Britta Blaschke

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas

Background  Basal cell carcinoma (BCC) of the skin is the most common human cancer. The genetic alterations underlying BCC development are only partly understood.

Alterations of the Tumor Suppressor Genes CDKN2A (p16INK4a), p14ARF, CDKN2B (p15INK4b), and CDKN2C (p18INK4c) in Atypical and Anaplastic Meningiomas

We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas.

Oligodendroglial tumors: refinement of candidate regions on chromosome arm 1p and correlation of 1p/19q status with survival.

Loss of heterozygosity (LOH) on the chromosome arms 1p and 19q is frequent in oligodendroglial tumors and has been correlated with chemosensitivity and good prognosis in anaplastic oligodendrogliomas. The oligodendroglioma‐associated tumor suppressor genes on 1p and 19q are as yet unknown. To narrow down candidate regions on 1p, we investigated oligodendroglial tumors from 89 patients for LOH at up to 30 polymorphic loci on 1p. In addition, all tumors were studied for LOH at 7 loci on 19q. Combined LOH on 1p and 19q was detected in 20 (83%) of 24 oligodendrogliomas, 15 (63%) of 24 anaplastic oligodendrogliomas, 10 (56%) of 18 oligoastrocytomas, and 12 (52%) of 23 anaplastic oligoastrocytomas. Five tumors demonstrated partial deletions on 1p, which allowed to define 3 distinct candidate regions at 1 p36.31 ‐pter distal to D1S2633, 1p36.22‐p36.31 between D1S489 and D1S2642, and 1p34.2‐p36.1 between D1S2743 and D1S482, respectively. No partial deletions were detected on 19q. Combined LOH on 1p and 19q was associated with prolonged time to progression (TTP), longer overall survival (OS), and a higher 5‐year survival rate. Depending on the presence or absence of combined LOH on 1p and 19q, patients with anaplastic oligodendroglial tumors treated with adjuvant radio‐ and/or chemotherapy showed a median TTP of 86 months versus 39 months, a median OS of 91 months versus 46 months, and a 5‐year survival rate of 80% versus 36%, respectively. Similarly, LOH on 1p and 19q was associated with longer survival in patients with low‐grade oligodendroglial tumors (TTP: 57 months versus 47 months; OS: 172 months versus 105 months; 5‐year survival rate: 92% versus 70%). Thus, our results refine the location of putative oligodendroglioma suppressor genes on 1p and support the significance of LOH on 1p and 19q as a favorable prognostic marker.

Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas

Ras signaling is important for the intracellular transduction of mitogenic stimuli from activated growth factor receptors. We have investigated 37 sporadic malignant melanomas (15 primary cutaneous melanomas and 22 melanoma metastases) and 6 melanoma cell lines for mutations in the 3 Ras genes NRAS, KRAS and HRAS. All tumors and cell lines were additionally analyzed for mutation and expression of BRAF, which encodes a Ras‐regulated serine/threonine kinase with oncogenic properties, as well as for expression of RASSF1A, which encodes a Ras‐binding protein with tumor suppressor properties. Mutational analyses identified somatic NRAS mutations in 2 primary melanomas, 4 melanoma metastases and 2 cell lines. One melanoma metastasis showed a somatic KRAS mutation whereas HRAS mutations were not detected. Eight primary melanomas, 6 melanoma metastases and 4 melanoma cell lines carried BRAF mutations affecting the known hot‐spot codon 599. None of the tumors or cell lines with BRAF mutation demonstrated NRAS or KRAS mutations. Real‐time reverse transcription‐PCR showed that 8 melanomas (3 primary tumors, 5 melanoma metastases) had reduced RASSF1A transcript levels of ≤50% relative to benign melanocytic nevi and normal skin. Three melanoma cell lines lacked detectable RASSF1A transcripts. The RASSF1A gene promoter was hypermethylated in these 3 cell lines as well as in 6 of 8 melanomas with reduced RASSF1A mRNA levels. Treatment of the cell lines with 5‐aza‐2′‐deoxycytidine and trichostatin A resulted in demethylation of the RASSF1A promoter and re‐expression of RASSF1A transcripts. Most tumors and all cell lines with RASSF1A promoter methylation additionally carried BRAF or NRAS mutations, suggesting a synergistic effect of these aberrations on melanoma growth. Taken together, 57% of the investigated melanomas and 100% of the melanoma cell lines carried mutations in either NRAS, KRAS or BRAF. In addition, 22% of the melanomas and 50% of the cell lines showed reduced RASSF1A transcript levels. Thus, alterations of Ras pathway genes are of paramount importance in the pathogenesis of sporadic melanomas.

Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC

Aberrant activation of the Wnt signaling pathway has been reported in different human tumor types, including malignant melanomas. We investigated 37 malignant melanomas (15 primary tumors and 22 metastases) for alterations of 4 genes encoding members of this pathway, i.e., CTNNB1 (β‐catenin gene, 3p22.1), APC (adenomatous polyposis coli gene, 5q22.2), BTRC (β‐transducin repeat–containing protein gene, 10q24.3) and ICAT (inhibitor of β‐catenin and Tcf‐4, 1p36.2). Mutational analysis of CTNNB1 identified somatic mutations in 1 primary melanoma and 1 melanoma metastasis from 2 different patients (5%). Both mutations affected the N‐terminal degradation box of β‐catenin, which is important for the regulation of β‐catenin homeostasis. Another primary melanoma carried a somatic APC missense mutation within the known mutation cluster region in exon 15. Fourteen tumors (40%) showed LOH at microsatellite markers on 1p36. None of the tumors had lost both copies of the ICAT gene, but 1 melanoma metastasis carried a somatic point mutation altering the translation start codon of ICAT. Real‐time RT‐PCR showed markedly reduced ICAT transcript levels (≤20% relative to normal skin and benign melanocytic nevi) in 28/36 malignant melanomas (78%), including 13/14 tumors with LOH on 1p36. Allelic loss on 10q was detected in 15 tumors (44%). We found neither mutations nor complete loss of expression of the BTRC gene in our melanoma series. Taken together, our results indicate that the Wnt pathway may be altered in malignant melanomas by different mechanisms, including rare somatic mutations in CTNNB1, APC or ICAT, as well as low or absent expression of ICAT transcripts.

Identification of novel genes associated with astrocytoma progression using suppression subtractive hybridization and real‐time reverse transcription‐polymerase chain reaction

To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low‐grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using real‐time reverse transcription‐PCR on a series of 10 pairs of primary low‐grade and recurrent high‐grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2‐qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5‐aza‐2′‐deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfite‐modified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells.

Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q

Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and have been associated with sensitivity to radio- and chemotherapy as well as favourable prognosis. By using microarray-based expression profiling, we found that oligodendroglial tumours with 1p and 19q losses showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to tumours without 1p and 19q losses. Mutational analysis showed no CITED4 mutations in gliomas. However, 1p and 19q losses as well as low expression of CITED4 transcripts were significantly associated with hypermethylation of the CITED4-associated CpG island. In line with the latter finding, treatment of CITED4 hypermethylated glioma cell lines with 5-aza-2′-deoxycytidine and trichostatine A resulted in a marked increase of the CITED4 transcript levels. Furthermore, CITED4 hypermethylation was significantly associated with longer recurrence-free and overall survival of patients with oligodendroglial tumours. Taken together, our results indicate that CITED4 is epigenetically silenced in the vast majority of oligodendroglial tumours with 1p and 19q deletions and suggest CITED4 hypermethylation as a novel prognostic marker in oligodendroglioma patients.

Absence of mutations in the putative tumor suppressor gene KLF6 in glioblastomas and meningiomas.

