Introduction: Increasing numbers of cases of mild asymptomatic pulmonary alveolar proteinosis (PAP) are being reported with the recent increase in chest computed tomography (CT). Bronchoscopic diagnosis of mild PAP is challenging because of the patchy distribution of lesions, which makes it difficult to obtain sufficient biopsy samples. Additionally, the pathological findings of mild PAP, particularly those that differ from severe PAP, have not been fully elucidated. This study aimed to clarify the pathological findings of mild PAP and the usefulness of optical biopsy using probe-based confocal laser endomicroscopy (pCLE). Methods: We performed bronchoscopic optical biopsy using pCLE and tissue biopsy in 5 consecutive patients with PAP (three with mild PAP and two with severe PAP). We compared the pCLE images of mild PAP with those of severe PAP by integrating clinical findings, tissue pathology, and chest CT images. Results: pCLE images of PAP showed giant cells with strong fluorescence, amorphous substances, and thin alveolar walls. Images of affected lesions in mild PAP were equivalent to those obtained in arbitrary lung lesions in severe cases. All 3 patients with mild PAP spontaneously improved or remained stable after ≥3 years of follow-up. Serum autoantibodies to granulocyte-macrophage colony-stimulating factor were detected in all 5 cases. Conclusion: Optical biopsy using pCLE can yield specific diagnostic findings, even in patients with mild PAP. pCLE images of affected areas in mild and severe PAP showed similar findings, indicating that the dysfunction level of pathogenic alveolar macrophages in affected areas is similar between both disease intensities.

Pulmonary alveolar proteinosis (PAP) is a relatively rare disease characterized by the abnormal accumulation of surfactant proteins within the alveoli. Most cases are of autoimmune PAP, in which anti-granulocyte macrophage colony-stimulating factor (anti-GM-CSF) antibody can be detected as a pathogenic factor [1]. Because the clinical course of immunogenic PAP is usually indolent, most cases are diagnosed at an advanced stage [2]. However, as computed tomography (CT) has become more popular for the screening of chest diseases, the diagnosis of cases of PAP without symptoms has increased. Miyashita et al. [3] reported a Japanese nationwide survey of patients with PAP that included the distribution of disease severity in Japan. They used the Disease Severity Score (DSS) proposed by Inoue et al. [4] and showed that approximately 18% of Japanese PAP patients are asymptomatic and without respiratory insufficiency (DSS 1). Bai et al. reported that patients with mild PAP (DSS 1 or 2) accounted for 62% of a multicenter cohort study in China [5]. Mild PAP often presents as multiple small patchy lung lesions, and therefore pathological diagnosis may be difficult. Transbronchial biopsy of multiple lesions may be risky. In addition, whether patchy lung lesions show pathological findings equivalent to those of severe PAP (abnormally enlarged macrophages and alveoli filled with lipoproteinaceous material) remains unclear. Probe-based confocal laser endomicroscopy (pCLE) is a relatively recently developed technique that has been increasingly applied in bronchoscopy [6]. Because pCLE can depict microscopic autofluorescent findings of the peripheral lung directly via bronchoscopy, pathological diagnosis of the observed lesions might be possible without a real tissue biopsy (instead, using what is termed an optical biopsy) [7, 8]. Herein, we report the pCLE findings of five consecutive cases of PAP: three cases of mild PAP exhibiting patchy sparse lesions that remained stable or spontaneously resolved, and two cases of severe PAP with diffusely distributed lesions. We analyzed the feasibility of optical biopsy for mild PAP and compared the pCLE images of mild PAP with those of severe PAP.

