Tissue eosinophilia and computed tomography features in paediatric chronic rhinosinusitis with nasal polyps requiring revision surgery.

BACKGROUND: Endoscopic sinus surgery (ESS) is an effective and safe treatment modality for medically recalcitrant chronic rhinosinusitis (CRS) in the paediatric population, especially in older children or those with nasal polyposis (CRSwNP). We aimed to elucidate the inflammatory pattern and clinical characteristics of CRSwNP related to revision surgery after ESS in a paediatric population. METHODS: We retrospectively enrolled 146 patients with bilateral CRSwNP. Twenty-two patients had recurrent nasal polyps that required revision surgery. The clinical characteristics, computed tomography (CT) features, tissue eosinophil count, and immunoactivity of signature cytokines in the two groups were analysed. RESULTS: Tissue eosinophil infiltration and immunoreactivity of eosinophilic cationic protein and IL-5 in the sinus mucosa were higher in patients that required revision surgery. The revision surgery group was significantly younger and had positive aeroallergen test results, higher total Lund-Mackay scores, and ethmoid/maxillary sinus ratio on CT images than those without revision surgery. A nomogram was developed to predict the probability of the requirement of revision surgery according to the logistic regression analysis results. CONCLUSIONS: We developed a nomogram model using clinical characteristics, tissue eosinophilia, and CT features for the preoperative identification of patients vulnerable to revision surgery in paediatric CRSwNP. This could help clinicians predict the probability of recurrence and perform intensive postoperative adjunct therapy and follow-up.

Characterization of 11p14-p12 deletion in WAGR syndrome by array CGH for identifying genes contributing to mental retardation and autism.

WAGR (Wilms tumor, Aniridia, Genitourinary malformations and mental Retardation) syndrome is a rare genomic disorder caused by deletion of the 11p14-p12 chromosome region. The majority of WAGR patients have mental retardation and behavioral problems, and more than 20% of the patients also have features of autism. While the Wilms tumor/genitourinary anomalies and aniridia are caused by deletion of WT1 and PAX6 respectively, the genomic cause of mental retardation and autism in WAGR syndrome remains unknown. Using oligonucleotide arrays, we have characterized the 11p14-p12 deletions in 31 patients and identified all the genes involved in each deletion. The deletions had sizes ranging from 4.9 to 23 Mb that encompass 18-62 genes (40 on average). In addition to WT1 and PAX6, all the patients had deletion of PRRG4 (transmembrane gamma-carboxyglutamic acid protein 4). The majority of them had deletion of BDNF (brain-derived neurotrophic factor) and SLC1A2 [solute carrier family 1 (glial high affinity glutamate transporter) member 2]. Deletion of BDNF and SLC1A2 occurred in patients with autism more frequently than in those without autism. Literature review on the functions of the genes suggests that haploinsufficiency of SLC1A2, PRRG4, and BDNF may contribute to mental retardation and behavioral problems. In particular, BDNF may modulate the risk of autism in WAGR patients as suggested by its link with Rett syndrome as a target of MECP2. We observed that all the de novo deletions occurred in the chromosome 11 inherited from the father in the families genotyped, implying a predisposition for de novo mutations occurring in spermatogenesis and possible involvement of imprinting in cognitive impairment in WAGR patients.

Characteristic Ber-EP4 and EMA expression in sebaceoma is immunohistochemically distinct from basal cell carcinoma.

AIMS: There is considerable overlap between the histological features of sebaceoma and basal cell carcinoma (BCC). The distinction between these two tumours is important due to the often more locally aggressive nature of BCC and the association of sebaceoma with the Muir-Torre syndrome. The aim of this study was to describe the immunohistochemical reactivity of the cells in sebaceoma to Ber-EP4 and epithelial membrane antigen (EMA) and investigate the utility of this panel to differentiate sebaceoma from basal cell carcinoma. METHODS AND RESULTS: Immunohistochemistry of 25 sebaceomas for Ber-EP4 and EMA revealed unequivocal negative expression of Ber-EP4 in 24 of 25 sebaceomas. A single case exhibited focal weak Ber-EP4 staining, predominantly in mature sebocytes and in < 10% of the tumour cells. EMA was not expressed in the germinative cells of sebaceoma, but was expressed strongly in approximately 50% of mature sebocytes in all cases and highlighted the cytoplasmic vacuoles. We reviewed the immunoreactivity of 51 cases of nodular BCCs and found moderate or strong BerEP4 expression in all cases with never less than 20% of the tumour staining. Expression of EMA was uncommon in BCC (moderate or strong in 8%) and was confined to keratotic or squamoid areas. CONCLUSION: The use of Ber-EP4 in combination with EMA, both widely used immunomarkers in histopathology, is a helpful aid in distinguishing sebaceoma from nodular BCC.

BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs.

BACKGROUND: Malignant melanoma arising from different body compartments may be associated with differing aetiological factors and clinical behaviour, and may manifest diverse molecular genetic profiles. Although many studies have focused on cutaneous melanoma, little is known of mucosal and other types of melanoma. In particular, malignant melanoma of soft parts is different from other melanomas in many respects, yet manifests a common melanocytic differentiation. Mutation of BRAF is now known to be common in cutaneous melanomas, and raises possible new therapeutic options of anti-RAF treatment for these patients. Few data are available for non-cutaneous melanomas. AIMS: To study the incidence of BRAF and NRAS mutations in melanomas arising in diverse internal organs. METHODS: Fifty one melanomas from various internal organs were investigated for BRAF and NRAS mutation by direct DNA sequencing. RESULTS: BRAF and NRAS mutations were found in two and five mucosal melanomas arising from the aerodigestive and female genital tracts (n = 36). Their occurrence is mutually exclusive, giving a combined mutation incidence rate of 19.4% in mucosal melanomas. Both BRAF and NRAS mutations were absent in malignant melanoma of soft parts (n = 7). BRAF mutation was also absent in uveal melanoma (n = 6), but was seen in two of five cutaneous melanomas. The incidence of BRAF or combined BRAF/NRAS mutations in all non-cutaneous groups was significantly lower than published rates for cutaneous melanomas. CONCLUSION: Each melanoma subtype may have a unique oncogenetic pathway of tumour development, and only a small fraction of non-cutaneous melanomas may benefit from anti-RAF treatment.

Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

Molecular cytogenetic characterization of a derivative chromosome 8 with an inverted duplication of 8p21.3-->p23.3 and a rearranged duplication of 8q24.13-->qter.

A derivative chromosome 8 was observed in a newborn boy who presented with low birth weight, multiple congenital anomalies, and dysmorphic face. The der(8) was further characterized at age 18 months by a high resolution G-banding analysis, spectral karyotyping, and fluorescence in situ hybridization (FISH) with multiple DNA probes. The karyotype was described as 46,XY,der(8)(qter-->q24.13::p21.3-->p23.3::p23.3-->qter), representing an inverted duplication of region 8p21.3-->p23.3 and a duplication of region 8q24.13-->qter, which attaches to the duplicated short arm segment at 8p21.3. Different from previously reported patients with an inverted duplication (8p), no deletion was detected in the distal region of 8p in this case. This young child had manifested a broad nasal bridge, micrognathia, cleft lip, hydrocephalus, partial agenesis of the corpus callosum, Dandy-Walker malformation, congenital heart defects, dysplastic kidneys, hydronephrosis, marked hypotonia, and significant psychomotor retardation. These features are compared with those commonly seen in cases with an inverted duplication of 8p and cases with a partial trisomy of 8q.

Sensitivity of multiple color spectral karyotyping in detecting small interchromosomal rearrangements.

Multiple color spectral karyotyping (SKY) has been proven to be a very useful tool for characterization of the complex rearrangements in cancer cells and the de novo constitutional structural abnormalities. The sensitivity of SKY in detecting interchromosomal alterations was assessed with 10 constitutional translocations involving subtelomeric regions. Among the 13 small segments tested, 9 were clearly visualized and 8 were unambiguously identified by SKY. Fluorescence in situ hybridizations (FISH) with subtelomeric probes confirmed the reciprocity in three of the four translocations in which a small segment was not detectable by SKY. On the basis of resolution level of G-banding and the information obtained from the FISH analysis, the minimum alteration that SKY can detect is estimated to be 1,000-2,000 kbp in size with the currently available probes. This study has demonstrated the power, but also the limitations, of SKY in detecting small interchromosomal alterations, particularly those in subtelomeric regions.