Dear Sir, Glioblastomas are the most common and most malignant primary brain tumors in adults.1 Molecular studies have identified a variety of genetic alterations in glioblastomas, with allelic losses on chromosome 10 being present in more than two-thirds of the cases.1 Although most glioblastomas have lost one entire copy of chromosome 10, partial losses have been detected in a subset of cases and suggest that several distinct tumor suppressor gene loci map to 10p, 10q23 and 10q24– qter, respectively,2,3 The PTEN tumor suppressor gene at 10q23 is altered by mutations or homozygous deletion in approximately one-third of primary glioblastomas.4 The glioblastoma-relevant tumor suppressor genes on 10p and 10q24– qter are still unknown. Among primary central nervous system tumors, chromosome 10 deletions are not restricted to glioblastomas but are also common in atypical and anaplastic meningiomas,5–7 with at least one meningioma-associated tumor suppressor gene region mapping to 10p12.1–pter, distal to the anonymous marker D10S89.7 Jeng and Hsu8 reported recently on somatic mutations in the KLF6 (Kruppel-like factor 6) gene at 10p15 in a subset of diffuse astrocytic gliomas including glioblastomas. Other authors proposed KLF6 as a tumor suppressor gene that is frequently mutated in prostate carcinomas.9 The latter group also showed that KLF6 suppresses cell growth by inducing a p53independent upregulation of the cyclin-dependent kinase inhibitor p21.9 We report on the analysis of the KLF6 gene for mutation and loss of expression in a series of 42 primary glioblastomas and 47 meningiomas (13 benign, 21 atypical and 13 anaplastic meningiomas). All tumors were histologically classified according to the World Health Organization (WHO) classification of tumors of the nervous system.1,10 High molecular weight DNA and RNA were extracted from frozen tumor tissue samples as reported elsewhere.11 All glioblastomas and 42 of 47 meningiomas had been studied before for deletions on chromosome 10 using microsatellite analysis or comparative genomic hybridization.5,6 Deletions involving sequences from 10p were found in 21 glioblastomas and 11 meningiomas (5 atypical and 6 anaplastic meningiomas). For mutational analysis of KLF6 in our series of 89 brain tumors, we amplified by PCR each of the 4 KLF6 coding exons and the flanking intronic sequences. The PCR products were then screened for mutations using non-radioactive single strand conformation polymorphism (SSCP) analysis as reported elsewhere.6 PCR products showing aberrant SSCP band patterns were sequenced in both directions by cycle sequencing using the ABI PrismTM dye terminator cycle sequencing kit and an ABI 377 semi-automated DNA sequencer (Applied Biosystems, Foster City, CA). Aberrant SSCP band patterns were detected in 5 tumors of our series. Sequencing of the respective PCR products showed 2 different alterations: c.603G A (R201R) in one glioblastoma and c. 4C A in 1 glioblastoma and 3 meningiomas. Sequencing of constitutional (leukocyte) DNA of the corresponding patients showed the same sequence variations, thus identifying these changes as DNA polymorphisms. To exclude, that SSCP analysis missed any mutations in exon 2, which carried all the mutations detected by Jeng and Hsu,8 we directly sequenced this exon in all 89 tumors. Direct sequencing verified the c.603G A (R201R) in the single glioblastoma but did not identify any other sequence changes. A recent study reported on the loss of KLF6 mRNA expression in 20% of prostate cancer cell lines and xenografts.12 To investigate the expression of KLF6 transcripts in primary glioblastomas and meningiomas, we carried out real-time reverse transcription-PCR analyses using the ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA) with continuous measurement of the PCR product amount by the detection of SybrGreen fluorescent dye incorporation into double-stranded DNA. The transcript level of KLF6 was normalized to the transcript level of the house-keeping gene ARF1 (ADP-ribosylation factor 1). As reference tissues we used non-neoplastic cerebral tissues samples or leptomeningeal tissue samples from 2 different individuals. None of the 42 glioblastomas showed reduced expression of KLF6 transcripts as compared to non-neoplastic brain tissue. KLF6 expression levels did not differ between glioblastomas with or without losses on 10p. In line with these findings, KLF6 transcript levels were not reduced relative to the reference tissue in 8 glioblastoma cell lines grown in monolayer cultures (T98G, TP365MG, U118MG, U138MG, U178MG, U251MG, U373MG and CCF-STTG1). Expression analysis of 45 meningiomas, however, showed KLF6 transcript levels reduced to 30% or less relative to normal leptomeningeal tissue in 14 tumors (1/13 benign, 8/20 atypical and 5/12 anaplastic tumors). Eight of 11 tumors with 10p deletions showed reduced KLF6 mRNA levels (p 0.05, Fisher’s exact test). To determine the potential role of KLF6 promoter hypermethylation in menin-