We encountered 5 consecutive patients with PAP who underwent diagnostic studies including bronchoscopy and chest CT between April 2015 and December 2017. The DSS was used to evaluate the severity of PAP [4, 9]. This DSS contains five categories based on PaO2 and symptoms at the initial assessment, as follows: (1): asymptomatic and PaO2 ≥70 mm Hg; (2): symptomatic and PaO2 ≥70 mm Hg; (3): PaO2 60–70 mm Hg; (4): PaO2 50–60 mm Hg; 5: PaO2 <50 mm Hg. We classified DSS 1 and 2 as mild cases, and DSS 4 and 5 as severe cases [5]. In all cases, we performed endobronchial ultrasound-guided transbronchial biopsy using a guide sheath (EBUS-GS-TBB), followed by pCLE. We recorded cellular video images using Cellvizio® (Mauna Kea Technologies, Paris, France), which generated fluorescent images with 488-nm excitation light. We did not use local or systemic fluorescein administration (only autofluorescent findings were observed). The pCLE probe (ALVEOFLEX®, Mauna Kea Technologies, France) was inserted into the guide sheath. Lung biopsies (EBUS-GS-TBB) were performed using the same guide sheath after obtaining the endobronchial ultrasound EBUS and pCLE images. EBUS-GS-TBB was performed using the standard method described elsewhere [10]. In brief, after careful evaluation of thin-section CT images, a guide-sheath (SG 200C Olympus, Tokyo, Japan) was introduced to the affected area under fluoroscopic assistance. EBUS images were obtained using a radial EBUS probe (UM-S20-17S, Olympus). Transbronchial biopsy was performed using 1.5-mm biopsy forceps (FB233D Olympus). We did not use virtual bronchoscopic planning before the procedure, and we did not routinely acquire pCLE images or EBUS images from non-affected areas. Finally, bronchoalveolar lavage (BAL) was performed at the corresponding lobe (three 50-mL instillations of saline). All pCLE images were analyzed using the corresponding software (Cellvizio Viewer®; Mauna Kea Technologies). Written informed consent was obtained from each patient in this study. This observational study was approved by our Institutional Review Board (Fujita Health University Hospital Review Board: HM18-030) and was registered in the National Clinical Trial Database (UMIN 000018518).

Table 1 shows the patient characteristics. All 3 patients with mild PAP were asymptomatic (DSS score 1), and their PAP was incidentally found on chest CT performed during follow-up studies for other diseases. Two patients with severe PAP complained of exertional dyspnea with respiratory failure (DSS score 4). Serum anti-GM-CSF antibodies were detected in all patients. The pCLE images of patients with severe PAP included giant cells with strong fluorescence, amorphous substances, and thin alveolar walls (Fig. 1d, e, i, m, n, o; online suppl. Video; for all online suppl. material, see https://doi.org/10.1159/000535992). BAL and transbronchial lung biopsies confirmed the pathological diagnosis of PAP (Fig. 1). In the 3 patients with mild PAP with a patchy distribution of crazy-paving infiltration on chest CT (Fig. 1a, f, k), we obtained pCLE findings by introducing the pCLE probe to the corresponding lesion area via a guide sheath. The pCLE microscopic fluorescence images were equivalent to those observed in severe PAP (Fig. 1r, s, v, w; online suppl. Video 2). A transbronchial biopsy confirmed the pathological diagnosis of PAP. EBUS images of the affected area (ill-defined low-echoic shadow, spotty echoic shadow, and radial linear shadow) were also observed (Fig. 1j). BAL samples from patients with mild PAP were less lactescent than those from patients with severe PAP. After comparing the pathological findings of the BAL cells and biopsy samples with the pCLE findings, we speculated that giant cells with strong fluorescence corresponded to enlarged macrophages that had engulfed the alveolar proteinaceous exudate within the cytoplasm (Fig. 1). All 3 patients with mild PAP spontaneously improved or remained stable after ≥3 years of follow-up. Two patients with severe PAP were transferred to another hospital for treatment.

Table 1.

