Wall disruption and cystic dilatation of interlobular septa in Birt-Hogg-Dubé syndrome

Abstract

Several hypotheses have been proposed to explain the mechanisms underlying pulmonary cyst formation in Birt–Hogg–Dubé syndrome (BHDS). We examined four patients with BHDS from two families and identified novel histopathological features potentially involved in cyst formation and pneumothorax. Genetic testing was performed in one patient from each family, and both harbored the same pathogenic variant of FLCN (c.1347_1353dup). Histological analysis revealed cystic dilatation and focal disruption of the interlobular septa, with communication between pulmonary cysts, alveoli, and septal structures. These findings suggest the possibility of air tracking into and along the interlobular septa and indicate that structural alterations of the interlobular septa may contribute to the development of pulmonary cysts and pneumothorax in BHDS.

Highlights

• •BHDS cysts communicate with alveoli and septal structures.
• •Cystic dilatation and focal disruption occur in interlobular septa.
• •Histological findings suggest possible air tracking along interlobular septa.
• •Findings provide insight into pneumothorax development in BHDS.

1. Introduction

Birt-Hogg-Dubé syndrome (BHDS) is a rare autosomal dominant genetic disorder characterized by pulmonary cysts, recurrent pneumothorax, cutaneous fibrofolliculomas, and renal tumors [1]. The causative gene is FLCN, which is located on chromosome 17 (17p11.2) and encodes folliculin [1]. While the full function of folliculin remains unclear, several functions in cellular metabolism, cell growth, and homeostasis have been identified [1].

Pulmonary cysts in BHDS distinctly differ histologically from those in spontaneous pneumothorax [2]. Pulmonary cysts in BHDS predominantly occur in the basal regions of the lung and often arise around the interlobular septa or in the subpleural area [3,4]. The cyst wall is lined by a single layer of alveolar epithelial cells accompanied by mild or absent inflammatory infiltrates and fibrous thickening [3,4]. Intracystic septa and protruding venules in the wall of pulmonary cysts have also been reported [2,4]. However, the exact mechanisms that contribute to pulmonary cyst formation in BHDS remain unknown. We examined four patients with BHDS to identify novel histologic features potentially associated with cyst formation and pneumothorax.

2. Materials and methods

We examined four consecutive patients with BHDS who underwent a surgical lung biopsy. Genetic testing was performed in Cases 1 and 4; Cases 2 and 3, who were members of the same family as Case 1, were not genetically tested. All obtained specimens were stained with hematoxylin and eosin and Elastica van Gieson (EvG). A pulmonary cyst was defined as a round, circumscribed space surrounded by an epithelial or fibrous wall of variable thickness [5,6]. All histological sections prepared at the time of diagnosis were reviewed in each case. The number, size, and distribution of pulmonary cysts, cyst wall characteristics, and associated architectural changes in the interlobular septa were evaluated on these sections.

For serial section analysis, tissue blocks containing cysts adjacent to the interlobular septa were selected. Serial 4-μm-thick sections were prepared and stained with EvG to examine the architectural communications between cysts, normal alveoli, and septal structures. All slides were converted to whole-slide images with a Slideview VS200 digital scanner (Olympus, Tokyo, Japan) at ×20 magnification. EvG-stained serial sections were converted into movies using Adobe Photoshop (Adobe Creative Cloud) and ImageJ (National Institutes of Health, Bethesda, MD, USA).

The study was approved by the Institutional Ethics Committee of Fukuoka University Chikushi Hospital (No. 25-03-04, April 9, 2025). Informed consent was obtained and documented in the medical record.

