Integrated bulk and single-cell RNA-sequencing reveals SPOCK2 as a novel biomarker gene in the development of congenital pulmonary airway malformation.

BACKGROUND: Congenital pulmonary airway malformation (CPAM) is the most frequent pulmonary developmental malformation and the pathophysiology remains poorly understood. This study aimed to identify the characteristic gene expression patterns and the marker genes essential to CPAM. METHODS: Tissues from the cystic area displaying CPAM and the area of normal appearance were obtained during surgery. Bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were performed for integrating analysis. Iterative weighted gene correlation network analysis (iWGCNA) was used to identify specifically expressed genes to CPAM. RESULTS: In total, 2074 genes were significantly differentially expressed between the CPAM and control areas. Of these differentially expressed genes (DEGs), 1675 genes were up-regulated and 399 genes were down-regulated. Gene ontology analysis revealed these DEGs were specifically enriched in ciliated epithelium and involved in immune response. We also identified several CPAM-related modules by iWGCNA, among them, P15_I4_M3 module was the most influential module for distinguishing CPAMs from controls. By combining the analysis of the expression dataset from RNA-seq and scRNA-seq, SPOCK2, STX11, and ZNF331 were highlighted in CPAM. CONCLUSIONS: Through our analysis of expression datasets from both scRNA-seq and bulk RNA-seq of tissues obtained from patients with CPAM, we identified the characteristic gene expression patterns associated with the condition. Our findings suggest that SPOCK2 could be a potential biomarker gene for the diagnosis and therapeutic target in the development of CPAM, whereas STX11 and ZNF331 might serve as prognostic markers for this condition. Further investigations with larger samples and function studies are necessary to confirm the involvement of these genes in CPAM.

Global interpretation of novel alternative splicing events in human congenital pulmonary airway malformations: A pilot study.

Little is known about differentially expressed genes (DEGs) and alternative splicing (AS) landscapes in congenital lung malformations (CLMs). We applied reference-based assembly of sequencing reads from RNA sequencing (RNA-seq) libraries to identify DEGs and AS landscapes in the lesions and normal lung tissue from the most common types of CLMs, including congenital pulmonary airway malformation-Ⅰ (CPAM-Ⅰ), CPAM-Ⅱ, intralobar sequestration (ILS), and ILS with CPAM (ILS-CPAM). We analyzed the expression profiles and related biological functions of AS events (ASEs). We further constructed a co-expression regulatory network between RNA binding protein (RBP) genes and corresponding ASEs to explore the related pathways in the regulated network. Ten DEGs were identified in the four types of CLMs, including eight upregulated genes and two downregulated genes. Additionally, 16 differential ASEs were detected, including the genes MACF1, RFX2, and FBXL4. Gene ontology (GO) enrichment was mainly observed in embryonic visual malformation and apoptotic process, and the KEGG pathway mainly enriched in the PI3K/AKT signaling pathway. We also detected 13 differentially expressed RBPs among 1979 DEGs in CPAM-I, in which ASEs in the MACF1 gene and RBP genes TLR8 and PTRH1 were closely associated. Moreover, we confirmed that the expression levels of PTRH1, NSUN7, and DZIP1L abundantly increased and the expression levels of TLR8, MEF2A, and NIPBL decreased in the CPAM-I lung tissue compared with the controls. It is suggested that ASEs in different types of CLMs is prominently different from normal controls, and ASEs differences occurring in CPAM-I malformation tissue are dramatically different from other types, which demonstrates the complex pathogenesis of CLMs and provides foundations for future studies to elucidate the mechanisms of developing CLMs.

Congenital cystic adenomatoid malformations of the lung: an epithelial transcriptomic approach.

