Marjut Puputti

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in Gliomas

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas. (Mol Cancer Res 2006;4(12):927–34)

Amplification of genes encoding KIT, PDGFRα and VEGFR2 receptor tyrosine kinases is frequent in glioblastoma multiforme

KIT, platelet‐derived growth factor receptors (PDGFRs) and vascular endothelial growth factor receptors (VEGFRs) are important clinical targets for tyrosine kinase inhibitors. The frequency of KIT and VEGFR2 amplification in glioblastomas is not known, and few data are available in any other human tumour type. We investigated 43 primary glioblastomas for KIT, VEGFR2, PDGFRA and EGFR amplification using fluorescence in situ hybridization. KIT was amplified in 47% and VEGFR2 in 39% of the glioblastomas, respectively, and PDGFRA in 29%. Thirty‐five (81%) of the tumours had either KIT or EGFR amplification. KIT, PDGFRA and VEGFR2 amplifications were strongly associated (p < 0.0001 for each pairwise comparison), suggesting co‐amplification, whereas no significant association was found with EGFR amplification. The four secondary glioblastomas arising from pre‐existing lower grade astrocytic tumours investigated had KIT amplification but none had EGFR amplification. No mutations were detected with denaturing high‐performance liquid chromatography in KIT exons 9, 11, 13 or 17, PDGFRA exons 12 and 18, or EGFR exons 18, 19 or 21. Glioblastomas with KIT, PDGFR or VEGFR2 amplification were associated with similar outcome to other glioblastomas. We conclude that KIT, PDGFRA and VEGFR2 are commonly amplified in primary glioblastoma and that they may also be amplified in secondary glioblastoma. Amplified kinases may be potential targets for tyrosine kinase inhibitor therapy. Copyright

Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma

While the phosphatidylinositol 3‐kinase (PI3K)/AKT signaling pathway is known to be activated in multiple sporadic cancers, the role of this pathway in familial tumors is mostly unknown. We searched for alterations in the catalytic domain of PI3K (PIK3CA), PTEN and KRAS, all of which may contribute to PI3K/AKT pathway activation, in a total of 160‐familial colorectal (CRC) and endometrial carcinomas (EC), stratified by the presence vs. absence of germline mutations in DNA mismatch repair (MMR) genes. PIK3CA alterations (consisting of point mutations or low‐level amplification, which were mutually exclusive with 1 exception) occurred in 10/70 (14%) of CRCs and 19/90 (21%) of ECs. Within ECs, amplification was significantly associated with the subgroup lacking germline mutations in MMR genes (familial site‐specific endometrial cancer) (p = 0.015). Decreased or lost PTEN expression was characteristic of endometrial tumourigenesis (51/81, 63%, in EC compared with 24/62, 39%, in CRC, p = 0.004) and KRAS mutations of colorectal tumourigenesis (19/70, 27% in CRC vs. 9/89, 10%, in EC, p = 0.006) regardless of the MMR gene mutation status. PIK3CA alterations frequently coexisted with PTEN or KRAS changes. Combined with published studies on sporadic tumors, our data broaden the understanding of the role for PI3K pathway genes in human tumorigenesis.

Epidermal growth factor receptor domain II, IV, and kinase domain mutations in human solid tumors

Mutations that may predict response to adenosine 5′-triphosphate (ATP)-mimetic epidermal growth factor receptor (EGFR) inhibitors occur in the EGFR kinase domain in lung adenocarcinomas and bronchioloalveolar carcinomas (BACs). Data on the frequency of EGFR mutations are sparse in other human tumors. Apart from the deletion mutant EGFRvIII, little is known about the frequency of mutations that encode for the EGFR extracellular domains II and IV that participate in receptor dimerization and formation of the tethered (autoinhibited) receptor conformation. We investigated 566 human neoplasms consisting of various histological types for mutations in exons 6, 7 (encode domain II), 14, 15 (domain IV), 18, 19, and 21 (the kinase domain) using denaturing high-performance liquid chromatography (DHPLC). Approximately 4,500 EGFR exons were screened for the presence of a mutation, and samples with an abnormal finding in DHPLC were sequenced. Only one mutation was found in the extracellular domain IV (glioblastoma), and none in domain II. Eight (11%) out of the 40 lung adenocarcinomas, or 33 BACs, investigated had exon 19 or 21 mutation in the kinase domain, but no mutations were found in other tumor types. Most of the lung cancers with mutated EGFR had three to six copies of the mutated gene in fluorescence in situ hybridization. We conclude that mutations of the EGFR kinase domain and the cysteine-rich extracellular domains are infrequent in most types of human cancer apart from lung adenocarcinoma. Mutated EGFR is usually not amplified in lung cancer.

