Bronchiolitis obliterans syndrome (BOS) is a pulmonary fibroproliferative disorder characterized by irreversible narrowing and obliteration of the small airways. (Barker et al., 2014). In the setting of allogeneic hematopoietic stem cell transplantation (allo-HSCT), BOS is the most common pulmonary manifestation of moderate/severe chronic graft versus host disease (cGVHD), and the overall prevalence of BOS is up to 14% among patients who develop cGVHD (Au et al., 2011). BOS remains a leading cause of late morbidity and mortality as well as impaired quality of life after allo-HSCT. (Dudek et al., 2003; Williams et al., 2009; Chien et al., 2010; Barker et al., 2014).

Historically, systemic corticosteroids have formed the backbone of therapy for BOS, but their side effect profile makes them disadvantageous for prolonged use. Combination of inhaled corticosteroids and bronchodilators, azithromycin and montelukast early in BOS therapy could reduce total corticosteroids exposure (Bergeron et al., 2015; Kim et al., 2016; Williams et al., 2016). Other regimens include calcineurin inhibitors (CNIs); extracorporeal photopheresis (ECP) (Lucid et al., 2011; Del Fante et al., 2016; Hefazi et al., 2018); rituximab; (Brownback et al., 2017); etanercept; (Yanik et al., 2012); and tyrosine kinase inhibitors, mainly for their inhibitory effect on fibrosis via the platelet-derived growth factor pathway (Watanabe et al., 2015; Bergeron et al., 2017; Watanabe et al., 2017). Young patients with refractory severe BOS despite all interventions and best supportive care, may be evaluated for lung transplantation (Vogl et al., 2013; Greer et al., 2018). However, no consensus has been reached regarding the optimal therapy for BOS because of suboptimal responses to medical therapies, and effective therapeutic strategies are therefore needed to stabilize lung function, reduce corticosteroid dosing, and improve quality of life for patients with BOS after HSCT.

Ruxolitinib, a Janus-associated kinase (JAK) 1/2 inhibitor approved for the treatment of patients with intermediate-2 or high-risk primary myelofibrosis (Harrison et al., 2012), has shown excellent response rates in steroid-refractory GVHD (Zeiser et al., 2015; Khoury et al., 2018; Zhao et al., 2020a; Escamilla Gómez et al., 2020; Wu et al., 2021). Presently, studies on ruxolitinib administration in patients with BOS are still in a preliminary stage, with only two published case series, which indicated ruxolitinib is an effective steroid sparing agent in BOS due to cGVHD (Schoettler et al., 2019; Streiler et al., 2020). However, the effect of ruxolitinib on pulmonary function, its toxicity profile, and factors affecting its response have not been fully determined. All these concerns must be addressed urgently. Thus, this retrospective study aimed to investigate whether ruxolitinib therapy would lead to stabilization or even improvement in pulmonary function in patients with steroid-refractory BOS. Additionally, it sought to observe the infectious events during ruxolitinib therapy. It also determined the effect of ruxolitinib in corticosteroid dosing in these patients and the clinical factors affecting the response to ruxolitinib.

Any grade hematological toxicities were observed in 4 (13.3%) patients, and for grade ≥3 cytopenia, only one patient had grade ≥3 anemia. Regarding the nonhematological toxicities, two patients developed grade 3 hypertriglyceridemia. Infection-related events were frequent in the present cohort. Of the 30 patients, 100% were cytomegalovirus (CMV) IgG positive, with a CMV-seropositive donor. CMV reactivation indicated as CMV DNAemia was observed in three patients (10%). Epstein–Barr virus (EBV) reactivation was observed in 11 patients (36.7%). None of the patients developed CMV disease or EBV post-transplant lymphoproliferative disorder. Hepatitis B virus was re-activated in two patients (6.7%), both of whom were hepatitis B surface antigen-negative (HBsAg-)/hepatitis B core antibody-positive (HBcAb+)/hepatitis B surface antibody negative (HBsAb-) without any hepatitis B prophylaxis. Fourteen patients (46.7%) were complicated with pulmonary infection ≥2 grade, including invasive pulmonary fungal diseases in 10 patients, pulmonary bacterial in three patients, and mixed infection (nocardiosis/Aspergillus fumigatus/Klebsiella pneumoniae) in one patient. The risk for pulmonary fungal infection was 54.5% in fluconazole prophylaxis group and 26.3% in voriconazole or posaconazole group (p = 0.238). The incidence of pulmonary infection was higher in the nonresponders than in the responders (89.0 vs. 46.7%, p = 0.019). No bacteremia occurred in this cohort.