BACKGROUND: The pathophysiology of congenital cystic adenomatoid malformations (CCAM) of the lung remains poorly understood. AIM: This study aimed to identify more precisely the molecular mechanisms limited to a compartment of lung tissue, through a transcriptomic analysis of the epithelium of macrocystic forms. METHODS: Tissue fragments displaying CCAM were obtained during planned surgical resections. Epithelial mRNA was obtained from cystic and normal areas after laser capture microdissection (LCM). Transcriptomic analyses were performed and the results were confirmed by RT-PCR and immunohistochemistry in independent samples. RESULTS: After controlling for RNA quality, we analysed the transcriptomes of six cystic areas and five control areas. In total, 393 transcripts were differentially expressed in the epithelium, between CCAM and control areas. The most highly redundant genes involved in biological functions and signalling pathways differentially expressed between CCAM and control epithelium included TGFB2, TGFBR1, and MAP 2 K1. These genes were considered particularly relevant as they have been implicated in branching morphogenesis. RT-qPCR analysis confirmed in independent samples that TGFBR1 was more strongly expressed in CCAM than in control tissues (p < 0.03). Immunohistochemistry analysis showed TGFBR1 (p = 0.0007) and TGFB2 (p < 0.02) levels to be significantly higher in the epithelium of CCAM than in that of control tissues. CONCLUSIONS: This compartmentalised transcriptomic analysis of the epithelium of macrocystic lung malformations identified a dysregulation of TGFB signalling at the mRNA and protein levels, suggesting a possible role of this pathway in CCAM pathogenesis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01732185.

Analysis of Wnt7B and BMP4 expression patterns in congenital pulmonary airway malformation.

BACKGROUND: Congenital pulmonary airway malformation (CPAM) is a rare disorder characterized by aberrant overgrowth of terminal bronchioles. The objective of this study was to describe wingless-type MMTV integration site family 7B (Wnt7B) and bone morphogenetic protein 4 (BMP4) expression patterns in human CPAM lesions and to explore the possible roles of Wnt7B and BMP4 in the pathogenesis of CPAM. METHODS: Fifteen tissue samples from patients with CPAM were obtained from the Pathology Department of Shengjing Hospital of China Medical University. Samples representing CPAM lesions and adjacent normal lung tissues were collected and Wnt7B and BMP4 expression was detected through immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. RESULTS: IHC revealed that Wnt7B immunopositive cells were only detected in epithelial cells, whereas BMP4 immunopositive cells were detected in epithelial and mesenchymal cells. Expression of Wnt7B and BMP4 immunopositive cells was higher in CPAM lesions than that in adjacent normal lung tissue. qRT-PCR and Western blotting showed that Wnt7B and BMP4 mRNA and protein expression were significantly higher in CPAM lesions than in adjacent normal lung tissue (P < .05). Overall, the level of BMP4 was higher than that of Wnt7B. CONCLUSIONS: Increased expression of Wnt7B and BMP4 appear to be related to the pathogenesis of CPAM and abnormal pulmonary development. Upregulation of Wnt7B and BMP4 could play an important role in the development of the bronchial-alveolar structures that characterize CPAM.

New insights on congenital pulmonary airways malformations revealed by proteomic analyses.

BACKGROUND: Congenital Pulmonary Airway Malformation (CPAM) has an estimated prevalence between 0.87 and 1.02/10,000 live births and little is know about their pathogenesis. To improve our knowledge on these rare malformations, we analyzed the cellular origin of the two most frequent CPAM, CPAM types 1 and 2, and compared these malformations with adjacent healthy lung and human fetal lungs. METHODS: We prospectively enrolled 21 infants undergoing surgical resection for CPAM. Human fetal lung samples were collected after termination of pregnancy. Immunohistochemistry and proteomic analysis were performed on laser microdissected samples. RESULTS: CPAM 1 and 2 express mostly bronchial markers, such as cytokeratin 17 (Krt17) or α-smooth muscle actin (ACTA 2). CPAM 1 also expresses alveolar type II epithelial cell markers (SPC). Proteomic analysis on microlaser dissected epithelium confirmed these results and showed distinct protein profiles, CPAM 1 being more heterogeneous and displaying some similarities with fetal bronchi. CONCLUSION: This study provides new insights in CPAM etiology, showing clear distinction between CPAM types 1 and 2, by immunohistochemistry and proteomics. This suggests that CPAM 1 and CPAM 2 might occur at different stages of lung branching. Finally, the comparison between fetal lung structures and CPAMs shows clearly different protein profiles, thereby arguing against a developmental arrest in a localized part of the lung.

Gene expression profiling reveals differential patterns between microcystic congenital cystic adenomatoid malformation and congenital lobar emphysema.