3. Results

Of the four patients with BHDS, Cases 1–3 were members of the same family (two males in their 20s and one female in her 40s), and Case 4 was an unrelated female. All patients had a history of multiple pneumothoraces. Genetic testing was performed in Cases 1 and 4, one patient from each family, and identified the same pathogenic variant of FLCN (c.1347_1353dup) in both patients. Cases 2 and 3 were not genetically tested. Case 4 was suspected to have BHDS because of skin fibrofolliculomas and a strong family history of pneumothorax. All patients underwent a surgical lung biopsy for pneumothorax. No air leak was found in the lung biopsies, and lung tissue was obtained for diagnostic purposes.

Lung tissue was obtained from the left lower lobe (Case 1), right middle lobe (Case 2), right upper lobe (Case 3), and left lower lobe (Case 4). Pulmonary cysts were evident in all four patients. Thirty-one cysts were observed and were variable in size, with a maximum diameter ranging from 0.7 to 10.5 mm. The distribution of cysts was 48% (15/31) in the subpleural area, 48% (15/31) around the interlobular septa, and 3% (1/31) without any obvious localization. There was minimal inflammatory cell infiltration in the cyst walls. The walls of the cysts in contact with the interlobular septum or visceral pleura were slightly thickened by connective tissue, but there was no fibrous thickening at the border between the cysts and normal-appearing alveoli. Among all cysts, intracystic septa were found in 23% (7/31) and protruding venules in the cyst wall were found in 10% (3/31).

Irregular cystic dilatation of the interlobular septa was observed in all four cases (Fig. 1A–C). Some of the dilated cystic lesions were connected in a chain extending to the pleura (Fig. 1B). Multiple communications (eight points in four cases) were found between the interlobular septa and pulmonary cysts (Fig. 1C and D) or normal-appearing alveoli. Inflammatory responses, such as hemorrhage and the appearance of hemosiderin-laden macrophages, were observed within the disrupted interlobular septa (Fig. 1D and E). Communication through disrupted interlobular septal walls between the pulmonary cyst and the interlobular septa was found in 10% (3/31) of pulmonary cysts. The connective tissue comprising the dilated interlobular septa was loose, but the venules and lymphatic vessels within the septa were well preserved (Fig. 1F and G). The dilated cystic lesions within the interlobular septa were partially separated by thin collagen fibers (Fig. 1F, inset). EvG-stained serial sections from Cases 1 and 4 demonstrated that the cysts were continuous with bronchioles and communicated with the interlobular septa (Fig. 1H and Supplemental Video 1).

Fig. 1.

A: Computed tomography image showing two adjacent cysts (asterisks) in the interlobular septa (white arrows). Blue arrows indicate pulmonary veins, and red arrows indicate the pulmonary artery.

B: Lower-magnification view demonstrates multiple cystic-like lesions within the cystically dilated interlobular septa (asterisks) extending to the pleura (Elastica van Gieson [EvG] staining).

C: Another lower-magnification view showing cystic dilatation of the interlobular septa (asterisks) (hematoxylin and eosin [HE] staining). The arrowhead indicates the site of communication through a disrupted interlobular septal wall between a pulmonary cyst and the interlobular septum.

D: High-magnification image of the communication site indicated by the arrowhead in C. The interlobular septal wall is disrupted, with communication between the pulmonary cyst and the irregularly dilated interlobular septum.

E: High-magnification image of the interlobular septum in D. Hemorrhage, hemosiderin-laden macrophages, and histiocytes are seen within the interlobular septum.

F and G: The solid square in C shows irregular dilatation of the interlobular septa with loose connective tissue and slit-like spaces between collagen fibers. Venules and lymphatics remain intact (F, EvG stain; G, podoplanin immunohistochemistry). The cyst-like lesions within the interlobular septa are partially separated by thin collagen fibers (inset of F).

H: A cyst adjacent to a bronchiole (dashed square in C). The arrow indicates the bronchiolar epithelium (EvG staining).