After a median follow-up of 318 (range, 53–730) days after ruxolitinib initiation, 4 out of 30 patients experienced a relapse of primary malignancy, including three bone marrow relapse and one with isolated extramedullary relapse. The cumulative 1-year incidence of relapse of the underlying malignancy was 14.7% (95%CI: 4.4–30.9%) after ruxolitinib. Eventually, eight patients died (26.7%), with causes of death predominantly resulting from infection (severe pulmonary infection/respiratory failure, n = 4; severe hepatitis B/hepatorenal syndrome, n = 1) and bone marrow relapse of original malignant disease (n = 3). The 2-years incidence of NRM and 2-years OS rate after ruxolitinib among patients with BOS were 25.1% (95%CI: 8.8–45.5%) and 62.6% (95%CI: 45.0–80.2%), respectively. There is no statistically significant difference in survival rate in ruxolitinib-responders and no responding patients (1-year OS rate: 72.9 vs. 42.9%, p = 0.27) (Figure 4).

Ruxolitinib, a potent anti-GVHD drug, might be a promising therapeutic option for BOS after HSCT. The present study added to a growing body of evidence for the use of ruxolitinib in SR-BOS related to cGVHD. In these heavily pretreated patients with BOS, ruxolitinib induced a relatively high response (ORR 66.7%), including FEV1 improvement and steroid sparing. The 2-years OS rate after ruxolitinib among patients with BOS was 62.6%. The present study suggests such heavily pretreated patients might benefit from salvage therapy with ruxolitinib. More importantly, relapse of the underlying malignancy in ruxolitinib-exposed patients was low (14.7%). This frequency is comparable to other currently applied immunosuppressive drugs, and other data from murine models proved that ruxolitinib does not impair the graft-vs.-leukemia (GvL) activity of alloreactive donor T cells, (Choi et al., 2014), indicating that ruxolitinib treatment is not linked to a higher relapse risk.

The pathophysiology of BOS following allo-HSCT is still not completely understood. Current evidence suggests that regulatory T (Treg) cells are effective in controlling chronic GVHD and BOS, as this cell subset can inhibit alloreactive T-cell and germinal center (GC) B-cell reactions to produce pathogenic antibodies for deposition in BOS lesion sites. (McDonald-Hyman et al., 2016). Due to the key role of JAK1/2-STAT3 pathways on T cells activation, JAK 1/2 inhibitor ruxolitinib may reduce donor alloreactive T-cell expansion and inflammatory cytokine production, and meanwhile preserve or even promote Treg recovery post-transplantation through sparing JAK3-STAT5 pathway (Spoerl et al., 2014), explaining the mechanism of action of ruxolitinib to ameliorate cGVHD related BOS. Furthermore, some components such as several chemokines, and other profibrotic factors accumulate in the airway, facilitate macrophage infiltration and collagen deposition, and hence lead to airway remodeling, and eventually, airway obstruction (Liu et al., 2001; Barker et al., 2014). In GVHD models, ruxolitinib was shown to decrease the production of chemokines and profibrotic cytokine (Spoerl et al., 2014), and in the neutrophilic asthma mouse model, it decreased the IL-17A level in bronchoalveolar lavage fluid (BALF), reduced the proportion of Th17 cells in lung tissue, and finally reduced airway inflammation. As for macrophages, ruxolitinib was shown to inhibit macrophage infiltration and prevent the upregulation of various proinflammatory cytokines in human macrophages (Maschalidi et al., 2016; Febvre-James et al., 2018). Collectively, the potent immunosuppressive and anti-inflammatory activities of ruxolitinib might be valuable for the management of BOS in the allogeneic stem cell transplantation setting.