INTRODUCTION: Congenital cystic adenomatoid malformation (CCAM), especially type-III, shares similar sonographic features with congenital lobar emphysema (CLE) in routine ultrasound scan. Thus, prenatal differentiation of CLE from a microcystic CCAM is challenging and difficult in practice. Discovery of molecular biomarkers has important clinical significance. METHODS: We profiled gene expression in lung tissue from four CCAM type-III and five CLE subjects by microarray. A bioinformatic tool was used for signal pathways enrichment analysis. Further, quantitative reverse transcriptase PCR (qRT-PCR) was used to verify the results. RESULTS: A total of 426 genes were identified to be significantly differentially expressed (fold-change >2.0, q value <0.05) between microcystic CCAM and CLE. Of these differentially expressed genes (DEGs), 392 were upregulated and 34 were downregulated in microcystic CCAM compared with CLE. Unsupervised clustering of the "expressed" genes could clearly delineate the CCAM and CLE samples. We also confirmed that eight randomly chose genes were differentially expressed at the mRNA level between CCAM and CLE. CONCLUSIONS: CCAM type-III and CLE have differential gene expression patterns. Our pilot study may gain a deeper understanding of the organogenetic origins and pathogenesis of these conditions. The suggestive candidates may serve as potential biomarkers for definitive diagnosis of congenital cystic lung lesions and eventually to treat them appropriately.

Elevated Krüppel-like factor 5 expression in spatiotemporal mouse lungs is similar to human congenital cystic adenomatoid malformation of the lungs.

Objective The study aimed to investigate the role of high Krüppel-like factor 5 (KLF5) expression on the pathogenesis of congenital cystic adenomatoid malformation of the lungs (CCAML) in mice. Methods A mouse model of high KLF5 expression in the lungs was established. KLF5 expression and the pulmonary lumen diameter were examined by immunohistochemistry to determine a successful model. Basement membrane damage and activity of matrix metalloproteinase-9 (MMP-9) were examined. After an adenovirus carrying KLF5 gene transfection in lung adenocarcinoma (H441) was created, changes in expression and activity of MMP-9 were determined. Results In a mouse model with high KLF5 expression, the pulmonary lumen was markedly enlarged, indicating establishment of CCAML. The basement membrane was degraded, and MMP-9 activity was significantly higher in the model group compared with the control group. Moreover, mice in a cellular model after transfection also showed higher MMP-9 activity than did controls. Conclusion High KLF5 expression may play a pivotal role in the pathogenesis of CCAML, partly through regulating the activity of MMP-9.

Lack of epithelial PPARγ causes cystic adenomatoid malformations in mouse fetal lung.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.

Sox2 regulates the emergence of lung basal cells by directly activating the transcription of Trp63.

Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.

FGF10 Signaling differences between type I pleuropulmonary blastoma and congenital cystic adenomatoid malformation.

BACKGROUND: Type I pleuropulmonary blastoma (PPB) and congenital cystic adenomatoid malformation of the lung (CCAM) are cystic lung diseases of childhood. Their clinical and radiological presentations are often similar, and pathologic discrimination remains difficult in many cases. As a consequence, type I PPB and CCAM are frequently confused, leading to delayed adequate management for type I PPB. Recent studies have suggested a role for fibroblast growth factor (FGF) 10 signal pathway in CCAM pathogenesis. The objective of our study was to determine whether FGF10 signaling differs between CCAM and type I PPB. METHODS: Immunohistochemical studies were performed for expression of FGF10, its receptor FGFR2b, and its inhibitor sonic hedgehog (SHH) in focal type I PPB (n=6), CCAM type I (n=7), CCAM type II (n=7), and control lungs (n=5). RESULTS: FGF10, FGFR2b, and SHH expressions differed markedly between type I PPB and both types of CCAM. Type I and type II CCAM cystic walls expressed FGF10, FGFR2b, and SHH, whereas staining was absent or poor in type I PBB cystic walls. Expression of FGF10, FGFR2b, and SHH did not differ between CCAM cystic walls and control airway walls. CONCLUSIONS: These findings show that immunohistochemistry with FGF10, FGFR2b, or SHH could be useful in differentiating CCAM from type I PPB, when a child presents with a focal cystic lung lesion. The absence of strong expression of FGF10, FGFR2b, and/or SHH makes the diagnosis of CCAM very doubtful.

