C-kit protein expression correlated with activating mutations in KIT gene in oral mucosal melanoma

C-kit is a trans-membrane receptor tyrosine kinase (RTK) encoded by the proto-oncogene KIT located at 4q11-12. Gain-of-function mutations arising to c-kit activation independent of its ligand were observed in various tumors related to germ cells, mast cells, and interstitial cells of Cajal. C-kit also participates in melanocyte development; hence, its involvement in oral mucosal melanoma (OMM) tumorigenesis was investigated. Immunohistochemistry and mutation analysis were performed using 18 cases of human primary OMM. Results revealed 16 cases positive to c-kit protein. Atypical melanocytes expressed c-kit. All in situ components expressed c-kit, but only four cases exhibited intense expression in the invasive component. Missense mutations were observed in four cases, and two of those correlated with increased protein expression. C-kit expression in atypical melanocytes suggests the role of c-kit in the early stage of OMM tumorigenesis. C-kit protein expression correlated with activating mutations indicating the pertinent role of the proto-oncogene KIT in the tumorigenesis of OMM.     

Clinicopathologic profile of gastrointestinal stromal tumors (GISTs) with primary KIT exon 13 or exon 17 mutations: a multicenter study on 54 cases.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms driven by oncogenic, mutational activation of KIT or platelet-derived growth factor receptor alpha (PDGFRA). GIST-specific KIT or PDGFRA mutations have been linked to tumor location, tumor cell morphology and clinical behavior. The purpose of this study was to evaluate the clinicopathologic profile of GISTs that have KIT exon 13 or exon 17 mutations. Through the collaboration of several GIST research groups, we gathered 54 cases from the pre-imatinib era that had such primary mutations. From our observations and those in the literature, we estimate that the frequency of these mutations is no higher than 1-2%. Almost all (32 of 33, 97%) of the KIT exon 13 mutations were the 1945A>G substitution leading to Lys642Glu. A majority (15 of 21, 71.4%) of the KIT exon 17 mutations were the 2487T>A substitution leading to Asn822Lys. Demographic and clinicopathologic data were available for 26 and 14 KIT exon 13 and exon 17 mutant GISTs, respectively. Median age and male to female ratio were similar to ones reported in other GIST studies. Small intestinal tumors were two times more frequent than gastric ones among KIT exon 17 mutants. Also, intestinal tumors were slightly overrepresented among KIT exon 13 mutants when compared with population-based studies. The majority of KIT exon 13 or exon 17 mutants had a spindle-cell morphology and only a few had epithelioid features. Tumor size varied from 1.2 to 25 cm and average mitotic rates were 9.5 and 4.2 for KIT exon 13 and exon 17 mutants, respectively. Gastric KIT exon 13 mutant GISTs tend to be slightly larger and more aggressive than gastric GISTs in average, whereas the behavior of small intestinal GISTs with KIT exon 13 mutations does not differ from other small intestinal GISTs. The latter is also true for all KIT exon 17 mutant GISTs.     

Presence of homozygous KIT exon 11 mutations is strongly associated with malignant clinical behavior in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of gastrointestinal tract. GISTs range from benign indolent neoplasms to highly malignant sarcomas. Gain-of-function mutations of tyrosine kinase receptors, KIT or PDGFRA, have been identified in most GISTs. In this study, we report 36 GIST patients whose tumors had homozygous KIT exon 11 mutations detected by direct sequencing of PCR products. Loss of heterozygosity in KIT locus and other chromosome 4 loci were documented in majority of these tumors. However, fluorescence in situ hybridization with KIT locus-specific probe and chromosome 4 centromeric enumeration probe showed no evidence of KIT hemizygosity in a majority of analyzed cases. These findings are consistent with duplication of chromosome 4 with KIT mutant allele. Homozygous KIT exon 11 mutations were found in 33 primary tumors and 7 metastatic lesions. In two cases, shift from heterozygosity to homozygosity was documented during tumor progression being present in metastases, but not in primary tumors. Among primary GISTs, there were 16 gastric, 18 intestinal and 2 from unknown locations. An average primary tumor size was 12 cm and average mitotic activity 32/50 HPFs. Out of 32 tumors 29 (90.6%) with complete clinicopathologic data were diagnosed as sarcomas with more than 50% risk of metastatic disease, and 26 of 29 patients with follow-up had metastases or died of disease. An average survival time among pre-imatinib patients, who died of the disease was 33.4 months. Based on these findings, we conclude that presence of homozygous KIT exon 11 mutations is associated with malignant course of disease and should be considered an adverse prognostic marker in GISTs.     

