Sequence analysis and high-throughput immunhistochemical profiling of KIT (CD 117) expression in uveal melanoma using tissue microarrays

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunhistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patient

KIT , PDGFRα and EGFR analysis in nephroblastoma

Nephroblastoma prognosis has dramatically improved, but an unfavourable prognostic subgroup warrants development of novel therapeutic strategies. Selective KIT, PDGFRα and epidermal growth factor receptor (EGFR) tyrosine kinase inhibition evolved as powerful targeted therapy for gastrointestinal stromal tumours and non-small-cell lung cancer. To investigate a potential role for tyrosine kinase inhibition, we analyzed 209 nephroblastomas for immunohistochemical KIT and EGFR expression, 63 nephroblastomas for mutations in KIT exons 9, 11, 13, EGFR exons 18, 19, 20 and 21, and all 209 nephroblastomas for PDGFRα exons 12, 14 and 18. Twenty-two tumours (10.5%) expressed KIT, 31 (14.8%) EGFR, and 10 (4.8%) both KIT and EGFR, respectively. KIT expression was relatively more common among high-risk tumours (6/27; 22.3%) compared to low-/intermediate-risk tumours (26/181; 14.4%). Nine patients deceased, four of which had high-risk tumours with KIT expression in two of four and EGFR expression in one of four. There were no KIT, PDGFRα or EGFR mutations. Our results suggest no significant contribution of KIT, EGFR or PDGFRα mutations to nephroblastoma pathogenesis. Despite a trend towards association of immunohistochemical KIT and EGFR expression with poor outcome in high-risk nephroblastomas, statistical analysis did not yield significant correlations in this subgroup. Therefore, it remains open if KIT, PDGFRα or EGFR tyrosine kinase inhibition constitute a therapeutic target in nephroblastoma in the absence of KIT, PDGFRα or EGFR mutation

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ? 4/5 methylated genes), MSI (MSI-H: ? 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p > 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p > 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p > 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer

In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high-level microsatellite instability (MSI-H). Cases with MSI-H or high-level CpG island methylator phenotype (CIMP-H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference

Ultrastructural and molecular analysis in fatal neonatal interstitial pneumonia caused by a novel ABCA3 mutation

Pulmonary surfactant is essential to maintain alveolar patency, and invariably fatal neonatal lung disease has been recognized to involve mutations in the genes encoding surfactant protein-B or ATP-binding cassette transporter family member ABCA3. The lipid transporter ABCA3 targets surfactant phospholipids to lamellar bodies that are lysosomal-derived organelles of alveolar type II cells. ABCA3-/- mice have grossly reduced surfactant phosphatidyl glycerol levels and die of respiratory failure soon after birth. We studied lung biopsy samples of two siblings with a novel homozygous ABCA3 mutation at nucleotide position 578 (c.578C<G), leading to a Pro193Arg amino-acid exchange, who died at 55 and 105 days of age. Light microscopy revealed thickened alveolar septa with abundant myxoid interstitial matrix, marked hyperplasia of type II pneumocytes, desquamation of alveolar macrophages and focal alveolar proteinosis. Surfactant protein-B was detected by immunohistochemistry after antigen retrieval. Transmission electron microscopy showed rare cytoplasmic inclusions with concentric membranes and eccentrically placed electron-dense aggregates. These 'fried-egg'-appearing lamellar bodies differed both from normal lamellar bodies and the larger, poorly formed composite bodies with multiple vesicular inclusions observed in surfactant protein-B deficiency. In conclusion, our findings underscore that the implications of interstitial lung disease in infant lungs differ from those in adults. In infants with a desquamative interstitial pneumonitis pattern, surfactant or ABCA3 mutations should be evaluated. Importantly, these findings support the notion that electron microscopy is useful in distinguishing between surfactant protein-B and ABCA3 deficiency, and has an important role in evaluating biopsies or autopsies of term infants with unexplained severe respiratory failure and interstitial lung disease

Wnt signaling pathway analysis in renal cell carcinoma in young patients

Renal cell carcinomas in young patients constitute a morphologically and genetically heterogeneous group. Twenty percent belong to the newly recognized Xp11.2 translocation-associated family and rare tumors arise from nephroblastoma. Aberrant Wnt signaling through beta-catenin mutation has been implicated in nephroblastoma pathogenesis and has been found to synergize with WT1 mutations. To characterize Wnt signaling activity in renal cell carcinomas in young patients, we gathered 34 tumors (three clear cell, ten Xp11.2 translocation associated, five papillary, two chromophobe, two collecting duct, one neuroblastoma associated, eight unclassified renal cell carcinomas, and three carcinomas combined with nephroblastoma) from patients less than 22 years. Expression of beta-catenin, its homologue gamma-catenin, and of WT1 was assessed by immunohistochemistry in 30 tumors, and sequence analysis of CTNNB1, CTNNG1, and WT1 genes was performed in 25 tumors. Cytoplasmic beta-catenin accumulation was demonstrated in two papillary carcinomas, one neuroblastoma-associated carcinoma, and two carcinomas arising from nephroblastoma. The pattern of gamma-catenin expression paralleled that of beta-catenin but its signal intensity was lower in 22, equal in 7, and stronger only in 1 tumor, respectively. Four tumors showed nuclear WT1 expression. One Xp11.2 translocation-associated carcinoma presented a rare intronic CTNNB1 single nucleotide polymorphism and cytoplasmic beta-catenin accumulation. There were no further CTNNB1 or CTNNG1 sequence alterations. A WT1 mutation was found in the nephroblastoma component of a carcinoma arising from nephroblastoma. These findings suggest Wnt signaling pathway activation only in a minority of renal cell carcinomas in young patients. CTNNB1 mutations are rare events. Cytoplasmic beta-catenin accumulation in an Xp11.2-associated carcinoma suggests potential interaction of Wnt signaling components with microphthalmia transcription factor family also in Xp11.2 translocation carcinomas. WT1 mutation in the nephroblastoma component of a mixed-type renal cell carcinoma provides direct evidence for clonal independence of nephroblastoma and carcinoma components in this exceptional tumor