[Congenital cystic adenomatoid pulmonary malformation and cancer of lung. Molecular-genetic and immunohistochemical investigation of p53 gene, a regulator of cellular cycle].

The loss of heterozygosity by p53 gene and the expression of p53 protein have been studied in cancerous pulmonary tissues and congenital cystic adenomatoid malformation by molecular-biologic and immunohistochemial methods. The loss of heterozygosity by p53 gene is confirmation of precocious molecular damage in epithelium of cystic adenomatoid pulmonary malformation.

The expression of vascular endothelial growth factor and its receptors in congenital bronchopulmonary cystic malformations.

BACKGROUND: Bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM) represent rare hamartomatous abnormalities of the lung. Dysregulation of cytokines that influence pulmonary vasculogenesis and epithelial growth, both known to be altered in BPS and CCAM, may play a role in their pathogenesis. OBJECTIVE: We hypothesized that expression of vascular endothelial growth factor (VEGF) or its receptors might be altered in CCAM and BPS, possibly distinguishing CCAM from BPS, or from controls. METHODS: Lung biopsy specimens obtained from infants who had undergone surgery for BPS (n = 4) or CCAM (n = 5) within the first month of life and normal lung autopsy samples (n = 4) serving as controls were investigated immunohistochemically for the protein expression levels of VEGF and its corresponding receptors. RESULTS: VEGF, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor receptor 3 (VEGFR3) staining was detected in CCAM and BPS specimens, as well as in control samples. VEGFR2 expression increased from controls to CCAM and from CCAM to BPS, the difference between controls and BPS being significant. The expression of VEGF, VEGFR1, and VEGFR3 was similar among the three groups. Consistent with a possible involvement of VEGFR2 in altered vasculogenesis-bronchiogenesis interaction, its expression was predominantly found in bronchial but not alveolar regions. CONCLUSIONS: The data suggest a possible role of VEGF-VEGFR2 interaction in the pathogenesis of congenital bronchopulmonary cystic malformations. However, VEGFR2 does not represent a suitable histochemical marker to distinguish between BPS and CCAM.

Overexpression of HOXB5, cyclin D1 and PCNA in congenital cystic adenomatoid malformation.

OBJECTIVE: We investigated the patterns of expression of HOXB5, cyclin D1 and proliferating cell nuclear antigen (PCNA) proteins in human congenital cystic adenomatoid malformation (CCAM) to establish the molecular basis of its etiology. METHODS: Immunohistochemistry was performed on frozen archival specimens of CCAM and normal lung tissue as controls using antibodies against HOXB5, cyclin D1 and PCNA proteins. RESULTS: Immunohistochemistry revealed a higher level of expression of HOXB5, cyclin D1 and PCNA predominantly in mesenchymal cells of the CCAM tissues as compared to normal adjacent control lung tissues. CONCLUSION: Elevated levels of immunohistochemical detection of HOXB5, cyclin D1 and PCNA were characteristic properties of lung tissue cells in CCAM. This elevated HOXB5 expression may correlate with the aberrant cellular differentiation observed in the CCAM disorder. Elevated expression of cyclin D1 and PCNA further suggests that increased cellular proliferation contributes to the overgrowth of lung tissue in CCAM.

Aberrant cell adhesion molecule expression in human bronchopulmonary sequestration and congenital cystic adenomatoid malformation.

In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on alpha(2)-, alpha(3)-, and beta(1)-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation. alpha(2)-, alpha(3)-, and beta(1)-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for alpha(2)-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected alpha(2)-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and alpha(2)-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas alpha(3) increased in CCAM compared with canalicular, but not with alveolar, stage lung. beta(1)-integrin levels were unchanged. We conclude that in BPS and CCAM, altered alpha(2)-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM.

Genetic analysis of congenital cystic adenomatoid malformation reveals a novel pulmonary gene: fatty acid binding protein-7 (brain type).

