Introduction

Late-onset noninfectious pulmonary complications (LONIPCs) after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) are a critical problem. Many studies have reported that the development of LONIPCs is closely associated with the occurrence of chronic GVHD (cGVHD).^{1, 2, 3} However, the pathogenesis of LONIPCs is unclear. LONIPCs are classified into bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans with organizing pneumonia and idiopathic pneumonia syndrome (IPS). BOS and IPS are difficult to cure, cause high mortality and decrease survival after allo-HSCT. The incidences of BOS and IPS reportedly vary widely from 1.7 to 26% and from 2.0 to 17% in allo-HSCT, respectively.^{4, 5} Early identification of patients with high risk for BOS/IPS and early treatment for them may be crucial in improving the outcome of allo-HSCT. Although many other risk factors for BOS/IPS have been reported,^{4, 5, 6, 7, 8} it remains difficult to detect patients at high risk for BOS/IPS.

Surfactant proteins A (SP-A) and D (SP-D) are collectins mainly synthesized by alveolar type II cells. The best-known function of surfactant (mainly SP-B) is to prevent the collapse of alveoli by decreasing surface tension at the alveolar air-lipid interface. SP-A and -D play an important role as mediators in innate immunity in the lung.^{9, 10} SP-D has diverse innate immune functions as follows:⁹ binding and agglutination of pathogens; enhancement of phagocytosis/killing of pathogens; rapid clearance of bacterial endotoxin; moderation of inflammatory response to infection and in allergy; antioxidant properties, clearance of apoptotic cells and Ag presentation. Furthermore, it has been reported that significant severe lung inflammation frequently occurs in SP-A-deficient mice given allogeneic donor BM plus spleen T cells followed by conditioning with CY and lethal irradiation.¹¹

Based on these previously reported studies, we hypothesized that individual constitutive levels of surfactant protein in allo-HSCT recipients might be associated with the development of BOS/IPS. In addition, Kerbs von Lungren 6 Ag (KL-6), which is a high-molecular-weight glycoprotein (molecular weight >1000K) classified in humans as MUC1 mucin, strongly expressed on type II alveolar pneumocytes and bronchiolar epithelial cells, and is a well-known marker for the activity of interstitial pneumonia, was also reported to increase in BOS after lung transplant.¹² The current study examined whether SP-A, -D or KL-6 in serum before transplant has a prognostic value for noninfectious severe complications, BOS/IPS after allo-HSCT.

Patients and methods

Patients

We retrospectively evaluated 56 patients with hematological malignancies who had undergone allo-HSCT between November 2001 and 2006 at our institute and survived more than 90 days after transplant. Patients who had obstructive lung disease before transplant or active pneumonia at blood sampling were excluded. The median age at transplant was 42 years old (range, 16–69) (Table 1). These patients included 31 with AML, 5 with ALL, 2 with CML, 6 with myelodysplastic syndrome (MDS), 7 with non-Hodgkin's lymphoma and 5 with adult T-cell leukemia (ATL) (Table 1). In total 25 patients (45%) received a transplant in the advanced phase of the disease. Standard disease status included MDS (refractory anemia), acute leukemia or lymphoma in the first or second remission or chronic leukemia in the first chronic phase. Advanced disease status included patients with MDS (refractory anemia with excess blasts), secondary acute leukemia, Ph chromosome-positive ALL, ATL and all patients beyond the second remission or the first chronic phase in acute and chronic leukemia or lymphoma. This study was approved by the Institutional Review Board. The concept, procedure and potential risks of the study were explained and written informed consent was obtained from all enrolled patients.

Table 1 - Characteristics of patients.