Characteristics of the patients with PAP

PatientAge, yearsSexSmoking statusSymptomsDSS*Disease severity**a-GM-CSF Ab,§ µg/mLOutcome (years of follow-up)
72 Female Never smoker Asymptomatic Mild 47.3 Stable (6.5 years) 
51 Female Never smoker Asymptomatic Mild 44.5 Improved (6.5 years) 
67 Male Ex-smoker Asymptomatic Mild 39.2 Improved (6 years) 
70 Male Current smoker Exertional dyspnea Severe 21.8 Transfer for WLL 
83 Male Never smoker Exertional dyspnea Severe 56.7 Transfer for GM-CSF inhalation 
PatientAge, yearsSexSmoking statusSymptomsDSS*Disease severity**a-GM-CSF Ab,§ µg/mLOutcome (years of follow-up)
72 Female Never smoker Asymptomatic Mild 47.3 Stable (6.5 years) 
51 Female Never smoker Asymptomatic Mild 44.5 Improved (6.5 years) 
67 Male Ex-smoker Asymptomatic Mild 39.2 Improved (6 years) 
70 Male Current smoker Exertional dyspnea Severe 21.8 Transfer for WLL 
83 Male Never smoker Exertional dyspnea Severe 56.7 Transfer for GM-CSF inhalation 

PAP, pulmonary alveolar proteinosis; a-GM-CSF, anti-granulocyte macrophage colony-stimulating factor; Ab, antibody; WLL, whole lung lavage.

*DSS; Disease severity score: 1: asymptomatic and PaO2 ≥ 70 mm Hg, 2: symptomatic and PaO2 ≥ 70 mm Hg, 3: PaO2 60–70 mm Hg; 4: PaO2 50–60 mm Hg, 5: PaO2 <50 mm Hg.

**Disease severity: mild PAP, cases with DSS score = 1 or 2. Severe PAP, cases with DSS score = 4 or 5.

§The cut-off for serum a-GM-CSF Ab was <1.65 μg/mL (measurement of serum a-GM-CSF Ab courtesy of Clinical and Translational Research Center, Niigata University Medical and Dental Hospital, Niigata, Japan).

Fig. 1.

a–e Patient 1. f–j Patient 2. k–o Patient 3. p–s Patient 4. t–w Patient 5. Patients 1 to 3 had mild PAP. Patients 4 and 5 had severe PAP. a, f, k Chest CT findings of mild PAP showed a crazy paving shadow in a small subpleural area. p, t Chest CT findings of severe PAP showed diffuse ground-glass opacity with consolidation in the bilateral lung field. b, g, l, q, u Pathological findings of transbronchial lung biopsy. All images show an eosinophilic amorphous substance filling the alveolar space, consistent with alveolar proteinosis. All but (l) (Patient 3: immunohistochemical staining using anti-SP-A antibody) show hematoxylin–eosin staining. c, h Giemsa stain findings of bronchial alveolar lavage samples from Patients 1 and 2, respectively, showing giant cells (macrophages that had dysfunctionally engulfed alveolar protein) with some relatively large macrophages. d, i, m, n, r, v pCLE images of highly fluorescent macrophages and giant cells; equivalent findings were obtained regardless of disease severity (d, i, m, n in mild PAP, r, v in severe PAP). e, o, s, w pCLE also showed amorphous fluorescent fluid in the alveolar space at the same level in both mild and severe disease (e, o in mild PAP, s, w in severe PAP). j Endobronchial ultrasound image of the PAP lesion in Patient 2.

Fig. 1.

a–e Patient 1. f–j Patient 2. k–o Patient 3. p–s Patient 4. t–w Patient 5. Patients 1 to 3 had mild PAP. Patients 4 and 5 had severe PAP. a, f, k Chest CT findings of mild PAP showed a crazy paving shadow in a small subpleural area. p, t Chest CT findings of severe PAP showed diffuse ground-glass opacity with consolidation in the bilateral lung field. b, g, l, q, u Pathological findings of transbronchial lung biopsy. All images show an eosinophilic amorphous substance filling the alveolar space, consistent with alveolar proteinosis. All but (l) (Patient 3: immunohistochemical staining using anti-SP-A antibody) show hematoxylin–eosin staining. c, h Giemsa stain findings of bronchial alveolar lavage samples from Patients 1 and 2, respectively, showing giant cells (macrophages that had dysfunctionally engulfed alveolar protein) with some relatively large macrophages. d, i, m, n, r, v pCLE images of highly fluorescent macrophages and giant cells; equivalent findings were obtained regardless of disease severity (d, i, m, n in mild PAP, r, v in severe PAP). e, o, s, w pCLE also showed amorphous fluorescent fluid in the alveolar space at the same level in both mild and severe disease (e, o in mild PAP, s, w in severe PAP). j Endobronchial ultrasound image of the PAP lesion in Patient 2.

