Csaba Galambos

A protocol for the handling of tissue obtained by operative lung biopsy: recommendations of the chILD pathology co-operative group.

This is the first of a series on pediatric pulmonary disease that will appear as Perspectives in Pediatric Pathology over the coming months. The series will include practical issues, such as this protocol for handling lung biopsies and another on bronchoalveolar lavage in childhood, as well as reviews of advances in various areas in pediatric pulmonary pathology. It has been 11 years since the last Perspectives on pulmonary disease. Much has happened since then in this area, and this collection will highlight some emerging and rapidly advancing areas in pediatric lung disease. These will include a review of molecular mechanisms of lung development, and another of mechanisms of pulmonary vascular development. The surfactant system and its disorders, as well as recent advances in the biology of the pulmonary neuroendocrine system and mechanisms of respiratory viral disease, will be addressed. Articles on pulmonary hypertension, pulmonary neoplasia, and pediatric lung transplantation, with their implications for the pediatric pathologist, are also planned. The contributors to this series are a diverse group with special interests and expertise in these areas. As Dr. William Thurlbeck noted in his foreword to the previous volume, Pulmonary Disease, volume 18 of Perspectives in Pediatric Pathology, pediatric pathology had been largely concerned with phenomenology, rather than with mechanisms, model systems, and experimental investigation. I think he would have been pleased to see the changes that have occurred over the past 10 years in pediatric lung biology and pathology in particular, because these were particularly favored interests of his later years.

Identification of lymphatic endothelium in pediatric vascular tumors and malformations.

The distinction between lymphatic and other vascular vessels on microscopic sections is a challenging task. D2-40, a novel antibody, has been reported to be selective for lymphatic endothelium. We studied the specificity and sensitivity of D2-40 in pediatric vascular tumors and malformations. Fourteen lymphatic and 11 vascular lesions were randomly selected and stained with D2-40 and CD31 antibodies. The lymphatic lesions included 6 lymphatic malformations, 5 cystic hygromas (macrocystic lymphatic malformation), 2 lymphovenous malformations, and 1 lymphangioma, and the vascular lesions comprised 3 infantile hemangiomas, 3 Kaposiform hemangioendotheliomas, 2 tufted angiomas, 1 pyogenic granuloma, 1 arteriovenous, and 1 venulocapillary malformations. The staining patterns of the vascular channels were compared. In all lesions D2-40 labeled only the endothelium of thin-walled vascular channels morphologically consistent with lymphatic vessels (25 of 25). No staining of the vascular lesions (0 of 11) or of arteries and veins (0 of 25) was observed. All lymphatic lesions had D2-40–positive vessels; however, the percentage of vessels that stained varied. Five lymphatic lesions showed more than 75% D2-40–positive channels, 5 lesions had approximately 50%, and 4 cases showed fewer than 25% D2-40–positive channels. There was a tendency of more consistent D2-40 staining of small versus large lymphatic channels. CD31 constantly labeled arteries, veins, capillaries, and lymphatics in all lesions and all endothelial cells in the vascular lesions. D2-40 is a very specific antibody for lymphatic endothelium, with variable sensitivity. CD31 more reliably identifies lymphatic endothelium. Currently, D2-40 appears to be a good marker to identify lymphatic vessels in pediatric vascular tumors and malformations.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Prox-1 and VEGFR3 antibodies are superior to D2-40 in identifying endothelial cells of lymphatic malformations--a proposal of a new immunohistochemical panel to differentiate lymphatic from other vascular malformations.

