Does the gene matter? Genotype-phenotype and genotype-outcome associations in congenital melanocytic naevi.

BACKGROUND: Genotype-phenotype studies can identify subgroups of patients with specific clinical features or differing outcomes, which can help shape management. OBJECTIVES: To characterize the frequency of different causative genotypes in congenital melanocytic naevi (CMN), and to investigate genotype-phenotype and genotype-outcome associations. METHODS: We conducted a large cohort study in which we undertook MC1R genotyping from blood, and high-sensitivity genotyping of NRAS and BRAF hotspots in 156 naevus biopsies from 134 patients with CMN [male 40%; multiple CMN 76%; projected adult size (PAS) > 20 cm, 59%]. RESULTS: Mosaic NRAS mutations were detected in 68%, mutually exclusive with BRAF mutations in 7%, with double wild-type in 25%. Two separate naevi were sequenced in five of seven patients with BRAF mutations, confirming clonality. Five of seven patients with BRAF mutations had a dramatic multinodular phenotype, with characteristic histology distinct from classical proliferative nodules. NRAS mutation was the commonest in all sizes of CMN, but was particularly common in naevi with PAS > 60 cm, implying more tolerance to that mutation early in embryogenesis. Facial features were less common in double wild-type patients. Importantly, the incidence of congenital neurological disease, and apparently of melanoma, was not altered by genotype; no cases of melanoma were seen in BRAF-mutant multiple CMN, however, this genotype is rare. CONCLUSIONS: CMN of all sizes are most commonly caused by mutations in NRAS. BRAF is confirmed as a much rarer cause of multiple CMN, and appears to be commonly associated with a multinodular phenotype. Genotype in this cohort was not associated with differences in incidence of neurological disease in childhood. However, genotyping should be undertaken in suspected melanoma, for guidance of treatment. What's already known about this topic? Multiple congenital melanocytic naevi (CMN) have been shown to be caused by NRAS mosaic mutations in 70-80% of cases, by BRAF mosaicism in one case report and by inference in some previous cases. There has been debate about genotypic association with different sizes of CMN, and no data on genotype-outcome. What does this study add? NRAS mosaicism was found in 68%, BRAF in 7% and double wild-type in 25% of cases of CMN. NRAS was the commonest mutation in all sizes of CMN, but was nearly universal in projected adult size > 60 cm. BRAF is often associated with a distinct multinodular clinical/histological phenotype. Adverse outcomes did not differ between genotypes on current numbers.

Histopathological reporting of paediatric cutaneous vascular anomalies in relation to proposed multidisciplinary classification system.

BACKGROUND: The terminology applied to vascular anomalies has been variable in previously published literature making interpretation suboptimal. The International Society for the Study of Vascular Anomalies (ISSVA) has proposed a revised classification based on clinical features and histopathological findings. This classification is increasingly being accepted as clinically useful and a platform for future studies. AIMS: To examine the extent to which the ISSVA classification can be practically applied to diagnostic histopathological specimens. METHODS: Cutaneous vascular lesions received in a single paediatric pathology unit during a 2-year period (2004-5) were reviewed, including glucose transporter protein 1 (GLUT1) immunostaining where required, and lesions were reclassified according to the ISSVA classification. RESULTS: 144 specimens were identified. Appropriate full clinical information was provided in only 17% of cases at submission. Infantile haemangiomas comprised 46% of cases, 18% of which were regressive type, initially inaccurately identified as vascular malformations before GLUT1 immunostaining. 30% of lymphatic malformations and all lymphovenous malformations were previously classified as vascular malformations, not otherwise specified. CONCLUSIONS: The ISSVA classification of vascular anomalies provides a useful framework for histopathologists to classify vascular anomalies. However, meaningful and appropriate use of such a system is dependent on the adequacy of clinical information provided and routine use of immunohistochemical markers.

Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation.

BACKGROUND: Recent reports have shown evidence of host derived parenchymal engraftment in several human allografts including the lung, leading to speculation that stem cell therapy may be useful for lung repair in diseases such as cystic fibrosis (CF). To date, previous studies have looked at single surgical or autopsy specimens and no longitudinal studies have been reported. The aim of this study was to assess whether transbronchial biopsies could be used to study the time course of chimerism following lung transplantation. METHODS: Specimens of archived transbronchial lung biopsies from five time points taken for clinical purposes from two boys who had received a sex mismatched heart-lung transplant for end stage CF were examined. Sections were dual stained for cytokeratin (epithelium) and a mixture of leucocyte common antigen and CD68 for inflammatory cells. Co-localisation of cells containing a Y chromosome was confirmed by fluorescent in situ hybridisation. RESULTS: Evidence of chimerism was found in up to 6.6% of epithelial cells in bronchial (median 1.4% (range 0-6.6)) and alveolar (median 3.6% (range 2.3-5.5) tissue without apparent evidence of fusion. This engraftment was seen as early as 3 weeks and remained relatively constant up to 37 months. CONCLUSIONS: This study has demonstrated proof of principle for long term chimerism in lung epithelium. Transbronchial biopsies may provide a new method for studying the kinetics of stem cell engraftment in the lung.

