ORIGINAL RESEARCH published: 30 July 2021 doi: 10.3389/fmed.2021.695919 Lung Transplant Improves Survival and Quality of Life Regardless of Telomere Dysfunction Lurdes Planas-Cerezales 1 , Elena G. Arias-Salgado 2,3 , Cristina Berastegui 4 , Ana Montes-Worboys 1 , Rafaela González-Montelongo 5 , José. M. Lorenzo-Salazar 5 , Vanesa Vicens-Zygmunt 1 , Marta Garcia-Moyano 6 , Jordi Dorca 1 , Carlos Flores 5,7,8,9 , Rosario Perona 2,3 , Antonio Román 4 and María Molina-Molina 1,9* 1 ILD Multidisciplinary Unit, Hospital Universitari Bellvitge, IDIBELL, Universitat de Barcelona, Hospitalet de Llobregat, Spain, 2 Edited by: Biomedical Research Institute CSIC/UAM, IdIPAZ, Madrid, Spain, 3 Centro de Investigación Biomédica en Red de Ramcés Falfán-Valencia, Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain, 4 Respiratory Department, Institute of Research, Hospital Instituto Nacional de Enfermedades Universitari Vall d’Hebrón, Universitat Autónoma de Barcelona, Barcelona, Spain, 5 Genomics Division, Instituto Tecnológico y Respiratorias-México (INER), Mexico de Energías Renovables, Santa Cruz de Tenerife, Spain, 6 Respiratory Department, Hospital Universitario Cruces, Barakaldo, Spain, 7 Research Unit, Hospital Universitario Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain, 8 Instituto de Reviewed by: Tecnologías Biomédicas, Universidad de La Laguna, Santa Cruz de Tenerife, Spain, 9 Centro Investigación Biomédica en Red Ernesto Juárez, de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Mexico Dmitri Churikov, Introduction: Fibrotic interstitial lung diseases (ILDs) are the first indication for lung INSERM U1068 Centre de Recherche en Cancérologie de Marseille transplantation (LT). Telomere dysfunction has been associated with poor post-transplant (CRCM), France outcomes. The aim of the study was to evaluate the morbi-mortality and quality of life in *Correspondence: fibrotic ILDs after lung transplant depending on telomere biology. María Molina-Molina mariamolinamolina@hotmail.com Methods: Fibrotic ILD patients that underwent lung transplant were allocated to two arms; with or without telomere dysfunction at diagnosis based on the telomere Specialty section: This article was submitted to length and telomerase related gene mutations revealed by whole-exome sequencing. Pulmonary Medicine, Post-transplant evaluation included: (1) short and long-term mortality and complications a section of the journal and (2) quality of life. Frontiers in Medicine Received: 15 April 2021 Results: Fifty-five percent of patients that underwent LT carried rare coding Accepted: 27 May 2021 mutations in telomerase-related genes. Patients with telomere shortening more frequently Published: 30 July 2021 needed extracorporeal circulation and presented a higher rate of early post-transplant Citation: hematological complications, longer stay in the intensive care unit (ICU), and a higher Planas-Cerezales L, Arias-Salgado EG, Berastegui C, number of long-term hospital admissions. However, post-transplant 1-year survival was Montes-Worboys A, higher than 80% regardless of telomere dysfunction, with improvement in the quality of González-Montelongo R, Lorenzo-Salazar JM, life and oxygen therapy withdrawal. Vicens-Zygmunt V, Garcia-Moyano M, Conclusions: Post-transplant morbidity is higher in patients with telomere dysfunction Dorca J, Flores C, Perona R, Román A and Molina-Molina M (2021) Lung and differs according to elapsed time from transplantation. However, lung transplant Transplant Improves Survival and improves survival and quality of life and the associated complications are manageable. Quality of Life Regardless of Telomere Dysfunction. Front. Med. 8:695919. Keywords: interstitial lung disease, pulmonary fibrosis, genetics, telomere shortening, telomere disorders, lung doi: 10.3389/fmed.2021.695919 transplantation Frontiers in Medicine | www.frontiersin.org 1 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD INTRODUCTION Clinical data were prospectively recorded from patient inclusion. Surgical, post-surgical procedure, and management Fibrotic interstitial lung diseases (ILDs) are comprised of protocols were blinded to the laboratory tests and followed parenchymal lung disorders with progressive, irreversible fibrosis national and international guidelines (32, 33). The Lung that present a devastating clinical course (1). Idiopathic Allocation Score (LAS) was used to assess the allocating process pulmonary fibrosis (IPF) is the most lethal fibrotic ILD. Although of donated lungs. The decision for unilateral vs. bilateral lung new anti-fibrotic medications slow disease progression, lung transplant was based on the presence of pulmonary hypertension transplantation (LT) remains the treatment option that improves or microbiological colonization. In those cases, a bilateral lung function and survival in those cases that can benefit from lung transplant was performed. A standard immunosuppression this procedure (2). After several years with an increasing trend, regimen was initiated after LT and adjusted as tolerated. The fibrotic ILD is often now reported as the first indication for prophylaxis for cytomegalovirus (CMV) was initiated after LT (3). the surgical procedures in all patients, using ganciclovir 3–5 In previous studies, pulmonary fibrosis has been linked to mg/kg/12 h during the Intensive Care Unit (ICU) stay, followed aging and repair defects, especially telomere attrition (4–14). by valganciclovir 900 mg/day when oral intake was tolerated. Rare coding mutations in telomere-maintenance genes (TERT, The treatment was maintained for 12 