Full table

Allogeneic hematopoietic stem-cell transplantation

In total, 15 patients received allo-HSCT from HLA-identical sibling donors, 6 from HLA-mismatched sibling donors, 20 from HLA-identical unrelated donors and 15 from HLA-mismatched unrelated donors including 10 cord blood transplants (CBT) (Table 1). HLA matching (HLA-A, -B and -DR) was determined by DNA genotyping in siblings and unrelated transplants except CBT, and only serologic typing was performed in CBT. We defined HLA mismatch as the presence of at least one serological or allele mismatch between recipient and donor. As a source of allogeneic stem cell grafts, BM cells were used for 28 patients, mobilized PBSCs for 18 patients and cord blood for 10 patients.

In total, 26 patients (46%) received the following myeloablative conditioning: BU/CY (n=10); CY/TBI (n=7); Ara-C/CY/TBI (n=8) and other (n=1) (Table 1). These conditioning regimens were selected individually based on the status of the underlying disease. On the other hand, reduced-intensity conditioning was employed for 30 patients (54%) ineligible for myeloablative conditioning because of advanced age, comorbidity, organ dysfunction or prior intensive chemotherapies. The reduced-intensity conditioning included fludarabine (Flu)/BU (n=19); Flu/BU/TBI (n=3); other Flu-containing regimens (n=3) and total lymphoid irradiation-containing regimens (n=5).

Acute GVHD prophylaxis and diagnosis

As a prophylaxis for acute GVHD (aGVHD), 44 patients received both CsA and short-term MTX; 8, CsA alone; 1, CsA and mycophenolate mofetil (MMF); 1, tacrolimus alone; 2, tacrolimus and short-term MTX (Table 1). The doses of CsA were adjusted to the target of trough level from 150 to 250 ng/ml until Day 100, whereas the doses of tacrolimus were adjusted to the target of trough level from 10 to 15 ng/ml until Day 100 and then these drugs were tapered unless GVHD occurred. Intravenous administration of MTX was performed at 10 mg/m² on Day 1, and 7 mg/m² on Days 3 and 5. aGVHD was diagnosed clinically, graded according to standard criteria and confirmed by appropriate biopsies. Chronic GVHD (cGVHD) was also defined according to the standard criteria. However, since BOS is generally incorporated as part of the manifestation of extensive cGVHD, there is inevitably a major confounding between BOS and extensive type of cGVHD. Therefore, we classified cGVHD into limited or extensive type except lung manifestation.

Pulmonary function tests

Pulmonary function tests (PFTs) were undergone pre- and post transplant. Pretransplant PFTs were performed within 30 days before transplant and post transplant PFTs were performed at a median time of 355 days (range, 90–1402) after transplant. The following parameters were evaluated: forced expiratory volume in 1 s (FEV₁), forced vital capacity (FVC) and FEV₁/FVC ratio.

Definition of BOS and IPS

The criteria used to diagnose BOS were as follows: clinical or radiological signs and symptoms of lung disease and/or abnormalities in PFTs, and a decline in FEV₁ to less than 80% of the predicted value and FEV₁/FVC less than 70% in PFT, without evidence of infectious causes.^{2, 13} IPS was diagnosed with the following criteria: widespread alveolar injury (multilobular infiltrates on chest radiography or computed tomography, with symptoms and signs of pneumonia and evidence of abnormal pulmonary physiology), absence of clinical or laboratory evidence of active lower respiratory tract infection (bacterial, fungal, viral or parasitic) and absence of malignancy.^{7, 14}

Collection and analysis of blood samples

Serum samples from peripheral blood were collected from 56 patients before allo-HSCT. Additionally, in five BOS and one IPS patients, we also evaluated post transplant SP-D, -A and KL-6 serum levels. The serum was separated by centrifuging the blood at 3500 r.p.m. for 10 min. The serum samples were then stored at -80 °C until they were used. According to the manufacturer's recommended protocol (SRL Inc.; Tokyo, Japan), levels of SP-D and -A in serum were determined using a commercially available enzyme immunoassay, and levels of KL-6 were measured using a commercially available electro chemiluminescent immunoassay. Since the detection sensitivity limit in the SP-D assay was 17.2 ng/ml, values less than 17.2 ng/ml in SP-D (6 in 49 non-BOS/IPS patients and 2 in 7 BOS/IPS patients) were statistically evaluated as 17.2 ng/ml.