The pathogenesis of congenital cystic adenomatoid malformation (CCAM) is unknown and its natural history is unpredictable. Fatty acid binding protein-7 (FABP-7) has been previously described in brain and breast development, but never before in the lung. We investigate gene expression in CCAM, and hypothesize that CCAM results from an aberration in the signaling pathway during lung development. Under IRB approval, tissue specimens of fetal CCAM, fetal control, postnatal CCAM, and postnatal control were examined and microarray analysis was performed. Candidate differentially expressed genes were selected with log-odds ratio (B) >0 and false discovery rate <0.05. Validation of differential expression was achieved at the RNA and protein levels. FABP-7 was underexpressed in fetal CCAM compared with fetal lung in both the microarray and by RT-PCR. Findings were duplicated by Western Blot analysis and immunohistochemistry. This is the first description of FABP-7 in the human lung. Decreased expression of FABP-7 in fetal CCAM compared with normal fetal lung at both the RNA and protein levels suggests FABP-7 may have a role in pulmonary development and in the pathogenesis of CCAM.

Cytogenetic and p53 profiles in congenital cystic adenomatoid malformation: insights into its relationship with pleuropulmonary blastoma.

Congenital cystic adenomatoid malformation (CCAM), a developmental anomaly of lung, shares many features with the pediatric tumor pleuropulmonary blastoma (PPB). Both may show benign epithelium-lined cysts and mesenchymal proliferation, often with skeletal muscle differentiation. Before its recognition as a distinct entity, PPB was described in several reports as "rhabdomyosarcoma arising in CCAM." Abnormal karyotypes in PPB often show excess material from chromosome 8. It has also been suggested that PPB may harbor p53 mutations. We examined the karyotype and searched for p53 mutations (via immunostaining and single-strand conformation polymorphism analysis) in 11 CCAM and in 2 PPB. Karyotypes were normal in all CCAM and showed clonal abnormalities in both PPB. There was marked and diffuse immunopositivity for nuclear p53 in the epithelial cells of CCAM and PPB. Strong staining was also observed in approximately 50% of the stromal cells in all PPB, but was seen in the stroma of only 2 of 10 CCAM, where it was faint and focal. TP53 mutations were not identified in CCAM or PPB. We conclude that CCAM does not contain the clonal chromosomal aberrations reported in PPB and shows less stromal p53 immunostaining than PPB. Since p53 mutations were not identified in either entity, the observed p53 immunoreactivity may be caused by another mechanism; its role in PPB and CCAM pathogenesis remains to be determined. Overall, these findings provide evidence that CCAM is nonneoplastic. Although some may view CCAM as a PPB precursor, it remains biologically distinct in terms of karyotype and p53 status.

Congenital pulmonary airway malformation (congenital cystic adenomatoid malformation) with multiple extrapulmonary anomalies: autopsy report of a fetus at 19 weeks of gestation.

Congenital pulmonary airway malformation, or congenital cystic adenomatoid malformation, is postulated to be a disorder of pulmonary airway morphogenesis and encompasses 5 different types with distinct levels or stages of tracheobronchial development. We present a unique case of type 2 congenital pulmonary airway malformation with a previously undocumented combination of multiple extrapulmonary anomalies, featuring ipsilateral multicystic renal dysgenesis, contralateral renal agenesis, and ovarian germ cell hypoplasia, diagnosed in a 19-week gestational age fetus by autopsy. Epithelial cells comprising the pulmonary lesions were positive for thyroid transcription factor-1, surfactant protein-B, and cytokeratin-7 but negative for cytokeratin-20 immunostainings, with the pattern seen in normal terminal bronchioles. Chromosomal analysis showed a normal female karyotype, despite a high estimated risk for Down syndrome suggested by the low maternal serum alpha-fetoprotein level.

Expression of Hoxb-5 during human lung development and in congenital lung malformations.