months in serum-negative TERC, PARN, RTEL1, DKC1, TINF2, NAF1, ACD, NOP10, pre-transplant cases and 6 months for those serum-positive. The NHP2) and telomere shortening have been identified in different post-transplant evaluation included: (1) type of complications fibrotic ILDs, irrespective of family aggregation (4–6, 9, 10, observed during the first 24 h, the first 30 days, and after the 12, 15, 16). Regardless of gene mutations and ILD diagnoses first month; and, (2) assessment of the quality of life after 6 (17), telomere shortening has been associated with disease months of LT based on an improvement of forced vital capacity progression, reduced survival, and poor LT outcomes (18, 19). (FVC) and health status questionnaires (K-BILD and ATAQ- Telomeric disease onset frequently appears in young adults; IPF), oxygen therapy withdrawal and declared independence for therefore, LT may be necessary in most patients (18). However, daily life activities. some reports have shown a high morbidity and mortality related to LT in fibrotic ILD cases with telomere defects (20– 25). Furthermore, increased immunosuppressive drug-related Telomere Genetic Studies toxicities have also been reported (26, 27), especially during The telomere genetic study was performed at fibrotic ILD post-transplant prophylaxis regimens (20–22), probably due to diagnosis and consisted of: (1) a telomere length screening of the associated T cell immunodeficiency (28). Nevertheless, most DNA isolated from mouth epithelial cells (oral swab) using a studies evaluating the effect of telomere dysfunction in LT lack commercial DNA isolation kit (Isohelix, Cell Projects Ltd.) (34) a control group that allow to differentiate those effects only and further validation based on DNA from peripheral blood; (2) attributed to this biological defect (20–24). a sequencing analysis of the known telomere related-genes (TRG) A systematic assessment of telomere length in the pre- described so far as a cause of pulmonary fibrosis. The assessment transplant study could be useful for optimizing LT protocol of telomere dysfunction in donors was unavailable in our study among fibrotic ILD cases. However, limited data exists about the due to the great inherent lung transplant technical difficulties. LT in pulmonary fibrosis depending on telomere biology and the different technical interventional approaches. The aim of the study was to compare the morbidity, mortality, and quality of Telomere Length Analysis life of fibrotic ILD patients that underwent LT with and without The relative telomere length was performed in the Instituto de telomere dysfunction. Investigaciones Biomédicas (CSIC/UAM) and initially assessed by quantitative polymerase chain reaction (qPCR), as previously described (19, 35, 36). Since telomere length changes with age, MATERIALS AND METHODS a Z-score value was obtained to allow the comparisons of the telomere length between individuals of different ages. The Z- Study Population score compared the Telomere Shortening T/S ratio value in each This prospective observational study included 20 patients from individual with the age-matched mean and standard deviation the ILD Unit of Hospital Universitari de Bellvitge (HUB) with (SD) of the values obtained in the controls. The Z-score below the any form of fibrotic ILD that underwent LT at the Hospital 25th percentile of a normal distribution was considered as “short Universitari Vall d’Hebrón, in Barcelona. The Ethics Committee telomere.” A severe telomere length reduction was identified of HUB approved the study and all patients provided written when Z-score was below the 10th percentile. informed consent before inclusion. A telomere genetic study was Telomere shortening was confirmed by telomere restriction performed at the moment of diagnosis. Diagnosis was performed fragment (TRF) assay (TeloTAGGG Telomere Length Assay, following the international clinical guidelines for ILDs (29–31). Roche) from peripheral blood DNA of each patient, as previously Patients were recruited at the time of referral to the LT evaluation, described (36, 37) (Figure 1). A positive correlation (r = with a post-transplant follow-up period of at least 3 months. 0.689) was observed between the two sets of telomere length Patients were excluded if they did not consent to the telomere measurements performed by either the qPCR on DNA extracted genetic test. from buccal cells or by the Southern Blot of blood DNA, Frontiers in Medicine | www.frontiersin.org 2 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD FIGURE 1 | (A) Telomere length and qPCR essential primary data of all study patients: telomere length was measured, at diagnosis, from oral epithelium DNA, through quantitative Polymerase Chain Reaction (qPCR) and Z-score was obtained as described in Methods section. The qPCR determines the ratio of telomere (T) repeat copy number to single-copy (S) gene (36B4) copy number (T/S ratio), as compared with a reference DNA sample. The Z-score below the 25th percentile of a normal distribution was considered “telomere shortening.” (B) qPCR validation through TRF Southern Blot: telomere shortening was validated in each patient from blood DNA by telomere restriction fragment (TRF) Southern Blot analysis (TeloTAGGG Telomere Length Assay, Roche), which is considered the gold standard to determine telomere length. A representative southern blotting image is shown. The absolute telomere length of the telomeres in blood DNA samples from controls (C lanes) and patients (F lanes) was measured (C) Distribution of the absolute telomere length (kb) obtained by TRF