Molecular genetic analysis of the REST/NRSF gene in nervous system tumors

The gene for RE1-silencing transcription factor (REST) alias neuron-restrictive silencer factor NRSF, acts as a transcriptional repressor in the neuronal differentiation pathways in non-neuronal cells, and plays an important role in neuronal development. Inactivating mutations or overexpression of REST have previously been reported in various types of cancer, but no data is available for the role of REST alterations in gliomas. REST gene was screened for mutations in 161 nervous system tumors consisting of astrocytomas, glioblastomas, oligodendrogliomas, oligoastrocytomas, medulloblastomas, meningiomas and schwannomas. REST exons 1–3 were analyzed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. The gene copy numbers of REST were investigated by chromogenic (CISH) and fluorescence in situ hybridization (FISH) techniques. Non-synonymous SNPs (P797L, P815S) were found in eight different brain tumor samples. No truncating or activating novel mutations of REST were discovered. Since REST is located at 4q12, a chromosome region implicated in brain tumorigenesis, we conducted gene copy number analyses in medulloblastomas and gliomas. The majority of gliomas (67%) demonstrated low-level amplifications of REST, and only one oligodendroglioma showed high-level amplification of the gene. In medulloblastomas, 38% of samples were determined as aneuploidic, no high-level amplifications were found. Our data suggests that REST is neither activated nor inactivated via mutations in gliomas, while high-level amplification may rarely occur.

Expression of KIT Receptor Tyrosine Kinase in Endothelial Cells of Juvenile Brain Tumors

KIT receptor tyrosine kinase is expressed in tumor endothelial cells of adult glioblastomas, but its expression in pediatric brain tumor endothelial cells is unknown. We assessed expression of KIT, phosphorylated KIT, stem cell factor (SCF) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) in 35 juvenile pilocytic astrocytomas and 49 other pediatric brain tumors using immunohistochemistry, and KIT messenger RNA (mRNA) using in situ hybridization. KIT and phospho‐KIT were moderately or strongly expressed in tumor endothelia of 37% and 35% of pilocytic astrocytomas, respectively, whereas marked SCF and VEGFR‐2 expression was uncommon. KIT mRNA was detected in tumor endothelial cells. Tumor endothelial cell KIT expression was strongly (P < 0.01) associated with endothelial cell phospho‐KIT and SCF expression, and with tumor KIT (P = 0.0011) and VEGFR‐2 expression (P = 0.022). KIT and phospho‐KIT were present in endothelia of other pediatric brain tumors, notably ependymomas. Endothelial cell KIT expression was associated with a young age at diagnosis of pilocytic astrocytoma or ependymoma, and it was occasionally present in histologically normal tissue of the fetus and children. We conclude that KIT is commonly present in endothelial cells of juvenile brain tumors and thus may play a role in angiogenesis in these neoplasms.

Cutaneous T-cell lymphoma-associated lung cancers show chromosomal aberrations differing from primary lung cancer.

Cutaneous T‐cell lymphoma (CTCL) patients have an increased risk of certain secondary cancers, the most common of which are lung cancers, especially small cell lung cancer. To reveal the molecular pathogenesis underlying CTCL‐associated lung cancer, we analyzed genomic aberrations in CTCL‐associated and reference lung cancer samples. DNA derived from microdissected lung cancer cells of five CTCL‐associated lung cancers and five reference lung cancers without CTCL association was analyzed by comparative genomic hybridization (CGH). Fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and loss of heterozygosity (LOH) analysis were performed for selected genes. In CTCL‐associated lung cancer, CGH revealed chromosomal aberrations characterizing both lung cancer and CTCL, but also losses of 1p, and 19, and gains of 4q and 7, hallmarks of CTCL. LOH for the CTCL‐associated NAV3 gene was detected in two of the four informative primary lung cancers. FISH revealed increased copy number of the KIT gene in 3/4 of CTCL‐associated lung cancers and 1/5 of primary lung cancers. PDGFRA and VEGFR2 copy numbers were also increased. IHC showed moderate KIT expression when the gene copy number was increased. CTCL‐associated lung cancer shows chromosomal aberrations different from primary lung cancer, especially amplifications of 4q, a chromosome arm frequently deleted in the latter tumor type. Copy numbers and expression of selected genes in chromosome 4 differed between CTCL‐associated and reference lung cancers. These preliminary observations warrant further prospective studies to identify the common underlying factors between CTCL and CTCL‐associated lung cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

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.