BACKGROUND: We have previously shown that the Hox gene Hoxb-5 is necessary for normal mouse lung branching morphogenesis. Abnormal Hoxb-5 regulation causes specific alterations in airway branching. We hypothesized that Hoxb-5 is similarly involved in human lung branching morphogenesis, and is abnormally expressed in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), both of which are congenital lung malformations with abnormal airway development. METHODS: The temporal, spatial, and cellular expression of the Hoxb-5 protein was evaluated in normal human lung and BPS and CCAM tissue using Western blot analysis and immunocytochemistry. RESULTS: The expression of Hoxb-5 during human lung development showed strong similarities to that during mouse lung development. Western blots showed high Hoxb-5 protein levels in the pseudoglandular period (PSG), decreased but sustained levels in the canalicular period (CAN), and negligible levels during the alveolar period (ALV). Immunocytochemistry showed Hoxb-5 protein expression in mesenchymal cells around branching airways in the pseuodglandular period, subepithelial fibroblast localization (especially at airway branch points) in the CAN and minimal expression in the ALV. In BPS and CCAM tissue, Hoxb-5 protein levels were increased compared to age- and developmentally-matched lung tissue, and were more similar to the PSG and CAN with Hoxb-5-positive cells in mesenchyme surrounding abnormally branched airways. CONCLUSIONS: Hoxb-5 expression during human lung branching morphogenesis, which is similar to that observed in mouse lung development, indicates that it plays a role in controlling airway patterning. This notion is supported by results from BPS and CCAM tissue, in which Hoxb-5 is maintained in a manner typical of an earlier developmental stage and is associated with development of abnormal lung tissue.

An assessment of the expanded classification of congenital cystic adenomatoid malformations and their relationship to malignant transformation.

The purpose of this study was to review cases of congenital cystic adenomatoid malformations (CCAMs) arising in children and adults, in order to assess the recently expanded classification system for these lesions and their association with malignant transformation. Of 28 CCAMs, there were 16 type 1, 4 type 2, and 8 type 4 lesions, 12 of which presented in adults. Five of 16 type 1 CCAMs were accompanied by microscopic foci of bronchioloalveolar carcinoma; two others showed focal mucous cell hyperplasia. In two further cases, foci of nonmucinous atypical adenomatous hyperplasia were identified in the adjacent lung parenchyma. The bronchioloalveolar carcinomas showed less cytologic atypia, proliferative activity (Ki-67), and p53 expression than a comparative group of bronchioloalveolar carcinomas arising de novo, but this was not statistically significant (p = 0.15). Neither bronchioloalveolar carcinomas nor hyperplasia was identified in type 2 or type 4 CCAMs. Four of the eight type 4 CCAMs showed focal stromal hypercellularity, and one case subsequently developed a pleuropulmonary blastoma. We conclude that classification according to the current system is of clinical value. Bronchioloalveolar carcinomas arise in association with type 1 CCAMs, but recurrence following resection is exceptional. Type 4 CCAMs show histologic overlap with grade 1 pleuropulmonary blastomas, and distinction between these entities may not be possible on histology alone. However, stromal cellularity in a type 4 CCAM should raise the possibility of blastomatous transformation.

Glial cell-derived neurotrophic factor expression in normal human lung and congenital cystic adenomatoid malformation.

CONTEXT: It has been recently suggested that dysregulation of developmental factors and disruption of cell turnover could play a role in the pathogenesis of congenital cystic adenomatoid malformation of the lung (CCAM). The glial cell-derived neurotrophic factor (GDNF) is a growth factor involved in organogenesis, and the temporal pattern of GDNF expression suggests that this factor may play a role in lung development. DESIGN: We studied GDNF expression by immunohistochemistry in postnatally resected CCAM of the lung (n = 10), normal fetal lung (n = 5), and normal postnatal lung (n = 5). We also studied the association between GDNF expression and both cell proliferation and apoptosis. RESULTS: GDNF was expressed in both epithelial and endothelial compartments of normal fetal lung, whereas no expression was found in normal postnatal lung. In contrast, in CCAM tissue, there was strong GDNF immunostaining that was restricted to epithelial cells. The percentage of proliferating epithelial cells was higher in CCAM tissue than in normal postnatal lung (6.3% vs 1.7%, P <.005). Apoptotic bodies were found in the mesenchyme of both normal fetal lung and CCAM tissue, whereas virtually no apoptotic bodies were detected in normal postnatal lung. CONCLUSIONS: Abnormal GDNF expression in CCAM suggests a dysregulation of the GDNF signaling pathway and argues in favor of a focal arrest in maturation during development. GDNF expression in lung tissue seems to be correlated with cell proliferation, suggesting that this factor could play a role in the growth of both fetal lung and CCAM.