Southern Blot in blood samples of controls and patients vs. age (years). in accordance with a previous study published by Demanelis Santa Clara, CA) and their concentration determined by the et al. (38). Qubit dsDNA HS Assay (Thermo Fisher, Waltham, MA). The control population for telomere Z-score calculation Samples were sequenced along with 1% of a PhiX control V3 to consisted of 243 healthy subjects, with no respiratory disease, an average depth >50X (after removal of duplicate reads) on a cancer, or any degenerative disorders such as diabetes, HiSeq 4000 instrument (Illumina Inc.) with paired-end 75-base hematological, liver, or kidney disease. Other characteristics reads. Reads were preprocessed with bcl2fastq v2.18 and aligned from the control population were described previously (19). (>99.70% of reads were mapped) to hg19 with BWA-MEM Oral swab and peripheral blood sample were also processed in 0.7.15-r1140 (39). BAM files were processed with SAMtools controls for DNA isolation. The same protocol described above v1.3 (40) and Picard v2.10.10, and small insertion/deletions was followed to process control DNA samples. (indels) and single nucleotide variants (SNVs) were identified with the HaplotypeCaller following the Best Practices workflow recommendations for germline variant calling in GATK Sequencing Analysis and Identification of (v3.8) (41). Detected variants were annotated for frequency in Rare Coding Mutations in TRG gnomAD, function and pathogenic potential with SnpEff v4.3 Patients with telomere shortening were subjected to whole- (42) and ANNOVAR v18.04.16 (43) based on data from different exome sequencing to obtain genetic variation at the telomere sources. We simplified the prioritization of pathogenicity maintenance genes. Whole-exome sequences were obtained potential by focusing only on the results in the previously- by the Instituto Tecnológico y de Energías Renovables (ITER). known TRG: TERT (chr5:1,253,262–1,295,184), TERC Briefly, DNA samples were processed with Nextera DNA (chr3:169,482,308–169,482,848), DCK1 (chrX:153,991,031– Exome Kit with dual indexes following the manufacturer’s 154,005,964), PARN (chr16:14,529,558–14,726,585), RTEL1 recommendations (Illumina Inc., San Diego, CA). Library sizes (chr20:62,289,163–62,328,416), TINF2 (chr14:24,708,849– were evaluated on a TapeStation 4200 (Agilent Technologies, 24,711,880), and TERF1 (chr8:73,921,097–73,960,357). Among Frontiers in Medicine | www.frontiersin.org 3 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD the TRG set, we prioritized the variants that were likely to Surgical Procedure and Immediate alter the function of the encoded proteins based on a CADD Post-surgery Requirements phred score >15 (standard threshold for deleteriousness) and Unilateral LT was performed in 14 (70.0%) cases. In both groups rareness in the population (<0.5% in genomAD). On those of patients, with and without telomere disorders, unilateral LT patients that these criteria did not evidence a prioritized variant, was the main choice. we opted for prioritizing one variant applying more liberal Although no statistically significant differences were found filters but showing a predicted damage potential based on the during the surgical procedure, more cases with telomere effect (frameshift, stop gain/loss, splice site, codon deletion, dysfunction required extracorporeal circulation (ECC); among non-synonymous change, etc.). Given the uncertainty in their four patients, three of them presented telomere dysfunction. A pathogenic potential, we will refer to the prioritized variants as longer time for ECC was registered for the telomere dysfunction rare coding mutations thereafter. group (median 150 min, range 122–244). The time of ischemia was also longer for patients with telomere disorders (median Statistical Analysis 6.88 h, range 5.25–7.69) (Table 3). Categorical variables were presented as the number of cases and Regarding the first 24 h after LT, 15% of overall cases percentages, while continuous variables were presented as the presented relevant complications, including hemodynamic mean and SD or median and the interquartile range (IQR). In instability, phrenic or diaphragmatic paresis, hypovolemic continuous variables, the group means were compared using shock, thrombocytopenia, and primary graft dysfunction the Student’s t-test and the group medians were compared with (Supplementary Table 2). No significant differences were the Mann-Whitney U-test. Fisher’s exact test or a Pearson’s X 2 - observed by telomere dysfunction (p = 0.495) (Table 3). test were applied to assess the relationship between categorical However, mild hematological complications during the first 24 h variables. Time until death was assessed using the Kaplan–Meier after LT were only reported in patients with telomere disorders estimator. Analyses were performed with R 3.4.0 (44). A p-value (Supplementary Table 2). <0.005 was considered statistically significant. Concerning the ICU admission period, the average stay was longer for patients with telomere dysfunction with a median of 30.0 days [(range 7.75–40.8 days, p = 0.352)] (Table 3). RESULTS Time of respiratory support required, difficult weaning, and tracheostomy did not differ significantly due to the presence of Patient Characteristics telomere dysfunction (p = 1.000) (Table 3). From 274 fibrotic ILD patients that had been tested for telomere attrition at diagnosis, 20 cases underwent LT from June 2013 to October 2018. Telomere attrition was identified in 12 (60%) Complications and Morbidity After Lung patients and rare coding mutations in