Statistics

Univariate analysis was performed by the Mann–Whitney U-test for metric variables and ²-test for categorical variables. The Mann–Whitney U-test was used to compare values of SP-D, -A, KL-6 levels and age between the BOS/IPS and the non-BOS/IPS groups. ²-test was used to compare disease status (high vs standard), donor type (HLA mismatched vs others), conditioning regimen (myeloablative vs reduced-intensity conditioning), TBI (high-dose TBI (12 Gy) vs others), BU (BU vs no BU), aGVHD (grades II–IV vs 0–I) and cGVHD (extensive type vs none and limited) in the BOS/IPS group with those in the non-BOS/IPS group. Multivariate analysis was performed with logistic analysis. Multivariate logistic analysis was conducted with covariates with P-values <0.10 on univariate analysis. All P-values were two-sided and a significance level of 0.05 was used.

Results

The median follow-up time of 56 patients was 615 days (range, 91–2040). Of 56 patients, 40 patients (71%) had aGVHD including 27 patients with grades II–IV (48%) and 12 patients with grades III–IV (21%), and 39 patients (70%) had cGVHD including 28 patients with extensive type (50%). In all patients, 2-year overall survival (OS) and event-free survival (EFS) were 70 and 66%. In total, 2-year OS and EFS in the BOS/IPS and the non-BOS/IPS patients were similar (2-year OS: 71 vs 69% and 2-year EFS: 71 vs 64%, respectively).

Characteristics of BOS/IPS patients

Of 56 patients, 5 (9%) developed BOS and 2 (4%) developed IPS (Table 2). Respiratory failure progressed rapidly in the two IPS patients, leading to death despite undergoing mechanical ventilation. Thus, it was difficult to perform transbronchial lung biopsy in these patients. However, in one of these patients, the autopsy was performed and the results of autopsy showed pulmonary congestion, fibrosis and diffuse alveolar damage, and did not show any evidence of viral, bacterial or fungal pulmonary infections. In the other patient, the results of bronchoalveolar lavage (BAL) examination showed no evidence of viral, bacterial and fungal pulmonary infections. Therefore, we diagnosed these patients as having IPS. The median time between the diagnosis of BOS/IPS and allo-HSCT was 303 days (range, 100–452) and 135 days (117 and 153), respectively. The median age of the BOS/IPS patients was 32 years (range, 18–67). Of seven BOS/IPS patients, there were no patients receiving allo-HSCT from matched related donors (3 matched unrelated donors, 2 one Ag-mismatched related donors, 1 one allele-mismatched unrelated donor and 1 one Ag and one allele-mismatched unrelated donor). All of the seven BOS/IPS patients had extensive type of cGVHD classified except lung manifestation. All patients received steroid-based therapies, but two IPS patients died. Among five BOS patients, BOS was stable in three patients and improved in two patients and all of the five BOS patients are still alive.

Table 2 - Characteristics of patients with BOS/IPS.

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Serum SP-A, -D and KL-6 levels pre- and post transplant

In all patients, median pretransplant SP-A, -D and KL-6 levels in serum were 27.2 ng/ml (range, 6.7–83.7), 45.3 ng/ml (range, 17.2–159) and 232 U/ml (range, 104–725), respectively (Figure 1). The median pretransplant values of SP-A, -D and KL-6 in serum were 28.1 ng/ml (range, 6.7–83.7), 54.4 ng/ml (range, 17.2–159) and 242 U/ml (range, 104–725) in 49 patients without BOS/IPS, whereas 26.4 ng/ml (range, 13.8–32.3), 27.6 ng/ml (range, 17.2–45.5) and 165 U/ml (range, 106–310) in 5 patients with BOS, and 24.9 and 26.3 ng/ml, 17.2 and 43.4 ng/ml and 223 and 135 U/ml in 2 patients with IPS, respectively (Table 2).

