfunction grade 3 at 72 hours was 2% in the ex vivo lung perfusion group and 8.5% in the control group (P = .14). One patient (2%) in the ex vivo lung perfusion group and 7 patients (2.7%) in the control group required extracorporeal lung support for primary graft dysfunction (P = 1.00). The median time to extubation, intensive care unit stay, and hospital length of stay were 2, 4, and 20 days, respectively, in the ex vivo lung perfusion group and 2, 4, and 23 days, respectively, in the control group (P > .05). Thirty-day mortality (4% in the ex vivo lung perfusion group and 3.5% in the control group, P = 1.00) and 1-year survival (87% in the ex vivo lung perfusion group and 86% in the control group, P = 1.00) were similar in both groups. CONCLUSIONS Transplantation of high-risk donor lungs after 4 to 6 hours of ex vivo lung perfusion is safe, and outcomes are similar to those of conventional transplants. Ex vivo lung perfusion improved our center use of donor lungs, accounting for 20% of our current lung transplant activity.

The Effect of Reflux and Bile Acid Aspiration on the Lung Allograft and Its Surfactant and Innate Immunity Molecules SP-A and SP-D

Gastro‐esophageal reflux and related pulmonary bile acid aspiration were prospectively investigated as possible contributors to postlung transplant bronchiolitis obliterans syndrome (BOS). We also studied the impact of aspiration on pulmonary surfactant collectin proteins SP‐A and SP‐D and on surfactant phospholipids—all important components of innate immunity in the lung. Proximal and distal esophageal 24‐h pH testing and broncho‐alveolar lavage fluid (BALF) bile acid assays were performed prospectively at 3‐month posttransplant in 50 patients. BALF was also assayed for SP‐A, SP‐D and phospholipids expressed as ratio to total lipids: phosphatidylcholine; dipalmitoylphosphatidylcholine; phosphatidylglycerol (PG); phosphatidylinositol; sphingomyelin (SM) and lysophosphatidylcholine. Actuarial freedom from BOS was assessed.

Heart-lung or lung transplantation for Eisenmenger syndrome.

BACKGROUND The optimal therapy for end-stage Eisenmenger syndrome (ES) is unknown. We analyzed the United Network for Organ Sharing/International Society for Heart and Lung Transplantation Joint Thoracic Registry to determine predictors of survival. METHODS Univariate analysis was performed using Kaplan-Meier survival curves. Groups were compared using the log-rank test. Multivariate analysis was performed using a proportional hazards model. RESULTS There were 605 transplants performed between 1988 and 1998. The causes of ES included atrial septal defect (ASD) in 171, ventricular septal defect (VSD) in 164, multiple congenital anomalies (MCA) in 68 and patent ductus arteriosus (PDA) in 32. Procedures included 430 heart-lung (HLT), 106 bilateral lung, and 69 single lung transplants (LT). Survival after HLT was better than after LT on univariate analysis (p = 0.002). For HLT, survival at 30 days and 1 year was 80.7% and 70.1% compared with 68% and 55.2% for LT. Diagnosis was also a significant predictor of survival (p = 0.011), being best for VSD and MCA (1-year survival 71.4% and 77.6%). There was a highly significant benefit of HLT over LT for VSD patients (p = 0.0001). Diagnosis, the combination of diagnosis and procedure, recipient age, recipient gender, donor age, ischemic time and recipient status were significant in a multivariate model. Multivariate analysis confirmed the superior prognosis of patients with VSD or MCA (p = 0.007 and p = 0.022, respectively) and suggested that the adverse effect of LT was predominately in patients with VSD (risk ratio 1.817, p = 0.035). CONCLUSIONS This analysis suggests that ES recipients are not a homogeneous group. Patients with VSD and MCA have the best prognosis. HLT appears to offer a survival benefit for patients with ES secondary to VSD and should be re-considered as the operation of choice.