4. Discussion

Several mechanisms have been proposed for pulmonary cyst formation in BHDS. Warren et al. showed that folliculin is expressed in type I alveolar epithelial cells, fibroblasts, and macrophages, suggesting that dysfunction of folliculin-expressing cells may be involved in pulmonary cyst formation [7]. Subpleural regions are subjected to mechanical stress due to significant changes in alveolar volume during respiratory cycles, which may contribute to pulmonary cyst formation in BHDS [8]. FLCN haploinsufficiency has also been associated with fibroblast dysfunction and impaired tissue repair [9], and increased apoptosis of alveolar epithelial cells has been implicated in cyst formation [10]. More recent studies have further supported the involvement of impaired reparative responses [11] and have highlighted a potential role of the pulmonary mesenchyme. Mesenchymal FLCN deletion results in extracellular matrix abnormalities, alveolar destruction, and cyst formation [12], while pulmonary interstitial glycogenosis-like changes have been described in the septal structures and cyst walls of BHD-associated lung cysts [13]. More recently, mTORC1 activation has been implicated in cyst formation associated with mesenchymal FLCN deficiency [14]. These mechanisms may not be mutually exclusive and may collectively contribute to structural vulnerability of the lung tissue, providing a possible background for the interlobular septal changes observed in our cases.

In this study, the connective tissue forming the interlobular septa was loose, and the interlobular septa showed cystic dilatation. Furthermore, cystic dilatation of the interlobular septa extended toward the pleura. In some areas, the walls of the dilated interlobular septa were disrupted and communicated with pulmonary cysts or normal-appearing alveoli. These morphological findings suggest the possibility of air tracking into and along the interlobular septa toward the pleura. However, because the presence and movement of air cannot be directly demonstrated histologically, this interpretation should be regarded as a proposed mechanism rather than a direct histological observation.

Although cystic lesions that appear to be encased in the interlobular septa have previously been reported in BHDS, it was unclear whether these cyst-like lesions were truly located within the interlobular septa or whether pulmonary cysts merely appeared to be surrounded by and embedded within the septa [3]. In the present study, reconstructed serial sections demonstrated multiple cystic-like spaces within the cystically dilated interlobular septa and their communication with pulmonary cysts or alveoli. These findings are consistent with, but do not directly prove, air tracking within the interlobular septa. Such cystic-like spaces within dilated interlobular septa may represent a useful pathological feature of BHDS.

Alternative explanations for the observed septal disruption should also be considered. The septal changes may reflect structural vulnerability associated with abnormalities of the pulmonary mesenchyme or extracellular matrix, impaired tissue repair, mechanical stress during respiration, or a combination of these factors. Therefore, the present histological findings do not establish whether septal disruption precedes or follows cyst formation.

This study has several limitations. Only four patients were examined, three of whom belonged to the same family. Genetic testing was performed only in Cases 1 and 4, one patient from each family, and both harbored the same pathogenic variant of FLCN, c.1347_1353dup, p.(Val452Profs*6); Cases 2 and 3 were not genetically tested. Although c.1347_1353dup is one of the common pathogenic FLCN variants identified in Asian patients with BHDS [15], the possibility that the observed septal changes represent a family-specific or variant-associated phenomenon cannot be excluded. Further studies involving larger numbers of genetically diverse patients are needed to determine whether interlobular septal dilatation, disruption, and communication with pulmonary cysts or alveoli are general pathological features of BHDS.

CRediT authorship contribution statement

Masayo Yoshimura: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Conceptualization. Yoshiaki Kinoshita: Writing – review & editing, Writing – original draft, Resources, Methodology, Conceptualization. Ryo Hayashida: Writing – review & editing, Validation, Methodology. Junya Fukuoka: Writing – review & editing, Conceptualization. Hiroshi Ishii: Writing – review & editing, Resources. Satoshi Nimura: Writing – review & editing, Supervision, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Serial sections showing that the cyst is continuous with the bronchiole and communicates with the interlobular septa (asterisks). Arrows indicate communication between the cyst and the bronchiole. Arrowheads indicate communication between the cyst and interlobular septa.Multimedia component 1