Figure 1.

Distribution of serum markers (SP-A (a), SP-D (b) and KL-6 (c)) before transplantation for patients with/without BOS/IPS. SP-D values before SCT were significantly lower in patients with BOS/IPS than in those without BOS/IPS. In SP-A and KL-6, no statistical significance existed between patients with BOS/IPS and those without BOS/IPS. The dashed lines indicate the upper limit of the normal range for each marker. SP-A=surfactant protein A; SP-D=surfactant protein D; BOS/IPS=bronchiolitis obliterans syndrome/idiopathic pneumonia syndrome and KL-6=Kerbs von Lungren 6 Ag.

Full figure and legend (22K)

In five BOS patients, post transplant SP-A, -D and KL-6 levels, when BOS was still active, specifically, obstructive abnormality in the examination of spirography sustained in all 5 patients, did not significantly change from the pretransplant values (median pre- vs post transplant values of SP-D, 27.6 vs 31.3 ng/ml; SP-A, 26.4 vs 36.55 ng/ml and KL-6, 165 vs 236.5 U/ml). On the other hand, in one IPS patient, respiratory dysfunction and abnormal opacity persisted during intubation, post transplant values of SP-A, -D and KL-6 apparently became higher than the pretransplant values (pre- vs post transplant SP-D in this patient, 43.4 vs 143 ng/ml; SP-A, 26.3 vs 136 ng/ml; KL-6, 135 vs 470 U/ml).

Risk factors for developing BOS and IPS

In the univariate analysis, pretransplant SP-D levels in serum were significantly lower and the incidence of extensive cGVHD was significantly more frequent in the patients with BOS/IPS as compared with the patients without BOS/IPS (P=0.03 and 0.03, respectively) (Table 3). Pretransplant KL-6 levels in serum tended to be lower in the BOS/IPS group (P=0.09). Thus, three factors, pretransplant SP-D and KL-6 level in serum and extensive type of cGVHD, were employed as covariates in multivariate logistic analysis. In multivariate analysis, the serum SP-D levels in the BOS/IPS group tended to be lower than those in the non-BOS/IPS group (P=0.08) (Table 4).

Table 3 - Comparison of risk factors between patients with and without BOS/IPS.

Full table

Table 4 - Multivariate logistic analysis of risk factor for BOS/IPS.

Full table

Discussion

The major finding in our study is that low steady-state serum concentration of SP-D before allo-HSCT was closely associated with BOS/IPS. The following risk factors for BOS, advanced recipient or donor age, preceding acute or cGVHD, PBSCT, MTX for aGVHD prevention, female donor to male recipient, BU containing or intensive conditioning, lower serum IgG levels, lower pretransplant FEV₁/FVC ratio, respiratory viral infections within 100 days post-HSCT and an episode of interstitial pneumonitis have been reported.⁵ Similar risk factors, greater patient age (>40 years), myeloablative regimens, aGVHD and high-dose TBI reportedly contribute to the development of IPS.^{7, 8} These reports suggest that the pathogenesis both of BOS and IPS involves compositive processes including non-allo-immune reaction affecting pulmonary function in allo-HSCT such as lung toxic conditioning, aging and a history of pulmonary infection and allo-immune reaction such as acute or cGVHD after allo-HSCT. However, in our study we found no significant association between age, donor source, conditioning regimen and aGVHD. Possible explanatory factors are (1) small population size, (2) various types of conditioning regimen and (3) only patients who survived more than 90 days after allo-HSCT in this study.