Jumping translocations of 11q in acute myeloid leukemia and 1q in follicular lymphoma.

Jumping translocation is a rare cytogenetic aberration in leukemia and lymphoma, and its etiologic mechanisms are not clearly known. We report two cases with jumping translocations. One had follicular lymphoma and jumping translocations of 1q onto the telomeric regions of 5p, 9p, and 15q in three cell lines, co-existing with the specific translocation t(14;18)(q32;q21). The second case had acute myeloid leukemia (AML) and jumping translocations of 11q as the sole aberration, onto multiple derivative chromosomes in each of the abnormal cells. A total of 17 telomeric regions were seen as the recipients of 11q in this case, and 9q was always involved as one of the recipients in all abnormal cells. Fluorescence in situ hybridization (FISH) confirmed the identification of 11q material in the derivative chromosomes. While 1q has been the most common donor of acquired jumping translocations, this is the first report on jumping translocations of 11q. Different from all previously reported jumping translocations which involve only one recipient in each cell line and lead to a mosaic trisomy, multiple recipients in most of the abnormal cells in this case had led to a tetrasomy, or a pentasomy of 11q. The pattern of chromosome involvement as the recipients of 11q appears to show a continuing evolutionary process of jumping, stabilization, and spreading of the donor material into other chromosomes. Somatic recombinations between the interstitial telomeric or subtelomeric sequences of a derivative chromosome and the telomeric sequences of normal chromosomes are believed to be the underlying mechanism of jumping translocations and their clonal evolution.

Interstitial deletion of 8(q13q22) in diffuse large B-cell gastric lymphoma.

An interstitial deletion in the long arm of chromosome 8 as the sole structural anomaly was detected in a primary gastric diffuse large B-cell lymphoma, high-grade mucosa-associated lymphoid tissue (MALT) type, from a 74-year-old man. Low-grade MALT lymphoma was not seen in the sections submitted for examination. Helicobactor pylori organisms were found in a biopsy performed prior to resection of the tumor. The karyotype was described as 45, X,-Y,del(8)(q13q22). No rearrangement between chromosome 8 and others was detected with fluorescence in situ hybridization using a whole chromosome 8 painting probe. Fluorescence in situ hybridization with the C-MYC gene showed its normal location at 8q24 on both chromosomes 8 without rearrangement. Amplification of C-MYC was not detected in interphase cells. Deletion of 8q may represent a unique genomic alteration in this particular subtype of primary gastric lymphoma.

A new complex variant Philadelphia chromosome, t(1;9;22)ins(17;22), characterized by fluorescence in situ hybridization in an adult ALL.

A new complex variant Philadelphia chromosome was detected in a 65-year-old man with acute, pre-B, lymphoblastic leukemia (ALL). The classic cytogenetic analysis identified an apparently balanced three-way translocation t(1;9;22)(q25;q34;q11.2). Fluorescence in situ hybridization (FISH) studies confirmed the translocation and showed bcr/abl fusion on the der(22). However, these studies revealed that the distal part of the bcr gene was not translocated onto chromosome 1 at 1q25, but inserted into chromosome 17 at 17p12-13. This complex variant translocation was described as a t(1;9;22)(q25;q34;q11.2)ins(17;22)(p12-13;q11.2q11.2). Secondary changes including +8, an inversion of the derivative chromosome 9, a translocation t(14;20)(q11;q13), and an additional derivative 22 were also identified in most of the abnormal cells. The patient died from systemic fungemia and multiorgan failure 9 months after the diagnosis of ALL. The clinical significance of complex variant Philadelphia chromosomes in ALL is reviewed and discussed.

Small terminal deletion of 1p and duplication of 1q: cytogenetics, FISH studies, and clinical observations at newborn and at age 16 years.

A small, extra chromosome segment added to 1p was found by Q-banding 16 years ago in a newborn baby with low birth weight, short stature, wide open fontanelle, small palpebral fissures, depressed nose bridge, and inguinal hernia. This chromosome abnormality has been characterized recently with G-banding and fluorescence in situ hybridization using multiple DNA probes. The karyotype is now described as 46,XY, der(1)(qter-->p36.13::q42.3-->qter), representing a small deletion of 1p36.13-pter and a small duplication of 1q42.3-qter. Re-examination of this patient at age 16 years showed marked psychomotor delay, severely accentuated dorsal kyphosis and scoliosis, pectus excavatum, and other anomalies but no clinical signs of neuroblastoma. Comparison of the clinical findings in this case with those described in the patients having either a deletion of 1p36-pter or a duplication of 1q42-qter further illustrated the complexity of the genotype-phenotype relationship.