We previously reported that D2–40 antibody identifies lymphatic endothelial cells (LECs) of lymphatic malformations (LM) with high specificity but only moderate sensitivity. The aim of this study was to compare the sensitivity and specificity of the markers Prospero-related homeobox gene–1 (Prox-1) and VEGFR3 to those of D2–40 for LECs in LM. Seventeen LM and 6 other vascular malformations with venous component (VM) were stained with D2–40, Prox-1, VEGFR3, CD31, and CD34 antibodies. The staining characteristics of vessels by each marker were assessed. Prox-1 and VEGFR3 specificity for LECs was examined by endothelial staining of VMs. Prox-1 and VEGFR3 stained substantially more large vessels than did D2–40 (15 of 17 cases). Small vessel staining was uniformly positive with all vascular markers except CD34, which did not stain lymphatic vessels. Eight cases had no or minimal D2–40 staining of large vessels, but the selected D2–40-vessels stained positive with VEGFR3, and 7 of 8 vessels were Prox-1 positive. Nine cases had variable D2–40 staining of large vessels; the selected D2–40 channels were all Prox-1 positive, and 8 of 9 were VEGFR3 positive. Two had rare vascular channels with no lymphatic marker stain but were positive for CD31 and CD34. Endothelial cells of VM had no Prox-1 but were VEGFR3/CD31/CD34 positive. VEGFR3 and Prox-1 antibodies have greater sensitivity for large lymphatic vessels than does D2–40. All lymphatic markers have high sensitivity for LECs of small lymphatic channels. Prox-1 has superior sensitivity and specificity to LECs. We recommend utilizing an immunohistochemical panel consisting of Prox-1, VEGFR3, CD31, and CD34 antibodies to differentiate lymphatic from venous malformations in pathologic practice.

Homozygous mutation of MYBPC3 associated with severe infantile hypertrophic cardiomyopathy at high frequency among the Amish

Background: Familial hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death among young and apparently healthy people. Autosomal dominant mutations within genes encoding sarcomeric proteins have been identified. An autosomal recessive form of HCM has been discovered in a group of Amish children that is associated with poor prognosis and death within the first year of life. Affected patients experienced progressive cardiac failure despite maximal medical treatment. Postmortem histology showed myofibre disarray and myocyte loss consistent with refractory clinical deterioration in affected infants. Objective: To conduct a genome-wide screen for linkage and try to identify an autozygous region which cosegregates with the infant cardiac phenotype Methods and results: An autozygous region of chromosome 11 which cosegregates with the infant cardiac phenotype was identified. This region contained the MYBPC3 gene, which has previously been associated with autosomal dominant adult-onset HCM. Sequence analysis of the MYBPC3 gene identified a splice site mutation in intron 30 which was homozygous in all affected infants. All surviving patients with the homozygous MYBPC3 gene mutations (3330+2T>G) underwent an orthotopic heart transplantation. Conclusions: Homozygous mutations in the MYBPC3 gene have been identified as the cause of severe infantile HCM among the Amish population.

Intrapulmonary vascular shunt pathways in alveolar capillary dysplasia with misalignment of pulmonary veins

Abstract Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal neonatal lung disease characterised by severe pulmonary hypertension, abnormal vasculature and intractable hypoxaemia. Mechanisms linking abnormal lung vasculature with severe hypoxaemia in ACD/MPV are unknown. We investigated whether bronchopulmonary anastomoses form right-to-left shunt pathways in ACD/MVP. We studied 2 infants who died of ACD/MPV postmortem with direct injections of coloured ink into the pulmonary artery, bronchial artery and pulmonary veins. Extensive histological evaluations included serial sectioning, immunostaining and 3-dimensional reconstruction demonstrated striking intrapulmonary vascular pathways linking the systemic and pulmonary circulations that bypass the alveolar capillary bed. These data support the role of prominent right-to-left intrapulmonary vascular shunt pathways in the pathophysiology of ACD/MPV.

Three-Dimensional Reconstruction Identifies Misaligned Pulmonary Veins as Intrapulmonary Shunt Vessels in Alveolar Capillary Dysplasia

Alveolar capillary dysplasia (ACD) with misalignment of pulmonary veins (MPV) is a lethal neonatal lung disease. Death from ACD/MPV is caused by hypoxia, but the role of the MPV is unknown. Using 3-dimensional reconstruction of ACD/MPV lung tissue, we report that the veins in MPV are intrapulmonary shunt vessels, and speculate that MPV contributes to the poor prognosis.

Prominent Intrapulmonary Bronchopulmonary Anastomoses and Abnormal Lung Development in Infants and Children with Down Syndrome