TRG in 11 of them (55% Transplant of the cohort) (Supplementary Table 1). Patients underwent We analyzed the complications reported and hospital admission LT at a younger age if telomere dysfunction was present. requirements after LT. More than one complication per patient Idiopathic pulmonary fibrosis and FPF were the most frequent was reported regardless of telomere dysfunction (p = 1.000) diagnoses. The anti-fibrotic therapy was prescribed in 12 patients (Table 4). During the first month, complications were present at diagnosis (Table 1). in 9 (81.8%) patients with telomere disorders, and in all A similar pre-transplant functional status was reported for patients (n = 8, 100%) without (p = 0.485) (Table 4). patients with or without telomere dysfunction (Table 2). Oxygen Renal failure was the main complication in the overall therapy was prescribed in all patients with telomere dysfunction population (Supplementary Table 3). A higher rate of primary (n = 12, 100%), with a median time of prescription of 12.5 graft dysfunction and hypovolemic shock was more frequently months (range 11.0–16.2); and in 7 (87.5%) patients without reported for patients with telomere dysfunction; while phrenic telomere disorders with a median prescription time of 9.0 months or diaphragmatic paresis and hemodynamic instability were (range 7.5–12.5) (p = 0.090). The pre-transplant health status more present in patients without. The main hematological questionnaires ATAQ and K-BILD were scored in the entire complication, thrombocytopenia, was only described in telomere patient series. Patients with telomere dysfunction presented a dysfunction group, although pre-transplant thrombocytopenia worse pre-transplant health status compared to the patients was only present in one of those cases (Supplementary Table 3). without telomere disorders, with a median ATAQ score of 108 After the first month of LT, all patients with telomere (8.27) and a median K-BILD score of 22.5 (16.0), (p = 0.032 and dysfunction (n = 11, 100%) and 7 (87.5%) patients without p = 0.036, respectively) (Table 2). telomere disorders had complications (p = 0.421). As shown in Regarding the LAS score, the patients with telomere disorders Table 4, endocrine dysfunction and infections were predominant had a median LAS of 43.1 (range 38.4–51.3) with no statistically in both groups. Chronic lung allograft dysfunction (CLAD) significant difference from patients without telomere dysfunction and post-transplant CMV replication were higher in patients (p = 0.283) (Table 2). Despite the median time from diagnosis to without telomere alterations [n = 5 (62.5%) and n = 7, (87.5%) the LT was lower in those cases with telomere dysfunction (p = respectively]. However, patients with telomere dysfunction 0.076), the time on the waiting list was similar among the patients showed a lower rate of CMV negativity after Valganciclovir (n with and without telomere disorders (p = 0.877) (Table 2). = 4, 44.4%) with a higher rate of hematological complications Frontiers in Medicine | www.frontiersin.org 4 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD TABLE 1 | Patient characteristics. All patients (n = 20) With telomere Without telomere P-value shortening (n = 12) shortening (n = 8) Age at lung transplant [years, mean (SD)] 56.6 (9.39) 51.8 (9.18) 63.8 (3.11) 0.001 Gender (n, %) - Males 15 (75.0) 9 (75.0) 6 (75.0) 1.000 - Females 5 (25.0) 3 (25.0) 2 (25.0) Smoking history at diagnosis (n, %): - Non-smoker 3 (15.0) 2 (16.7) 1 (12.5) 1.000 - Current smoker 3 (15.0) 2 (16.7) 1 (12.5) - Former smoker 14 (70.0) 8 (66.7) 6 (75.0) Diagnosis (n, %): - IPF 8 (40.0) 4 (33.3) 4 (50.0) 0.961 - FPF 8 (40.0) 5 (41.6) 3 (37.5) - HP 1 (5.0) 0 (0.0) 1 (12.5) - CPFE 1 (5.0) 1 (8.3) 0 (0.0) - DIP 1 (5.0) 1 (8.3) 0 (0.0) - CTD-ILD 1 (5.0) 1 (8.3) 0 (0.0) Pre-transplant treatment (n, %): - Pirfenidone 7 (35.0) 2 (16.6) 5 (62.5) 0.062 - Nintedanib 5 (25.0) 3 (25.0) 2 (25.0) 1.000 - Corticosteroids 4 (20.0) 2 (16.6) 2 (25.0) 1.000 - Corticosteroids + immunosuppressive drug 5 (25.0) 5 (41.6) 0 (0.0) 0.055 Telomere shortening (n, %): - n, below 1st percentile 3 (25.0) 3 (25.0) 0 (0.0) NA - n, below 10th percentile 4 (33.3) 4 (33.3) 0 (0.0) - n, below 25th percentile 5 (41.7) 5 (41.7) 0 (0.0) Relative telomere length [mean, (SD)] 1.13 (0.33) 0.95 (0.29) 1.40 (0.17) 0.001 Z-score [mean, (SD)] −1.15 (1.22) −1.83 (1.04) −0.006 (0.44) 0.001 Likely causal variants in telomere-maintenance genes (n =1 8) (n, %): 11 (55.0) 10 (83.3) 1 (12.5) NA - RTEL1 5 (45.5) 5 (50.0) 0 (0.0) - DKC1 2 (18.2) 2 (20.0) 0 (0.0) - PARN 1 (09.1) 0 (0.0) 1 (100) - TERT 3 (27.3) 3 (30.0) 0 (0.0) IPF, idiopathic pulmonary fibrosis; FPF, familial pulmonary fibrosis; HP, hypersensivity pneumonitis; CPFE, combined pulmonary fibrosis and emphysema; DIP, desquamative interstitial pneumonia; CTD-ILD, connective tissue disease-associated interstitial lung disease; NA, not applicable. due to CMV treatment (n = 3, 33.3%) (Table 4). Long-term two deaths reported. One subject died after bilateral LT due to complications are summarized in Supplementary Table 4. The multi-organic failure in the first 24 h; and the other patient died type of complications according to telomere dysfunction and after 17 months of post-transplant follow-up due to stage IIIB time after LT are summarized in Table 5. cervical squamous-cell carcinoma. Both had a telomere length Regarding hospital admissions after LT, patients with telomere below the 10th percentile and one of them carried a rare coding dysfunction presented more hospitalizations (p = 0.033) and 2.9 RTEL1 gene mutation (chr20:62317210). No TRG mutations times higher risk for readmission [Incidence Rate Ratio of 2.91, were identified in the other patient. The median post-transplant 95%CI = 1.11–7.59, (p = 0.029)] (Table 4). Infections were the follow-up period in patients without telomere dysfunction was main cause of readmission in both groups of patients (Table 6). 