Endoreduplication and telomeric association in a choroid plexus carcinoma.

Cytogenetic studies showed a hyperhaploid stemline, (32,XY,+1,+7,+9,+12,+13,+14,+19,+20) in a patient with choroid plexus carcinoma. Endoreduplication and doubling of the stemline to 200-400 chromosomes per cell and variation in numerical changes were also noted. Telomeric association was present in most cells. The 12p and 20q were by far the most frequently involved chromosome arms. Telomeric association is believed to have triggered further structural changes in this case since the 12p and 20q were always involved in the few structural abnormalities identified. A review of the literature suggests that hyperhaploidy may characterize choroid plexus carcinoma and hyperdiploidy choroid plexus papilloma.

Cytogenetic evidence that a tumor suppressor gene in the long arm of chromosome 1 contributes to glioma growth.

In a patient with a rare subtype of glioma, pleomorphic xanthoastrocytoma, cytogenetic studies revealed that both homologues of chromosome 1 were involved in translocations at the same band 1q42 but with different partner chromosomes. In addition, 5 glioblastomas out of 25 gliomas karyotyped in our laboratory had lost at least one copy of band 1q42 through deletions, unbalanced rearrangements, or chromosome losses. Twenty-one gliomas that had lost at least one copy of chromosome band 1q42 were identified in the literature; all were astrocytic tumors and the majority were glioblastomas. It indicates a covert tumor suppressor gene in the region that is involved in astrocytic gliomas.

Correction of mucolipidosis III in vitro by gene transfer.

Mucolipidosis II (ML II, I-cell disease) and mucolipidosis III (ML III, pseudo-Hurler polydystrophy) are human autosomal recessive genetic disorders resulting from deficient UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase (GNPT) activity. Normally, this enzyme is involved in the processing of most lysosomal enzymes. Cultured fibroblasts from individuals with either disorder are deficient in a broad array of lysosomal enzymes as a result of the diminished GNPT activity. We report the correction of this phenotype by fusing transformed ML III cells generated for this study to lethally irradiated rodent cells. This method of gene transfer does not require selection for the gene of interest, animal models, nor any knowledge of the gene product except a screening method for its presence. It has generated corrected cell hybrids that contain approximately 1% hamster-derived sequences. These cell lines, which contain the hamster analogue to the human phosphotransferase gene, are useful for the molecular cloning of the gene defective in ML II and ML III.

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes.

A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders.

Human islet amyloid polypeptide gene: complete nucleotide sequence, chromosomal localization, and evolutionary history.

The gene-encoding human islet amyloid polypeptide (hIAPP), a recently discovered 37 amino acid hormone-like polypeptide which is expressed in the insulin-producing beta-cells of the endocrine pancreas, has been isolated and characterized. The coding region of the gene is interrupted in the 5'-untranslated region and NH2-terminal propeptide by introns of 330 and 4808 base pairs (bp), respectively. Exon 1 (104 bp) encodes most of the 5'-untranslated region of the mRNA; exon 2 (95 bp) encodes 15 nucleotides of 5'-untranslated region, the putative 22 amino acid signal peptide and five residues of the NH2-terminal propeptide; exon 3 (1246 bp) encodes the remainder of the NH2-terminal propeptide (residues 6-9), the IAPP moiety and its processing signals and the 16 amino acid COOH-terminal propeptide, as well as the 3'-untranslated region of the mRNA (1059 bp). Analysis of the nucleotide and predicted amino acid sequence of intron 2 of the hIAPP gene did not reveal any homology with the structurally related calcitonin/calcitonin-gene-related peptide genes and indicated that, in contrast to these latter genes, the hIAPP gene apparently gives rise to only a single hormonal product. The transcriptional initiation site was identified about 28 bp downstream from a TATAA sequence. The hIAPP gene was localized to the p12.3 region of chromosome 12.