Key words : Acute cellular rejection, Hemorrhage, Immunosuppression, mTOR inhibitors, Wound healing

Introduction
Lung transplantation is a life-saving procedure for patients with end-stage lung disease, but management of postoperative immunosuppression remains a challenging process. Standard triple immunosuppressive regimens often include corticosteroids, mycophenolate mofetil (CellCept), or mycophenolate sodium (Myfortic) and calcineurin inhibitors (CNI) such as tacrolimus.¹

Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) and derivative of sirolimus, can be considered in several scenarios in lung transplant recipients. First, everolimus is often used in patients who experience intolerance to mycophenolate mofetil or mycophenolate sodium, such as those with abdominal side effects or severe leukopenia or neutropenia.^2-4 In addition, everolimus can be used as a CNI minimization/elimination agent and is an important option for patients with renal insufficiency, as it allows for a reduction in tacrolimus dosage by either replacing it or by use in combination with low-dose tacrolimus,^5-9 although a clear long-term benefit on renal function could not be demonstrated in some studies.^4,10 Moreover, in cases where lung transplant recipients continue to develop donor-specific antibodies despite receiving standard triple immunosuppressive therapy, everolimus may be added as part of quadruple therapy, to prevent antibody-mediated rejection.¹¹ Everolimus can also be used in the prevention^12,13 and treatment¹⁴ of chronic lung allograft dysfunction (CLAD) as add-on therapy to the standard triple immunosuppression. Everolimus has also been recommended for recurrent cytomegalovirus infections¹⁵ and, because of its antiangiogenic properties, as a CNI alternative option in posttransplant de novo malignancies.¹⁶

These broad applications of everolimus highlight the versatility of this agent in addressing both immunosuppressive needs and complications after lung transplant. Everolimus has been shown to reduce the risk of chronic rejection, especially in patients who form donor-specific antibodies, which may indicate a higher risk of graft rejection. Everolimus treatment has also been shown to prevent decline in forced expiratory volume in 1 s in patients with CLAD.¹⁷ Everolimus exerts its immunosuppressive effects by suppressing lymphocyte proliferation, reducing the expression of proliferative cytokines, and inhibiting fibroblast proliferation. However, concerns regarding the use of everolimus in lung transplant recipients also exist. A rare but potentially severe adverse event could be everolimus-induced pneumonitis, presenting with an organizing pneumonia, a nonspecific interstitial pneumonitis-like pattern, or both, and can be difficult to differentiate from CLAD.^18-21

Although everolimus has proven effective in preventing rejection, its inhibition of fibroblast proliferation raises concerns about wound healing. In particular, in the early (<3 mo) postoperative phase, the mTOR inhibitor sirolimus has been associated with anastomotic dehiscence.^20,22,23 Thus, initiation or mTOR inhibitors, such as sirolimus and everolimus, should be delayed until 3 months after transplant.²³

This concern is especially relevant for lung transplant recipients who undergo frequent diagnostic procedures, such as surveillance bronchoscopies, including transbronchial forceps, but sometimes also cryobiopsies, within the first postoperative year. Cryobiopsies are associated with larger tissue extraction compared with traditional transbronchial forceps biopsies, which raises concerns about complications, like pneumothorax, bleeding, and impaired tissue repair. Existing literature highlights that mTOR inhibitors, such as everolimus and sirolimus, can impede wound healing by inhibiting collagen synthesis, deposition, and angiogenesis and thus may contribute to wound complications. Despite these potential risks, studies on safety of everolimus during minimally invasive procedures such as bronchoscopy, gastroscopy, or colonoscopy are scarce.

In this study, we investigated whether the use of everolimus in lung transplant recipients correlates with an increased incidence of procedural complications, such as massive bleeding complications or persistent pneumothorax from delayed wound healing, particularly in the context of cryobiopsy. In our hospital, we do not discontinue everolimus before patients receive a surveillance bronchoscopy with cryobiopsy. Our aim was to provide critical insights into the safety profile of everolimus in lung transplant recipients undergoing minimally invasive procedures, specifically bronchoscopy with cryobiopsy. These findings may inform clinical guidelines on the use of everolimus in transplant patients at risk for procedural complications.