15 months (range of 12–19) with one death reported after 17 months of unilateral LT due to CLAD. The time from diagnosis Survival and Quality of Life After Lung to LT was shorter in those cases with telomere dysfunction. Transplant Therefore, we also analyzed survival from diagnosis to the end of At the end of the study, 17 of 20 subjects (85%) survived after the study period. No significant differences were found between transplantation: 10/12 with telomere dysfunction (83.3%) and 7/8 both groups; 5 years (range 5–6 years) vs. 6 years (range 4–7 (87.5%) without. Survival after LT is represented in Figure 2A. years), with and without telomere dysfunction, respectively. Patients with telomere shortening had a median post-transplant Post-transplant quality of life assessment reported a significant follow-up period of 39 months (range of 27–50 months) with improvement in respiratory functional and health status for Frontiers in Medicine | www.frontiersin.org 5 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD TABLE 2 | Pre-transplant functional status. All patients (n = 20) With telomere Without telomere P-value shortening (n = 12) shortening (n = 8) LAS [median, (Q1;Q3)] 40.3 (37.3;46.0) 43.1 (38.4;51.3) 39.1 (36.9;41.2) 0.283 Time from diagnosis to transplant [months, mean (SD)] 42.1 (32.1) 36.2 (36.7) 51.0 (23.3) 0.284 Time from diagnosis to transplant [months, median (Q1;Q3)] 33.1 (24.0;50.3) 26.5 (21.9;36.3) 51.2 (30.8;67.6) 0.076 Waiting list time [months, median (Q1;Q3)] 5.00 (1.75;8.00) 5.00 (2.75;7.25) 4.00 (1.00;9.25) 0.877 FVC [L, mean (SD)] 1.87 (0.44) 1.96 (0.39) 1.73 (0.50) 0.294 FVC [%, mean (SD)] 45.3 (11.1) 45.9 (13.8) 44.5 (5.92) 0.764 DLCO [%, mean (SD)] 22.8 (5.71) 21.2 (6.06) 24.7 (5.10) 0.255 Basal SatO2 <90% in WT6m [n, mean (SD)] 79.2 (5.76) 79.2 (5.14) 79.0 (6.97) 0.932 6-min walk test distance [n, mean (SD)] 292 (83.5) 274 (77.7) 319 (89.9) 0.267 Oxygen therapy (n, %) 19 (95) 12 (100) 7 (87.5) 0.400 Time with oxygen therapy before transplant [months, mean (SD)] 12.8 (5.55) 14.3 (6.02) 10.1 (3.63) 0.076 Time with oxygen therapy before transplant [months, median (Q1;Q3)] 12.0 (9.00;15.5) 12.5 (11.0;16.2) 9.00 (7.50;12.5) 0.090 ATAQ score [mean (SD)] 102 (13.3) 108 (8.27) 92.6 (14.4) 0.032 K-BILD score [mean (SD)] 28.4 (15.1) 22.5 (16.0) 36.7 (9.20) 0.036 LAS, lung allocation score; FVC, forced vital capacity; DLCO, diffusion capacity for carbon monoxide; LT, lung transplant. TABLE 3 | Surgical technique and immediate post-operative requirements. All patients (n = 20) With telomere Without telomere P-value shortening (n = 12) shortening (n = 8) Type of lung transplant (n, %): 1.000 - Unilateral 14 (70.0) 8 (66.7) 6 (75.0) - Bilateral 6 (30.0) 4 (33.3) 2 (25.0) Ischemia time [h, mean (SD)] 6.61 (2.82) 6.86 (2.55) 6.23 (3.33) 0.658 Ischemia time [h, median (Q1;Q3)] 6.42 (4.75;7.69) 6.88 (5.25;7.69) 5.42 (4.22;6.83) 0.354 Extracorporeal circulation need (n, %) 4 (20.0) 3 (25.0) 1 (12.5) 0.619 Extracorporeal circulation time [min, median (Q1;Q3)] 122 (88.0;197) 150 (122;244) 70.0 (70.0;70.0) 0.180 Stay in ICU [days, median (Q1;Q3)] 22.0 (7.50;36.5) 30.0 (7.75;40.8) 17.0 (7.00;22.5) 0.352 Post-operative complications in the first 24 h (n, %) 18 (90.0) 10 (83.3) 8 (100) 0.495 Difficult weaning (n, %) (n = 19) 10 (52.6) 6 (54.5) 4 (50.0) 1.000 Tracheostomy (n, %) (n = 19) 10 (52.6) 6 (54.5) 4 (50.0) 1.000 all patients. The mean FVC increase after LT for the entire Lung Transplant in Familial Pulmonary study population was 0.55 L (p = 0.0003) [95%CI: 0.21–0.89]. Fibrosis As shown in Figure 3, pre-transplant FVC-values (L) and the Family aggregation was reported in 8 (40.0%) patients and absence of family aggregation significantly influenced the post- telomere shortening was present in 5 (62.5%) of them. Rare transplant FVC-value (L) (p = 0.007, respectively). Regarding coding TRG mutations were identified in 4 out of 8 (50.0%) the health status, a significant improvement was reported overall patients. A similar pre-transplant functional status was reported through ATAQ-IPF and K-BILD scores, with p < 0.001 for both according to family aggregation without differences in the questionnaires. No differences in ATAQ-IPF and K-BILD post- time from diagnosis to the LT (Supplementary data Table 5). transplant scores were found in telomere dysfunction patient FPF cases presented a non-significant higher rate of difficult group (Table 7). Likewise, no differences were found for declared weaning and a greater need for tracheostomy. No differences daily life activity independence and active life after LT among were found in the ratio of the other complications during patients with and without telomere disorders. Nine out of 10 the first 24 h and the first month. Long-term complications (90%) patients with telomere dysfunction reported daily life were reported in all patients. Those cases without family activity independence, and 6 out of 10 (60%) had an active life aggregation presented a trend of higher rate for mechanical, (p = 1.000). In addition, oxygen therapy withdrawal was possible nephrological, hematological, endocrine, and infectious in 16 out of 17 (94.1%) patients of the overall population: 9 out complications (Supplementary Table 5). Survival after of 10 (90.0%) with telomere dysfunction