Objectives To determine the frequency of histologic features of impaired lung vascular and alveolar development and to identify the presence of intrapulmonary bronchopulmonary anastomoses (IBA) in infants and children who died with Down syndrome. Study design A retrospective review of autopsy reports and lung histology from 13 children with Down syndrome (ages: 0‐8 years) was performed. Histologic features of abnormal lung development were identified and semiquantified, including the presence of IBA. Three‐dimensional reconstructions of IBA were also performed. Comparisons were made with 4 age‐matched patients without Down syndrome with congenital heart defects who underwent autopsies during this time period. Results Of the 13 subjects with Down syndrome, 69% died from cardiac events, 77% had a congenital heart defect, and 46% had a clinical diagnosis of pulmonary hypertension. Lung histology from all subjects with Down syndrome demonstrated alveolar simplification, and 92% had signs of persistence of a double capillary network in the distal lung. The lungs from the subjects with Down syndrome frequently had features of pulmonary arterial hypertensive remodeling (85%), and prominent bronchial vessels and IBA were observed in all subjects with Down syndrome. These features were more frequent in subjects with Down syndrome compared with control subjects. Conclusions Children with Down syndrome who died of cardiopulmonary diseases often have histologic evidence of impaired lung alveolar and vascular development, including the presence of prominent IBA and pulmonary hypertension. We speculate that children with Down syndrome are at risk for reduced lung surface area and recruitment of IBA, which may worsen gas exchange in subjects with Down syndrome.

Dux4 Immunohistochemistry Is a Highly Sensitive and Specific Marker for Cic-dux4 Fusion-positive Round Cell Tumor.

The histologic differential diagnosis of pediatric and adult round cell tumors is vast and includes the recently recognized entity CIC-DUX4 fusion-positive round cell tumor. The diagnosis of CIC-DUX4 tumor can be suggested by light microscopic and immunohistochemical features, but currently, definitive diagnosis requires ancillary genetic testing such as conventional karyotyping, fluorescence in situ hybridization, or molecular methods. We sought to determine whether DUX4 expression would serve as a fusion-specific immunohistochemical marker distinguishing CIC-DUX4 tumor from potential histologic mimics. A cohort of CIC-DUX4 fusion-positive round cell tumors harboring t(4;19)(q35;q13) and t(10;19)(q26;q13) translocations was designed, with additional inclusion of a case with a translocation confirmed to involve the CIC gene without delineation of the partner. Round cell tumors with potentially overlapping histologic features were also collected. Staining with a monoclonal antibody raised against the C-terminus of the DUX4 protein was applied to all cases. DUX4 immunohistochemistry exhibited diffuse, crisp, strong nuclear staining in all CIC-DUX4 fusion-positive round cell tumors (5/5, 100% sensitivity), and exhibited negative staining in nuclei of all of the other tested round cell tumors, including 20 Ewing sarcomas, 1 Ewing-like sarcoma, 11 alveolar rhabdomyosarcomas, 9 embryonal rhabdomyosarcomas, 12 synovial sarcomas, 7 desmoplastic small round cell tumors, 3 malignant rhabdoid tumors, 9 neuroblastomas, and 4 clear cell sarcomas (0/76, 100% specificity). Thus, in our experience, DUX4 immunostaining distinguishes CIC-DUX4 tumors from other round cell mimics. We recommend its use when CIC-DUX4 fusion-positive round cell tumor enters the histologic differential diagnosis.

Chronic intrauterine pulmonary hypertension increases main pulmonary artery stiffness and adventitial remodeling in fetal sheep

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that is characterized by high pulmonary vascular resistance due to changes in lung vascular growth, structure, and tone. PPHN has been primarily considered as a disease of the small pulmonary arteries (PA), but proximal vascular stiffness has been shown to be an important predictor of morbidity and mortality in other diseases associated with pulmonary hypertension (PH). The objective of this study is to characterize main PA (MPA) stiffness in experimental PPHN and to determine the relationship of altered biomechanics of the MPA with changes in extracellular matrix (ECM) content and orientation of collagen and elastin fibers. MPAs were isolated from control and PPHN fetal sheep model and were tested by planar biaxial testing to measure stiffness in circumferential and axial vessel orientations. Test specimens were fixed for histological assessments of the vascular wall ECM constituents collagen and elastin. MPAs from PPHN sheep had increased mechanical stiffness (P < 0.05) and altered ECM remodeling compared with control MPA. A constitutive mathematical model and histology demonstrated that PPHN vessels have a smaller contribution of elastin and a greater role for collagen fiber engagement compared with the control arteries. We conclude that exposure to chronic hemodynamic stress in late-gestation fetal sheep increases proximal PA stiffness and alters ECM remodeling. We speculate that proximal PA stiffness further contributes to increased right ventricular impedance in experimental PPHN, which contributes to abnormal transition of the pulmonary circulation at birth.