Materials and Methods
Study design and setting: This retrospective cohort study was conducted at the University Hospital Zurich in Zurich, Switzerland. We reviewed medical records of patients who underwent lung transplant between January 2022 and December 2023. We included patients who had undergone at least 1 surveillance bronchoscopy with transbronchial forceps and/or cryobiopsy during the first postoperative year. The study included 2 groups: patients who received everolimus as part of their immunosuppressive regimen and patients who were on standard CNI-based immunosuppression without everolimus. In the treatment protocols, everolimus could be used either as a substitute for antimetabolites or CNIs, to allow a reduction in the dosage of antimetabolites or CNIs, or as an addition to the conventional triple immunosuppression regimen (typically including a CNI, an antimetabolite, and corticosteroids). We identified 105 bronchoscopic procedures for our analysis. All patients provided written informed consent, and the regional Ethics Committee of Canton Zurich approved the protocol of the current study (BASEC No. 2024-02121).

Data collection: We collected baseline demographic and clinical data for all included patients, including age at transplant, sex, and immunosuppressive medications (Table 1). We used descriptive statistics to summarize baseline characteristics and presented categorical variables as frequencies and percentages and continuous variables as means ± SD. To minimize confounding factors, we compared baseline demographic and clinical characteristics, including age, sex, and underlying medical conditions, between the 2 groups. We reviewed perioperative and procedural protocols, such as antibiotic prophylaxis, sedation, and anticoagulation management. We also reviewed pre-bronchoscopy coagulation profiles and laboratory assessments to ensure standardization.

Bronchoscopic procedure: All bronchoscopies were performed as in-patient procedures, which is the standard of care for lung transplant recipients at our hospital. Patients routinely received prophylactic intravenous antibiotics, starting 1 day before the procedure and continuing until the day after. Laboratory assessments were conducted the day before bronchoscopy, and the procedure was canceled for patients with leukopenia, neutropenia, thrombocytopenia (<100 G/L), or elevated C-reactive protein levels. Bronchoscopy was also contraindicated in patients with a fever, defined as a body temperature exceeding 38.4 °C.

Vitamin K antagonists were discontinued 5 to 7 days before the procedure. If a patient’s international normalized ratio exceeded 1.3, vitamin K was administered to reduce the international normalized ratio to ≤1.3 on the day of the bronchoscopy. Local anesthesia and moderate sedation with propofol were used for all procedures. Flexible Olympus bronchoscopes (190 series) were introduced through a 7.5-mm uncuffed tracheal tube (BronchoFlex, Rüsch) to prevent laryngeal injury during cryobiopsy sample extraction. Each procedure began with a standardized bronchoalveolar lavage of 150 to 200 mL of normal saline, followed by both forceps biopsy and cryobiopsy during the same session. Forceps biopsy and cryobiopsy were performed unilaterally in different lung segments, with 5 forceps biopsies and 2 cryobiopsy specimens collected. In cases of moderate to severe bleeding, xylometazoline (2 mg) and tranexamic acid (500 mg) were administered via the bronchoscope. A bronchial blocker (Rüsch, size Ch.6) was kept on hand but not routinely used.

The severity of bleeding was graded by the Nashville Bleeding Scale.²⁴ Grade 1 bleeding requires suctioning of blood for <1 minute, whereas grade 2 bleeding necessitates suctioning for >1 minute, along with repeated wedging or the instillation of vasoactive substances or thrombin. Grade 3 bleeding is characterized by the premature interruption of the procedure, insertion of a balloon blocker for <20 minutes, or selective intubation. Grade 4 bleeding is defined as selective intubation or balloon blocker insertion lasting >20 minutes, in addition to red blood cell transfusion, selective bronchial artery embolization, admission to the intensive care unit, surgical intervention, or resuscitation.^24,25

Outcomes and variables: The primary outcome of this study was the incidence of bleeding complications (bleeding: yes/no) following bronchoscopy. This binary outcome was used to compare the incidence of bleeding between patients on everolimus and those on a standard triple immunosuppression regimen without everolimus.

In a secondary subanalysis, we examined the severity of bleeding complications based on the Nashville classification system: grade 0 (no bleeding), grade 1 (very light), grade 2 (moderate), grade 3 (severe), and grade 4 (very severe). This subanalysis allowed us to assess differences in the distribution of specific bleeding grades between the everolimus and non-everolimus groups.

Statistical analyses: To evaluate differences in the incidence of bleeding (yes/no) between the everolimus and non-everolimus groups, we used the X² test. For the subanalysis comparing the distribution of specific bleeding grades (Nashville grades 0 through 4) between the groups, we also applied the X² test. P < .05 was considered significant for all analyses. We used SPSS software (IBM SPSS Statistics, version 29.0.2) for statistical analyses.