and 7 out of 7 (100%) transplantation according to family aggregation is represented without it (Table 7). in Figure 2B. Frontiers in Medicine | www.frontiersin.org 6 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD TABLE 4 | Long-term complications after lung transplant. All patients (n = 20) With telomere Without telomere P-value shortening (n = 12) shortening (n = 8) >1 complication after transplant (n, %)* (n = 20) 18 (90.0) 11 (91.7) 7 (87.5) 1.000 Post-operative complications in the first 24 h (n, %) 18 (90.0) 10 (83.3) 8 (100) 0.495 Complications during the first month (n, %) 17 (89.5) 9 (81.9) 8 (100) 0.485 Complications after the first month (n, %) (n = 19) 18 (94.7) 11 (100) 7 (87.5) 0.421 Allograft dysfunction (n, %) 8 (42.1) 3 (27.3) 5 (62.5) 0.181 Nephrological complications (n, %) 7 (35.0) 4 (33.3) 3 (37.5) 1.000 Positive pre-transplant CMV serology (n, %) 18 (90.0) 11 (91.7) 7 (87.5) 1.000 Post-transplant CMV replication (n, %) (n = 19) 14 (73.7) 7 (63.6) 7 (87.5) 0.338 Hematological alterations due to CMV treatment (n, %) (n = 15) 4 (26.7) 3 (33.3) 1 (16.7) 0.604 CMV negativity after treatment (n, %) (n = 16) 9 (56.2) 4 (44.4) 5 (71.4) 0.358 Infectious complications (n, %) 12 (60.0) 6 (50.0) 6 (75.0) 0.373 Hematological complications (n, %) 6 (30.0) 4 (33.3) 2 (25.0) 1.000 Endocrine complications (n, %) 15 (75.0) 8 (66.7) 7 (87.5) 0.603 Hospital admissions after transplant [median (Q1;Q3)] (n = 19) 1.0 (0.00;3.00) 2.00 (1.00;3.00) 0.00 (0.00;1.25) 0.033 * >1 complication at any time after lung transplant. TABLE 5 | Post-transplant complications according to telomere dysfunction and time after lung transplantation. Type of post-transplant complications First 24 h During the first month After the first month Telomere No telomere Telomere No telomere Telomere No telomere shortening shortening shortening shortening shortening shortening Endocrine (n, %) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 8 (67) 7 (88) Post-transplant CMV replication (n, %) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 7 (58) 7 (88) Infectious (n, %) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 6 (50) 6 (75) Allograft dysfunction (n, %) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3 (25) 5 (62) Hemodynamic dysfunction (n, %) 4 (33) 3 (38) 3 (25) 3 (38) 0 (0.0) 0 (0.0) Hematological (n, %) 4 (33) 0 (0.0) 2 (17) 0 (0.0) 4 (33) 2 (25) Mechanical (n, %) 3 (25) 3 (38) 2 (17) 3 (38) 0 (0.0) 0 (0.0) Primary graft dysfunction (n, %) 2 (17) 1 (12) 2 (17) 1 (12) 0 (0.0) 0 (0.0) Donor lung infection (n, %) 0 (0.0) 1 (12) 0 (0.0) 1 (12) 0 (0.0) 0 (0.0) Atrial fibrillation (n, %) 2 (17) 0 (0.0) 1 (8) 0 (0.0) 0 (0.0) 0 (0.0) Renal (n, %) 1 (8) 1 (12) 0 (0.0) 0 (0.0) 4 (33) 3 (38) Miopathy of critical patient (n, %) 1 (8) 0 (0.0) 1 (8) 0 (0.0) 0 (0.0) 0 (0.0) DISCUSSION Our main finding was the benefit of LT in survival of fibrotic ILD regardless of telomere dysfunction. We report a good Previous studies have analyzed LT morbidity and mortality LT outcome in the overall study population with a survival in patients with telomere abnormalities with controversial rate >80% and a similar post-transplant follow-up. Previous results (19–25). Our study reveals for the first time a long- studies have also evaluated the impact of transplantation for term positive LT outcome in overall fibrotic ILD population pulmonary fibrosis in ILD patients carrying telomere disorders. regardless of telomere dysfunction. Furthermore, we report Swaminathan et al. reported a significantly higher risk of death the order in appearance and the frequency of the subsequent for patients carrying variants in TERT, RTEL1, or PARN (25); complications according to elapsed time from transplantation while Newton et al. showed the association between telomere and presence of telomere dysfunction. Therefore, while a length below the 10th percentile and worse post-transplant higher rate of LT complications has previously been reported survival (23). Although this poor outcome was related to systemic in patients with telomeropathy (19–25), our study highlights complications of telomere dysfunction, Borie et al. reported no the long-term benefits of LT despite telomere dysfunction differences in survival between controls and telomerase mutation by optimizing the knowledge and the management of carriers with higher risk for hematological complications (21). transplant morbidity. Similarly, Silhan et al. also described a reasonable post-transplant Frontiers in Medicine | www.frontiersin.org 7 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD TABLE 6 | Causes for hospital admission after lung transplant. Patient Family Percentile Likely causal Hospital admissions Infectious cause Other causes aggregation telomere length gene(s) after transplant PATIENTS WITHOUT TELOMERE SHORTENING 1 Yes 75–50th Not tested 1 Urinary sepsis None 2 No 50–25th Not tested 1 Lower right lobe pneumonia (P. None aeruginosa) 3 No 50–25th Not tested 1 Respiratory infection (Rhinovirus) CLAD PATIENTS WITH TELOMERE SHORTENING 4 No <25th TERT 1 None Cervical lymphadenectomy (M1 squamous cell carcinoma of unknown primary origin) 5 Yes <25th TERT 3 Respiratory infection (MRSA) Suspected unconfirmed CLAD Empyema (S.aureus) 6 No <25th RTEL1 3 Acute gastroenteritis None Elbow abscess Influenza A virus infection 7 Yes <25th DKC1 3 CMV viremia Fever of unknown origin Lower right lobe pneumonia + non-complicated parapneumonic pleural effusion 8 No <10th RTEL1 1 None CLAD 9 No <10th Not found 2 None Pulmonary embolism Cervix squamous cell carcinoma stage IIIB 10 No <10th RTEL1 3 Respiratory infection (Respiratory None syncytial virus) Bacteremia (P.aeruginosa) Influenza b virus infection 11 No <1th DKC1 6 Respiratory infection (x2) Acute ILD exacerbation in native Perianal condylomatosis lung ILD progression in native lung (X2) 12 Yes <1th Not found 1 Cavitated pneumonia None (P.aeruginosa) + bacteriemia (P.aeruginosa) survival despite a higher rate of post-transplant complications overall study population despite the younger mean age of the (20). In addition, LT feasibility was also assessed for other telomere dysfunction group of patients, the fibrotic ILD and/or telomeropathies like dyskeratosis congenital (45) and in the case the pre-transplant treatment prescription. of multiple solid organ transplantation (46) with a favorable An important issue when considering survival is to investigate long-term post-transplant outcome. the quality of patient survival. Although morbidity and mortality The controversial results reported so far are probably due have previously been assessed in fibrotic ILD with telomeropathy to a wide dispersion of methods and lack of control group in (20–25), knowledge is lacking about outcomes in terms of quality most studies (19–25). Moreover, a broad variety of transplant of life. Our study describes for the first time the post-transplant centers were involved in the previous studies with great benefits in quality of life despite telomere disorders and their heterogeneity of national organ transplant programs and center- complications. The enhancement of post- transplant quality of specific pre-surgical, surgical, and post-surgical procedures. Very life was reported for the overall study population through the few randomized studies have been conducted in LT for the improvement of ATAQ and K-BILD scores and the feasibility for general population leaving some topics with a lack of evidence independent and active life with oxygen withdrawal. Hence, our and outcomes far from optimal for many patients (47). Hence, study shows quality of life gains for transplanted ILD patients making decisions in LT is sometimes highly controversial and regardless of telomere dysfunction. requires an evidence-experience balance. Considering these However, this favorable effect needs to be balanced arguments, we prospectively assessed LT outcomes according to with the higher rate of post-transplant morbidity reported telomere dysfunction as the only differential biological marker for telomeropathies. Therefore, our research thoroughly among recipients in our single-transplantation center cohort. No investigates the morbidity profiles according to elapsed time differences were found in pre-transplant functional status for the from transplantation. The post-transplant appearance and Frontiers in Medicine | www.frontiersin.org 8 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD FIGURE 2 | (A) Post-transplant survival according to presence or absence of telomere dysfunction. Patients with telomere dysfunction had a greater post-transplant follow up period due to an earlier requirement of lung transplant after pulmonary fibrosis diagnosis. As previously described in Table 2, a shorter time from diagnosis to transplantation was reported for patients with telomere dysfunction. (B) Survival after lung transplant according to family aggregation. A worse post-transplant survival was reported for family aggregation patients group during the first year. However, at 24 months, post-transplant survival was ≤50% regardless of family aggregation. Since genetic studies have not yet been implemented in routine clinical practice in most centers, further studies are needed to evaluate family aggregation as an outcome predictor regardless of the underlying genetic defect. FIGURE 3 | Post-transplant FVC (L) models. We analyzed the impact in post-transplant FVC of the following variables: the pre-transplant FVC, the age at the time of lung transplantation and the absence of telomere dysfunction and pulmonary fibrosis family aggregation. A statistically significant impact in post-transplant FVC was reported for pre-transplant FVC and the absence of family aggregation. The absence of telomere dysfunction did not statistically impact in post-transplant FVC. Frontiers in Medicine | www.frontiersin.org 9 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD TABLE 7 | Quality of life after lung transplant. All patients (n = 20) With telomere Without telomere P-value shortening (n = 12) shortening (n = 8) Post-transplant FVC [ L, mean (SD)] 2.42 (0.77) 2.31 (0.51) 2.57 (1.05) 0.524 Post-transplant FVC [%, mean (SD)] 60.4 (17.6) 53.0 (12.7) 70.4 (19.0) 0.045 Post-transplant ATAQ score [mean (SD)] 58.2 (12.9) 60.6 (15.0) 54.7 (9.16) 0.333 Post-transplant K-BILD score [mean (SD)] 60.1 (9.14) 60.1 (9.66) 60.0 (9.10) 0.983 Oxygen withdrawal after transplantation (n, %) 16 (94.1) 9 (90.0) 7 (100) 1.000 Independence for daily life activities after transplantation (n, %) 15 (88.2) 9 (90.0) 6 (85.7) 1.000 Active life after transplantation (n, %) 11 (64.7) 6 (60.0) 5 (71.4) 1.000 Independence for daily life activities: feeding, personal hygiene and dressing tasks without help. Active life: to be able to return to work and/or hobbies. predominance of complications could be chronological and ETHICS STATEMENT change according to successive clinical requirements after transplantation and patient susceptibility. Telomere dysfunction The studies involving human participants were reviewed is associated with immunodeficiency (28) and poor outcomes and approved by Ethics Committee of Hospital Universitari of immunosuppressive drugs (26, 27). Thereby, it could