Abstract 3800: HES6 gene is a strong glioma biomarker and a key transcriptional regulator needed for cancer cell growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Malignant glioma is the most common brain tumor with 16,000 individuals diagnosed annually in the United States. We performed a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the GeneSapiens database (www.genesapiens.org) to systematically identify genes that are most glioma-specific. We searched for genes that were highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared to 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (Hairy and Enhancer of Split 6) emerged as one of the most glioma-specific genes. Immunostaining of a tissue microarray showed HES6 expression in 335 (98.8%) out of the 339 glioma samples. HES6 was also expressed in endothelial cells in normal and glioma tissues. Recurrent grade 2 astrocytomas and grade 2-3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Functional studies implied a critical role for HES6 in supporting survival of glioma cells, as evidenced by reduction of cancer cell proliferation and induction of Caspase 3/7 activity after HES6 silencing in A172 and LN405 cells. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which implicated a role for HES6 in p53, c-myc, and NF-κB transcriptional networks. We conclude that HES6 has a critical role in sustaining glioma cell growth, survival, migration and possibly angiogenesis. HES6 is a potential therapeutic target and biomarker for glioma. This work was supported by funding under the 6th FP of the European Union, Project RIGHT (LSHB-CT-2004-005276), and the Academy of Finland. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2011-3800

Abstract 4708: Hes6 as a glioma biomarker with functional significance for cancer growth

Malignant gliomas are the most common type of primary brain tumors affecting 16,000 new patients every year in the United States. In this study, we undertook a large-scale transcriptomics mining study of Affymetrix data from 9,783 samples from the genesapiens database (www.genesapiens.org) focusing on glioma biomarkers. We searched for genes that were specifically expressed in Glioblastoma Multiforme (GBM) and anaplastic astrocytoma using gene expression data from 475 different types of glioma samples (including 322 GBM, 66 anaplastic astrocytomas) and 425 samples from different normal anatomical locations of central nervous system. One of the prominent biomarkers arising from this bioinformatic search was the transcription cofactor HES6 (Hairy and enhancer of split 6), which was highly expressed in gliomas compared to normal brain. Since the role of HES6 in glioma pathogenesis is poorly understood, we chose to validate its expression by immunostaining and functional role by siRNA knockdown studies in glioma cell lines. HES6 protein levels were studied in a glioma tissue microarray material that consisted of 414 samples as well as in normal brain tissue controls. Positive HES6 immunoreactivity was present in 99 % of available gliomas. Recurrent tumors of grade 2 astrocytomas and grade 2-3 oligodenrogliomas showed higher levels of HES6 immunoreactivity than the primary tumors of these classes. Endothelial cells that aligned the blood vessels within the tumors were also stained in 75 % of gliomas. HES6 immunoreactivity was also present in glial and endothelial cell nuclei in non-neoplastic cerebral white matter and cortex. Cell viability was reduced by 60 % after HES6 silencing in A172 and LN405 cells and by ∼20-25 % in the U87MG and SVG p12 cell lines. HES6 silencing also increased apoptosis 2-3 fold in two cell lines as measured by Apo-ONE Homogeneous Caspase-3/7 Assay. We then studied the biological consequences of HES6 silencing by genome-wide gene expression studies. Gene ontology analysis implicated genes involved in cellular movement, development and RNA post-transcriptional modification. Also, aberrant expression around key regulator genes, such as p53, c-myc and SP1 was seen. In conclusion, these results suggest that HES6 is a possible glioma marker that also has critical functional role in glioma growth. This work was supported by funding under the 6 th FP of the European Union, Project RIGHT (LSHB-CT-2004-005276), and the Academy of Finland. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4708.