Close modal

Several reports on pCLE imaging of parenchymal lung disease have been published [11]. These studies indicated that pCLE may show specific imaging patterns in selected lung parenchymal diseases, patterns that can be partly attributed to the diagnosis [12]. Salaün et al. [13] first described pCLE imaging of PAP, finding highly fluorescent globular and granular structures and fluorescent alveolar macrophages. Yserbyt et al. [14] also described similar pCLE images in another case of PAP. Notably, most of the PAP cases in these reports were smokers. In contrast, the patients with PAP in our report were non-smokers. Indeed, a Japanese cohort study of PAP reported that 32.7% of PAP patients were never smokers [3]. Our pCLE images of PAP might indicate strong autofluorescent giant cells, which may be true PAP-specific findings that were not altered by smoking. Danilevskaya et al. [15] reported pCLE images of advanced-stage PAP, including highly fluorescent intra-alveolar complexes of different sizes, along with giant alveolar macrophages. They showed that the pCLE images of PAP corresponded to findings on high-resolution CT images; however, some degree of fluorescent complex images could be observed even in lung areas without a crazy-paving pattern on high-resolution CT. This might indicate that malfunctional macrophages with strong autofluorescence are part of the fundamental pathophysiology of the disease and are not related to disease severity. Some investigators proposed a PAP severity score (Disease Severity Score; DSS) based on respiratory symptoms and PaO2 [4, 9]. Among the five cases in this report, 3 cases of mild PAP were assigned a DSS of 1 (asymptomatic and PaO2 >70 mm Hg). In these three mild cases, the pulmonary lesions improved spontaneously or remained stable after 2 or 3 years. According to retrospective studies, the natural history of PAP varies from spontaneous remission in the best-case scenario to progression through the entire spectrum of disease severity toward fatal respiratory failure [16]. In a nationwide Japanese retrospective study, approximately 15% of patients with PAP exhibited no symptoms [3, 17]. Our study also indicated that pathological macrophages of PAP exhibit the same morphology in both spontaneous remission and progressive disease, and it remains unclear whether mild PAP is likely to improve spontaneously. One intriguing case report described a patient with severe autoimmune PAP who also developed lung cancer and left lower pulmonary artery obstruction [18], although infiltration was not observed in the left lower lobe. The authors suggested that pulmonary circulation of the anti-GM-CSF antibody might be crucial for the development of PAP lesions (malfunction of alveolar macrophages). As the titer for a-GMCSF antibody was equivalent in mild and severe cases in our study, we speculate that malfunctional macrophage development is a part of the fundamental pathology in PAP and that disease severity and remission might be dependent on other factors related to alveolar space clearance.

In conclusion, pCLE observation of multiple mild PAP lesions showed findings equivalent to those in severe diffuse PAP, suggesting that macrophage dysfunction may be the same in mild and severe disease.

We thank Angela Morben, DVM, ELS from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

This study was conducted in accordance with the World Medical Association Declaration of Helsinki guidelines. Written informed consent was obtained from all patients before the bronchoscopic procedure and pCLE examination. The study protocol was approved by our Institutional Review Board (Fujita Health University Hospital Review Board: HM18-030) and was registered in the National Clinical Trial Database (UMIN 000018518).

The authors have no conflicts of interest to declare.

This work was supported by JSPS KAKENHI (Grant Number JP21K08216).

Takuya Okamura, Sayako Morikawa, Tomoya Horiguchi, Yasuhiro Goto, Sumito Isogai, Naoki Yamamoto, Shotaro Okachi, Naozumi Hashimoto, and Kazuyoshi Imaizumi were involved in study design and data interpretation. Kumiko Yamatsuta, Toshikazu Watanabe, Aki Ikeda, Yuri Maeda, Takuma Ina, Hidetaka Takahashi, and Ryoma Moriya were involved in data collection. All authors critically revised the report, commented on the drafts of the manuscript, and approved the final report.

All data generated or analyzed during this study are included in this article and its online supplementary files. Further inquiries can be directed to the corresponding authors.

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