Neuroblastoma in monozygotic twins--a case of probable twin-to-twin metastasis.

Concordance for neuroblastoma in monozygotic twins has been reported only rarely, and the cause of the shared pathology has not been established. We describe a case of infant monozygotic twins developing tumours that were morphologically, clinically and molecularly indistinguishable, but with a delay of 6 months between times of presentation. Both tumours were metastatic and had amplification of MYCN and deletion at 1p36. Twin 1, who developed neuroblastoma first, had constitutional karyotype abnormalities in at least 5% of peripheral blood mononuclear cells involving 1p and 3p, and a deletion of 1q44 in 21% of cells. Twin 2 had a normal constitutional karyotype and lacked rearrangement or deletion of these regions. We propose an acquired neuroblastoma predisposition specific for twin 1, and in utero metastatic spread of tumour cells to twin 2 via the shared placental circulation.

Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.

AIM: At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens. METHODS: All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison. RESULTS: All the antibodies except delta sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer. CONCLUSION: A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy.

Desmin expression in rhabdomyosarcoma: influence of the desmin clone and immunohistochemical method.

AIM: To determine which, if any, of five commercially available desmin clones is most reliable at labelling desmin filaments and whether the enhanced polymer one step (EPOS) method of labelling is of any advantage in the routine diagnostic laboratory. METHODS: Thirty four rhabdomyosarcomas from the files at The Hospital for Sick Children, Great Ormond Street, London, were studied. Four different desmin clones, DE-R-11, D33, DE-U-10, and PDE, were applied to each using the conventional extravidin biotin peroxidase method. The D33 clone was also applied using the EPOS method. RESULTS: The EPOS method incorporating D33 persistently scored more cells as desmin positive and was positive in four cases which were negative on staining with the other clones. CONCLUSIONS: The D33 desmin clone used with the EPOS method is more reliable for identifying desmin filaments in tumours than other desmin antibodies tested. Different desmin clones using a routine technique label different rhabdomyosarcoma cells and therefore it is justifiable to use more than one clone.

Localized Leishmania lymphadenitis. Immunohistochemical studies.

Tissues from 10 patients with localized leishmania lymphadenitis were stained by immunoperoxidase technique for leishmanian antigens, immunoglobulins (Ig), complement, components, alpha 1-antitrypsin, and fibrin and by routine and chloroacetate esterase techniques. Lymph nodes with leishmania lymphadenitis showed a granulomatous process with a varying degree of necrosis, leishmanian amastigotes, abundant plasma cells, and focal fibrosis. Leishmanian antigens stained by rabbit anti-Leishmania tropica antiserum was detected in lymph nodes with granulomatous lymphadenitis (eight cases examined) and in a skin lesion but not in a lymph node showing only nonspecific hyperplasia. Positive antigen staining was seen in leishmanian amastigotes, granulomata, and (in three cases) vascular smooth muscles. Dominant plasma cell classes were IgG and IgE. Macrophages and granulomata stained positively for IgG and IgE (but not for IgM or IgA), faintly for C3, and strongly for alpha 1-antitrypsin. Mast cells were few. IgG and IgE may be involved in a specific way in this condition; formation of immune complexes with leishmanian antigens leads to immunologically mediated lysis of the host macrophages. This finding is important for the understanding of the pathogenesis of leishmania lymphadenitis and for effective elimination of the parasites.

Immunocytochemical study of 12E7 in small round-cell tumours of childhood: an assessment of its sensitivity and specificity.

12E7 is a monoclonal antibody to the MIC2 gene product and can be applied to formalin-fixed, paraffin-embedded tissue. The diagnostic utility of 12E7 as a marker of Ewing's sarcoma and peripheral neuroectodermal tumour was assessed. Immunocytochemical studies were performed on 120 small round-cell tumours from children and adolescents. Immunoreactivity for 12E7 was seen in 13 of 15 Ewing's sarcomas. 14 of 15 peripheral neuroectodermal tumours, four of 14 embryonal rhabdomyosarcoma, seven of 11 T-lymphoblastic lymphomas and one T-cell acute lymphoblastic leukaemia. Immunoreactivity was located on the cell-membrane of Ewing's sarcomas, peripheral neuroectodermal tumours and lymphoid tumours while rhabdomyosarcomas showed weak, cytoplasmic staining in differentiated rhabdomyoblasts. Studies on alveolar rhabdomyosarcomas (n = 10), acute myeloid leukaemias (3), B-lymphoblastic lymphomas (8), blastema-rich nephroblastomas (9), neuroblastomas (20) and retinoblastomas (10) as well as single examples of B-cell acute lymphoblastic leukaemia, Ki-1 anaplastic lymphoma of indeterminate phenotype and intra-abdominal desmoplastic tumour with divergent differentiation were negative. 12E7 is a sensitive marker for the Ewing's sarcoma/peripheral neuroectodermal group of tumours and is useful in distinguishing them from neuroblastoma and blastema-rich nephroblastoma. However, immunopositivity for 12E7 should be interpreted in conjunction with the results of neural and lymphoid markers.