Results
The incidence of bleeding complications following bronchoscopy showed no significant difference between patients who were on everolimus therapy and those who were not (Table 2). In the subanalysis, the distribution of bleeding severity grades, according to the Nashville classification system, did not reveal significant differences between the everolimus and non-everolimus groups. Among the 105 reviewed bronchoscopies, 41 procedures were in patients on everolimus therapy (205 forceps biopsies and 82 cryobiopsies) and 64 procedures were in patients not on everolimus (320 forceps biopsies and 128 cryobiopsies), totaling 525 forceps biopsies and 210 cryobiopsies. A minor pneumothorax was observed in 1 patient not receiving everolimus; this instance did not require treatment and was managed with in-hospital observation and supplemental oxygen. No intensive care unit admissions or unscheduled hospitalization days were associated with any of the bronchoscopies.

Discussion
Our study found that procedure-related complications during surveillance bronchoscopy with forceps and cryobiopsies in lung transplant recipients did not differ significantly between those on everolimus and those not on it. Specifically, 68.3% of patients in the everolimus group and 60.9% in the non-everolimus group experienced no bleeding, a difference that was not significant. In cases where bleeding did occur, bleeding was not life-threatening and predominantly Nashville grade 2, with 26.8% of patients on everolimus and 34.4% without everolimus experiencing this level of bleeding, also without significant difference. Severe (Nashville grade 3) bleeding occurred in 2 patients (1 from each group), without statistical significance, and no Nashville grade 4 bleeding was observed in any patients.

Delayed wound healing is a serious adverse event of everolimus in solid-organ transplant recipients. This adverse event is the rationale for not using everolimus within the first 3 months after lung transplant. After this period, everolimus can be a valuable drug in certain lung transplant recipients. Two prior studies specifically explored the effects of sirolimus, an mTOR inhibitor like everolimus, on lung transplant recipients and showed significant issues related to bleeding complications.^20,23 Sirolimus, or rapamycin, is the original mTOR inhibitor derived from Streptomyces hygroscopicus, and everolimus is a derivative with slight modifications with enhanced solubility and distinct pharmacological properties. These differences might partially explain why our findings for everolimus did not replicate the severe complications observed with sirolimus. Sirolimus has a longer half-life (~60 h), leading to prolonged systemic exposure and delayed dose adjustments, whereas everolimus, with a half-life of ~30 hours, is eliminated more rapidly, enabling quicker titration. Everolimus is also more commonly used in lung transplant due to a more favorable safety profile, including lower rates of pulmonary complications and wound healing issues, compared with sirolimus.

In the studies by Groetzner and colleagues²³ and King-Biggs and colleagues,²⁰ early administration of sirolimus after lung transplant was shown to be linked to severe complications. In their 2004 pilot study, Groetzner and colleagues found that, among 4 patients on sirolimus immediately posttransplant, 3 experienced significant wound healing problems and 2 developed fatal bronchial dehiscence.²³ In their 2003 study, involving 15 lung transplant recipients, King-Biggs and colleagues showed that 4 experienced airway complications, leading to the death of 3 transplant recipients, prompting the cessation of the trial.²⁰ Animal models might provide further insights into the mechanistic effects of everolimus on tissue healing and bleeding risk.

To further investigate the potential effects of everolimus on tissue healing, we propose conducting a histopathological comparison of biopsy materials from groups with and without everolimus. This would allow differences in tissue architecture, inflammation, and healing responses to be assessed between transplant recipients with everolimus and without everolimus treatment. However, such an analysis was beyond the scope of our study.

Although delayed wound healing is a well-documented adverse event of everolimus, other mechanisms may also contribute to bleeding risk. Everolimus affects cellular proliferation and angiogenesis, which can influence vascular integrity and platelet function. For example, in Kirchner and colleagues, the pharmacological effects of everolimus on coagulation pathways and endothelial health were highlighted.²⁶ These mechanisms may exacerbate the risk of bleeding during invasive procedures, independent of tissue healing delays.

An intriguing question not addressed in our study is the use of sirolimus in patients with lymphangioleiomyomatosis (LAM) who are listed for lung transplant. Since the publication of the Multicentre International LAM Efficacy of Sirolimus (MILES) trial in 2011,²⁷ sirolimus has become the standard treatment for LAM and was approved by the Food and Drug Administration in 2015, marking it as the first effective therapy for these patients.²⁸ Consequently, many patients with LAM remain on sirolimus therapy at the time of transplant. Previous studies have shown that the use of sirolimus in the early postoperative period significantly increases the risk of complications, including delayed wound healing and severe airway issues. This presents a unique challenge in management of immunosuppression for patients with LAM undergoing transplant.