Length analysis of polymerase chain reaction products: a sensitive and reliable technique for the detection of mutations in KIT exon 11 in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors are the most common mesenchymal neoplasms of the digestive tract. These tumors express the c-kit receptor tyrosine kinase, and many have activating mutations in the juxtamembrane region coded by the exon 11 of KIT. Detection of these mutations has prognostic and therapeutic impact. The aim of the study was to compare a new detection method by length analysis of polymerase chain reaction products (LAPP) to direct sequencing. The detection of either deletion or insertion mutations within the exon 11 of KIT was performed on genomic DNA extracted from 40 paraffin-embedded samples from 38 patients. Double-strand direct sequencing revealed a mutation in 25 of 40 samples. In two additional samples, a mutation was suspected but could not be determined by sequencing. LAPP revealed a mutation in 27 samples, corresponding to the 25 determined and 2 suspected samples. One of these latter samples contained three different alleles. Mutations corresponded to either deletions (n = 24) or insertion (n = 1) and had the same size with sequencing and LAPP. Our results show that LAPP is as accurate and more sensitive than direct sequencing for the detection of deletion or insertion mutations of exon 11 of KIT in gastrointestinal stromal tumors.     

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11

In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.     

A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.

Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.     

KIT expression in fetal, normal adult, and neoplastic renal tissues

BACKGROUND: KIT is a transmembrane tyrosine kinase receptor, expressed in high amounts in various normal cells. In addition, c-kit mutation or activation is a major pathogenetic event in certain tumours (such as gastrointestinal stromal tumours). There are only limited data in the literature on the expression of KIT in normal and neoplastic renal tissues. AIMS: To investigate KIT expression in normal and neoplastic renal tissues. METHODS: KIT expression was evaluated by means of immunohistochemistry in paraffin wax embedded sections from 67 tissue samples. RESULTS: Eight of eight fetal kidneys, and 10 of 10 normal adult kidneys revealed cytoplasmic staining of renal tubules. The three cases of renal dysplasia studied expressed KIT in their normal and aberrant tubules. Two of 13 conventional renal cell carcinomas (RCCs), two of seven papillary type RCCs, four of seven chromophobe type RCCs, none of six nephroblastomas, seven of seven oncocytomas, two of two mesoblastic nephromas, and two of four angiomyolipomas were positive. CONCLUSION: KIT is expressed in normal fetal and adult renal tubules, and in a subset of renal tumours. The expression of KIT in these renal tumours may prove to have diagnostic relevance and/or therapeutic implications.     

KIT codon 558 insertions in gastrointestinal stromal tumors. Analysis of 17 rare KIT mutants

Gastrointestinal stromal tumors are the most common mesenchymal neoplasms of gastrointestinal tract often driven by oncogenic KIT exon 11 mutations. Although deletions and substitutions are most frequent KIT exon 11 mutations, duplications and insertions have been reported as well. In contrast to duplications, which cluster in 3′KIT exon 11, insertions affect 5′KIT, particularly codon 558. Clinicopathologic profile of gastrointestinal stromal tumors with insertions in codon 558 is not known. In this study, 17 gastrointestinal stromal tumors with codon 558 insertions are reported. Fifteen (88.2%) KIT codon 558 insertions consisted of 1694_1695insTCC leading to Lys558delinsAsnPro. However, 2 variant mutants Lys558delinsAsnGln and Lys558delinsAsnAsn were also identified. Based on analysis of inserted and flanking sequences, the insertions contain inverted DNA sequences of the opposite strand. Therefore, these insertions may develop due to a DNA strand switch during replication by DNA polymerases and by the effects of several different DNA repair processes. Patient median age was 61 years, and male-to-female ratio was 1:1.8. gastrointestinal stromal tumors were diagnosed in stomach (n = 4), small intestine (n = 7), and rectum (n = 3). Three tumors were disseminated and primary location could not be established. Fourteen tumors had spindle cell morphology, and epithelioid cell features were seen in 2 intestinal and 1 disseminated gastrointestinal stromal tumor. Based on size and mitotic activity, 2 (50%) of 4 gastric and 3 (48.9%) of 7 small intestinal gastrointestinal stromal tumors had more than 50% risk of metastases according to previous studies of gastrointestinal stromal tumor prognosis. All 3 rectal gastrointestinal stromal tumors were malignant. Metastases were verified in 8 (66.7%) of 12 patients with known clinical and follow-up data. In summary, KIT codon 558 insertions are rare mutations accounting for less than 1% of all KIT mutants. Gastrointestinal stromal tumors with these mutations appear to have predilection to female patients and intestinal location. Moreover, KIT codon 558 insertions might indicate an increased risk of malignant behavior for gastric gastrointestinal stromal tumors.     