explain de Bellvitge. The patients/participants provided their written our higher rate of hospital admissions after transplantation informed consent to participate in this study. for the telomere dysfunction group due to a wide variety of infections. The post-transplant antibiotic prophylaxis in these AUTHOR CONTRIBUTIONS cases is a hypothesis that still needs to be evaluated. The CMV post-transplant prophylaxis performed could explain a similar The participation level of each author is the following: LP-C and rate of CMV relapsing viremia in those cases with or without MM-M had full access to all of the data in the study, and take telomere dysfunction, deviating from previous reports (24). responsibility for the integrity of the data and the accuracy of Although a similar effect, CMV prophylaxis toxicity was mainly the data analysis, including and especially any adverse effects, and reported in telomere dysfunction cases as thrombocytopenia or contributed to statistical analysis and interpretation. LP-C served leukopenia. Hematological complications are highly associated as principal author. MM-M served as corresponding author. with telomeropathy (20, 21) and may complicate further due LP-C, EA-S, CB, RP, AM-W, CF, AR, and MM-M contributed to ECC in the early post-transplant period and long-term to the study concept and design. LP-C, CB, VV-Z, MG-M, drug toxicities. AR, and MM-M contributed to identification and inclusion of The main limitations of the present study are the small sample patients and clinical data collection. LP-C, EA-S, AM-W, RG-M, size of our series and the lack of a validation cohort. However, JL-S, VV-Z, MG-M, JD, CF, RP, AR, and MM-M contributed to LT experience and protocols may differ among centers, which patient’s sample collection and processing. LP-C, EA-S, AM-W, represents a major difficulty for homogeneously increasing the RG-M, JL-S, CF, RP, and MM-M contributed to analysis and number of cases or finding a proper validating cohort with interpretation of data for the work. LP-C, EA-S, CB, RG-M, JL-S, patients screened for telomere length at diagnosis. AM-W, CF, RP, AR, and MM-M contributed to the writing of Hence, increasing the knowledge about when and why the manuscript. All authors contributed to review critically of the each complication appears would set specific prevention and draft and approved the final manuscript. management strategies in order to tip the balance in favor of survival benefits. Pre-transplant work-up considerations should include telomere disorders in order to establish variations FUNDING in care (48). However, wider evidence is clearly needed This study was funded by Instituto de Salud Carlos III to identify interventions that work best for this subset of through project PI18/00367 (Co-funded by European Regional ILD patients. Development Fund, ERDF, a way to build Europe), Spanish In conclusion, our results support the survival gains of lung Society of Respiratory (SEPAR), Barcelona Respiratory Network transplant regardless of telomere disorders. (BRN), and Fundació Ramón Pla Armengol. RP laboratory was funded by grants PI20-00335 (Fondo de Investigaciones DATA AVAILABILITY STATEMENT Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds). MM-M was funded by grants PI18/00367 The original contributions presented in the study are (Fondo de Investigaciones Sanitarias, ISCIII, Spain, supported publicly available. This data can be found in the European by FEDER funds), AC19/00006 (Projects of International Variation Archive (EVA), accession numbers PRJEB46414 Programs, ISCIII, Spain, supported by FEDER funds), Cohorte and ERZ2875159. FPI CIBERES-ISCIII, Barcelona Respiratory Network-Fundation Frontiers in Medicine | www.frontiersin.org 10 July 2021 | Volume 8 | Article 695919 Planas-Cerezales et al. Transplant Gains in Telomere-Related ILD Ramon Pla Armengol, Spanish Society of Respiratory (SEPAR), HUB-ICO-IDIBELL (PT17/0015/0024) integrated in the and Catalan Society of Respiratory (SOCAP-FUCAP). CF was Spanish National Biobanks Network for their collaboration; funded by Ministerio de Ciencia e Innovación (grant RTC- and CERCA Programme/Generalitat de Catalunya. We are 2017-6471-1; AEI/FEDER, UE), and by Cabildo Insular de also grateful to all the patients involved in the study and Tenerife (CGIEU0000219140). their families. ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL The authors thank Josep M Palma, nurse specialist The Supplementary Material for this article can be found for ILD Unit of Hospital Universitari de Bellvitge, for online at: https://www.frontiersin.org/articles/10.3389/fmed. his contribution obtaining blood samples; the Biobank 2021.695919/full#supplementary-material REFERENCES 17. Newton CA, Batra K, Torrealba J, Kozlitina J, Glazer CS, Aravena C, et al. Telomere-related lung fibrosis is diagnostically 1. Cottin V, Wollin L, Fischer A, Quaresma M, Stowasser S, Harari S. Fibrosing heterogeneous but uniformly progressive. Eur Respir J. 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DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, distribution or reproduction in other forums is permitted, provided the original et al. A framework for variation discovery and genotyping using next- author(s) and the copyright owner(s) are credited and that the original publication generation DNA sequencing data. Nat Genet. (2011) 43:491–8. doi: 10.1038/ in this journal is cited, in accordance with accepted academic practice. No use, ng.806 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Medicine | www.frontiersin.org 12 July 2021 | Volume 8 | Article 695919