These outcomes shown in Groetzner and colleagues²³ and King-Biggs and colleagues²⁰ highlight the risks associated with sirolimus’s pharmacokinetics, suggesting that its slower clearance and longer systemic presence may contribute to higher rates of complications compared with everolimus. Our study supported that everolimus, with its unique pharmacological profile, does not show these severe bleeding risks during bronchoscopy, making it a safer alternative posttransplant compared with sirolimus.

The question of whether everolimus should be temporarily discontinued for nonemergency diagnostic procedures, such as routine surveillance bronchoscopies, is still under consideration. The standard approach for surveillance bronchoscopy, as recommended by the International Society of Heart and Lung Transplantation (ISHLT), typically involves forceps biopsies.²⁹ However, at our hospital, we also use cryobiopsies. According to Steinack and colleagues, cryobiopsies improve the diagnostic yield for detecting acute cellular rejection.²⁵

The macroscopic differences between forceps biopsies and cryobiopsies are illustrated in Figure 1, which depicts cryobiopsies on the left and forceps biopsies on the right. Histological specimens corresponding to the cryobiopsies and forceps biopsies are presented in Figure 2 and Figure 3, respectively. These images clearly demonstrate that cryobiopsies are larger and contain a greater number of alveoli compared with forceps biopsies. Notably, in this example, acute cellular rejection (ISHLT grade A3) was identified exclusively in the cryobiopsy, whereas it was undetectable in the forceps biopsy. Both specimens were stained with hematoxylin and eosin. This emphasizes the potential of cryobiopsy to provide superior diagnostic accuracy in lung transplant recipients. However, one of the most commonly reported complications associated with transbronchial lung cryobiopsy is bleeding, along with pneumothorax.

Several studies have highlighted the risk of severe, potentially life-threatening hemorrhage associated with cryobiopsy.^30,31 Initial reports have suggested that up to 12% of patients experience significant bleeding immediately after the procedure, including life-threatening hemorrhages. However, in high-volume centers with experienced practitioners, cryobiopsy has been shown to have an acceptable safety profile, with complications such as hemodynamic instability, intensive care unit admission, blood transfusion, embolization, or death being rare.³²

In line with these findings, our study observed no evidence of life-threatening bleeding, despite a large number of procedures performed on immunocompromised patients, many of whom were also taking everolimus. As a high-volume academic lung transplant and interventional center, our results suggest that the use of everolimus may be safe in the context of these procedures.

Our study had some limitations. First, the study was conducted in a single center, which could limit the generalizability of the results to other institutions with different patient populations, clinical practices, and procedural expertise. Second, a retrospective study may have inherent biases, such as selection bias or incomplete data capture, which can affect the reliability of the conclusions. Third, the number of biopsies performed may not be large enough to detect rare complications or events. Fourth, our study focused specifically on lung transplant recipients, which may not apply to other patient populations undergoing bronchoscopy for different indications. Fifth, although our study demonstrated that everolimus did not increase the risk of complications during surveillance bronchoscopy (with forceps and cryobiopsy), these findings may not be directly applicable to lung transplant recipients undergoing other types of biopsy procedures, such as those performed during colonoscopy (eg, for polyp removal). Finally, the safety profile of everolimus could vary based on the type of tissue sampled, the procedure involved, and the associated risks.

Conclusions
The use of everolimus in lung transplant recipients did not appear to increase the risk of procedure-related complications during surveillance bronchoscopy, including both forceps and cryobiopsies. The incidence of bleeding, whether minor or severe, was similar between patients on everolimus and those not on the drug, with no observed cases of life-threatening bleeding. These findings suggest that, when used appropriately, everolimus does not pose additional risks for patients undergoing these procedures.

Given the absence of significant adverse events in our study and considering the established safety profile of everolimus, our findings support its continued use in lung transplant recipients, even during procedures like surveillance bronchoscopy with forceps and cryobiopsy.

In summary, our study contributed to the growing body of evidence that everolimus is a safe and effective treatment for lung transplant recipients, including those undergoing routine diagnostic procedures like surveillance bronchoscopy and cryobiopsy. From our perspective from a high-volume academic center specializing in lung transplant, we believe that everolimus can be safely maintained in these patients without compromising their safety during such procedures.

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