In clinical practice, elevated serum SP-D and KL-6 levels have been used as a marker of various lung diseases and activity of interstitial pneumonia.^{10, 12} Significant elevation in the serum level of SP-D and KL-6 in pulmonary inflammation is considered to be evoked by an activated secretion from the bronchiolar epithelium and/or an increase in the permeability of the lung/blood interface partly caused by the destruction of the bronchiolar epithelium and vascular endothelial cells. Also in this study, SP-A, -D and KL-6 were apparently elevated in IPS but not in BOS. Although we cannot conclude that surfactants and KL-6 increased in IPS but not in BOS based on the results in only one patient with IPS, in IPS the rapid and diffuse destruction of lung tissue may promote the release of SP-D into the circulation, while in BOS, which is a kind of chronic lung disease, the serum SP-D levels remained low. Although very little is known about the role of SP-D in chronic lung diseases, these results suggest that IPS and BOS have distinct etiologies.

Low production of surfactant protein and secretary protein in the alveoli is considered an important pathogenesis of certain types of lung disease such as adult respiratory distress and BOS.^{15, 16, 17} SP-D is synthesized by non-ciliated Clara cells as well as pulmonary type II pneumocytes. A decrease in the serum level of Clara cell secretary protein (CC16) was observed in BOS in both lung¹⁶ and SCT.¹⁷ The latter study also suggests that decreased serum levels of CC16 might permit early diagnosis of BOS. It was also reported that there is a protective effect of exogenous surfactant instillation to donor lungs before retrieval on post-lung transplantation surfactant function.¹⁸ In addition, it was observed that absence or low levels of SP-D augment inflammation by acute oxidative stress in the mice model¹⁹ and severe lung inflammation frequently occurred in SP-A-deficient mice receiving myeloablative allogeneic BM and spleen T-cells transplant.¹¹ Furthermore, in mice transtracheal human SP-A treatment attenuated the manifestation of IPS probably as a result of suppressed IFN-production by allo-activated lung-infiltrating T cells, and thereby, early survival was improved.²⁰

The molecular weight of SP-D and -A is smaller than KL-6.²¹ SP-D leaks more easily than SP-A because most of SP-A tightly bind to surfactant lipid aggregates in alveoli but SP-D appears to be lipid free.²² In these points, a decrease in the serum SP-D level might reflect low production of SP-D in the alveoli of chronically damaged lung more than SP-A and KL-6. However, this is just speculation because it is unclear how the serum SP-D concentration is regulated and whether steady-state serum values of SP-D exclusively reflect constitutive synthesis levels of SP-D in the lung.

It has recently been shown that the constitutional SP-D serum levels are largely determined genetically based on the twin studies.^{23, 24} Moreover, it has been reported that, among the nine haplotypes of the human gene coding surfactant protein-D (SFTPD), the third most common haplotype of SFTPD (allele frequency 13.53%) is associated with a low serum level of SP-D.²⁵

Therefore, we speculate that patients who have a certain SFTPD polymorphism will be put at higher risk of BOS/IPS when additive allo-immunological reaction or pulmonary infection occurs to them in allo-HSCT.

This study has the following limitations: (1) This study was retrospective in a single institution, (2) consisted of heterogeneous patient characteristics, diseases, conditioning regimens and GVHD prophylaxes and (3) we did not show decreased secretion or production of surfactant protein in the alveoli of the lung. To prove a causal linkage between the level of surfactant protein secretion in the alveoli and the pathogenesis of BOS/IPS in allo-HSCT patients, we need to directly measure the surfactant protein production level in the alveoli by BAL in allo-HSCT patients. However, measurement with BAL is an invasive examination for routine practice and has artifacts related to the instillation of fluid into the airway. Therefore, it may be a better alternative to address the association between single nucleotide polymorphism in SP-D in allo-HSCT recipients and LONIPCs.

In conclusion, pretransplant serum SP-D level may be a noninvasive and useful predictive marker for the development of BOS/IPS following allo-HSCT. To establish the usefulness of constitutive serum SP-D in allo-HSCT, a prospective trial in a large number of patients is needed.

References

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Acknowledgements

This work was supported in part by grant 19591128 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.