This study demonstrated that while many patients showed an improvement in PFT with ruxolitinib, a substantial proportion of patients failed to respond to this treatment. Identifying potential responders may be more important than implementing ruxolitinib across the board in patients with BOS. Theoretically, early “inflammatory” phase might be more prone to respond to ruxolitinib than late “fibrotic” phase due to the potent anti-inflammatory effect of the drug. Actually, in our retrospective study, there is no significant difference in ORR between early ruxolitinib group and delayed ruxolitinib group. Only one element was identified to be independently related to achieving responses: FEV1%pred mild-decline before ruxolitinib with the FEV1%pred decline ratio ≤15%. It was consistent with a previous study by Kwok et al., who found that patients with BOS who manifested initial rapid lung function decline within 3 months of BOS diagnosis had significantly poorer lung function and worse OS compared with those with a gradual decline (Kwok et al., 2019). Unfortunately, despite interventions, a rapid persistent decline in FEV1 was associated with poor prognosis (Ahn et al., 2015). The ability of ruxolitinib to reverse the natural history of BOS in the FEV1 rapid decline cohort was not observed. Considering that the majority of early ruxolitinib group who moved quickly to ruxolitinib therapy belonged to the BOS progressive population, with a severe deteriorating pulmonary function, thereby the advantage of early inflammatory nature might be obscured by the more important element: rapid lung function decline. And the causes of the rapid deterioration of lung function need to be clarified and other treatment strategies need to be explored.

Ruxolitinib might increase the incidence of infections, which remained a major concern. Increasing evidence suggests that ruxolitinib may impair both innate and adaptive immunity. In detail, ruxolitinib was shown to reduced and functionally impaired dendritic cell (DC) leading to impaired T-cell activation (Heine et al., 2013a). Inhibition of JAK1 impairs cytokine production, which may result in reduced control of silent infections and increased risk of reactivation of latent infections (Heine et al., 2013b) In particular, ruxolitinib drastically reduces the quantity of natural killer (NK) cells (Schönberg et al., 2015). All these effects may result in varying degrees of immunodeficiency, leading to high risks for infectious complications. Besides, it's worth noting that the incidence of pulmonary infection (46.7%) in patients with BOS in the present study was obviously higher than that in patients with myeloproliferative neoplasms receiving ruxolitinib form Comfort II/JUMP (5.3–13.1%) and that in another cohort receiving ruxolitinib as GVHD prophylaxis (Lussana et al., 2018; Zhao et al., 2020b). indicating this high rate of coinfections was not only attributed to the effects of JAK1/2 inhibition on immunosurveillance but also associated with the innate characteristics of BOS. Like patients with bronchiectasis from other causes, patients with BOS can be colonized with Gram-negative organisms and fungus (mostly Aspergillus) (Hayes et al., 2013). Interestingly, in the present study, the incidence of pulmonary infection was higher in the nonresponders than in the responders. For nonresponders who relied on long-term high-dose steroids, ruxolitinib might worsen the infection status, inciting chemokines, accelerating BOS progression, and finally forming a vicious circle (Shino et al., 2013). Also, in our study, patients receiving fluconazole as antifungal prophylaxis might be even more at risk from fungal infection, indicating strong consideration should be given to voriconazole or posaconazole for prophylaxis, as mentioned by Williams, K.M (Williams, 2017). Taken together, the present study highlighted the importance of the assessment of risk factors for infectious events, along with anti-infective prophylaxis prior to initiation of ruxolitinib in patients who were already immunocompromised.

As it is considered standard of care, that every patient is required to monitor PFTs after allogeneic SCT, and in the long time, this implementation is highly recommended, thus the main limitation of the study is insufficient monitoring of PFTs in asymptomatic patients due to inadequate medical resources. The second imitation is that a small number of subjects and a retrospective nonrandomized design of this study might make a bias. However, our data still indicated ruxolitinib as a promising treatment option to improve the prognosis of post-HSCT BOS, with pulmonary function stabilization or even improvement and steroid sparing. This approach might provide a reference for the treatment of BOS in other causes including autoimmune and post lung transplantation. Also, the substantial clinical benefits and efficacy of ruxolitinib on BOS need to be balanced against infection risks. Further, hopefully prospective randomized controlled trials, are needed to definitely prove the efficacy of ruxolitinib in treatment of cGVHD-related BOS.