Up-regulated expression of ADAM17 in human colon carcinoma: co-expression with EGFR in neoplastic and endothelial cells

The ADAM17 metalloproteinase (a disintegrin and metalloprotease 17) controls epidermal growth factor receptor (EGFR) activation through regulated shedding of EGFR ligands. With the advent of new therapeutic options targeting EGFR signalling in colon carcinoma, it was decided to determine ADAM17 status in relation to clinico-pathological parameters and EGFR status. To this end, a series of 39 colon carcinomas were analysed. Immunohistochemistry and immunofluorescence were used to localize ADAM17, EGFR, and the activated forms of EGFR. The activated form of ADAM17 was assessed in primary cancers and colon cell lines by immunoblotting. ADAM17 and EGFR mRNA levels were assessed by quantitative RT-PCR. Chromogenic in situ hybridization (CISH) was used to quantify the HER1 gene. ADAM17 was strongly expressed in all tumours, by both neoplastic and endothelial cells. It was expressed both as a pro- and as an active form in tumours and colonic cancer cell lines. ADAM17 mRNA was up-regulated in 90% of colon carcinomas relative to the paired normal mucosa, whatever the tumour grade or stage. When present, activated EGFR was co-expressed with ADAM17 by colon carcinomas, although at a variable level among tumour cells, and by endothelial cells. EGFR mRNA was overexpressed in 77% of colon carcinomas compared with the paired normal mucosa. One case showed high-level amplification of HER1. In conclusion, this study is the first demonstration that ADAM17 is overexpressed in human primary colon carcinoma, whatever the tumour stage and differentiation and whatever the level of EGFR expression. Its co-expression with EGFR, in both neoplastic and endothelial cells, suggests a role for ADAM17 in tumour growth and angiogenesis.     

Tumour microvessel endothelial cell KIT and stem cell factor expression in human solid tumours

Aims:  To assess KIT receptor tyrosine kinase and stem cell factor (SCF, KIT ligand) expression in tumour microvessel endothelial cells.
Methods and results:  KIT and SCF expression were studied in 248 human tumours consisting of 15 different histological types using immunohistochemistry and in situ hybridization for KIT messenger RNA. Moderate to strong intratumoral endothelial cell KIT expression was present in 11 of the 15 tumour types, and was most common in glioblastoma (58%), mixed embryonal carcinoma with teratoma (33%) and renal clear cell carcinoma (29%). Results of in situ hybridization were in line with those obtained with immunohistochemistry in the cases studied ( n  = 9). Marked SCF expression was uncommon in tumour endothelial cells, but frequent in perinecrotic tumour cells. Patients with glioblastoma with moderate to strong endothelial cell KIT expression had more favourable survival than those whose tumour showed little or no expression ( P  = 0.024). Glioblastoma patients whose tumour expressed SCF had an unfavourable outcome compared with those with tumour with weak or no expression ( P  = 0.034).
Conclusions:  Intratumoral microvessels of several types of human malignant tumours express KIT. Tumour cell SCF expression and absence of marked endothelial cell KIT expression are novel adverse prognostic features in glioblastoma.     

KIT protein expression and mutational status of KIT gene in pituitary adenomas

KIT protein expression and mutational status of KIT gene in different types of tumours have been intensively studied since Imatinib Mesylate, KIT/PDGFRA tyrosine kinase inhibitor became available. However, only one immunohistochemical study on KIT expression in pituitary adenomas has been published. There are currently no reports on mutational status of KIT gene in pituitary adenomas. We have immunohistochemically investigated KIT expression in 252 pituitary adenomas and found cytoplasmic reactivity in 52.4% and membranous reactivity in 8.3% of all adenomas. There was statistically significant difference in KIT expression between clinically non-functioning, growth hormone- and adrenocorticotroph hormone-producing adenomas. The group with membranous expression was dominated by somatotropinomas and clinically non-functioning adenomas. KIT expression in a subset of adenomas was also confirmed by western blot analysis of 48 adenomas. Immunohistochemical KIT expression was correlated with basic clinical data and in a cohort of acromegalic patients with additional data (somatostatin receptor type 2A expression, response to somatostatin analogue treatment and mutational status of gsp oncogene). Exons 9, 11, 13 and 17 of KIT gene were searched for mutations in the tumours with membranous KIT expression and in a minority of tumours with cytoplasmic KIT expression using denaturing high-performance liquid chromatography and in suspected cases sequencing of one or more exons. No mutations in the examined exons were found. Our results may suggest a role of KIT in the pathogenesis of a subset of pituitary adenomas and point out the need for further research to find out if KIT-reactive adenomas could be sensitive to Imatinib Mesylate.     

Primary mediastinal seminomas: evidence of single and multiple KIT mutations

Primary mediastinal seminomas (MS) are rare tumors that are histologically similar to their testicular counterparts. Reports document KIT mutations in gastrointestinal stromal tumors, mastocytosis, and germ cell tumors. Although rare exon 17 mutations have been reported in gonadal seminomas, their mediastinal counterparts have not been studied. To determine whether primary MS harbor KIT mutations, eight formalin-fixed, paraffin-embedded primary MS were microdissected; KIT exons 11 and 17 were sequenced. Four (50%) of the eight cases demonstrated KIT exon 17 mutations. Two of the cases showed single monoallelic base pair alterations; one of these mutations were silent. The other two cases each demonstrated two monoallelic point mutations. In each case, one of these mutations results in protein sequence alteration, and the other is silent. Sequencing of cloned PCR products showed both the silent and amino acid-altering mutations to be found on the same allele (cis). The codon 816 mutation previously identified in mastocytosis and gonadal germ cell tumors was not observed. Non-neoplastic tissues from these patients did not demonstrate KIT mutations; exon 11 mutations were not seen in either tumors or normal tissues. Only the three cases in which amino acid-altering mutations were observed showed a predominantly cytoplasmic CD177 KIT immunohistochemical staining, whereas the one non-amino acid mutating and all wild-type cases were immunonegative. Our findings demonstrate a unique KIT sequence and expression pattern among MS. KIT sequencing may assist in differentiating primary from metastatic MS.     

Enhanced detection of mutations in BRCA1 exon 11 using restriction endonuclease fingerprinting-single-strand conformation polymorphism

A novel approach to mutation screening in the large exon 11 (comprising 3427 bp) of the human BRCA1 gene is presented. Restriction endonuclease fingerprinting single-strand conformation polymorphism (REF-SSCP) is based on repeated detection of DNA sequence variants in different restriction endonuclease fragments, and we evaluated the method using blood samples from 25 Norwegian patients with hereditary breast/ovarian cancer. We compared REF-SSCP to constant denaturant gel electrophoresis (CDGE) and to the protein truncation test (PTT). REF-SSCP detected 12 different DNA variants. Four of these were not detected by CDGE, and only one variant detected by CDGE was missed by REF-SSCP. PTT detected 4 of these 13 variants. REF-SSCP was subsequently applied to a second patient series (Swedish, n=20). A total of 14 different DNA variants were detected by REF-SSCP, 6 of which were truncating mutations (PTT detected only 4). Nonsense and frameshift mutations that are putative breast/ovarian cancer mutations, were detected in 7 of the 25 Norwegian and 9 of the 20 Swedish patients.     

Different immunoreactivity of endothelial markers in well and poorly differentiated areas of angiosarcomas

This study evaluated the immunohistochemical staining of four endothelial cell markers in well differentiated and poorly differentiated areas of angiosarcomas. Formaldehyde-fixed, paraffin-embedded sections from eight angiosarcomas were studied using the antibodies anti-factor VIII-related antigen (FVIII-RA), Ulex europaeus I agglutinin, anti-CD34 (QBEND/10) and anti-CD31 (JC70). The immunostaining of the angiomatous (well differentiated) and solid (poorly differentiated) areas was separately analysed and specificity was evaluated in 20 non-vascular tumours. The antibody anti-CD31 and Ulex europaeus were the most sensitive markers staining well differentiated vasoformative structures and poorly differentiated solid areas. Anti-FVIII-RA and anti-CD34 did not stain undifferentiated malignant endothelial cells from solid areas. Ulex europaeus and anti-CD34 showed very low specificity; in contrast, none of the non-vascular tumours expressed CD31 or FVIII-RA. JC70 (anti-CD31) appears to be the most useful marker in elucidating the vascular nature of angiosarcomas. Is important to emphasize the lack of specificity of Ulex europaeus and the low sensitivity of anti-CD34 and anti-FVIII-RA for poorly differentiated lesions.