Loss of Heterozygosity on Chromosome 10 Is More Extensive in Primary (De Novo) Than in Secondary Glioblastomas

Fujisawa, Hironori; Reis, Rui Manuel; Nakamura, Mitsutoshi; Colella, Stefano; Yonekawa, Yasuhiro; Kleihues, Paul; Ohgaki, Hideaki

doi:10.1038/labinvest.3780009

Cited by 151 publications

(118 citation statements)

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“…Recent evidence suggests that complete losses of one copy of chromosome 10 are more frequent in primary GBM and partial losses in secondary GBM. 2 More often deletions affect the long arm of chromosome 10 but in a large fraction of patients they are also present on the short arm. 3 A comparable frequency of deletions on 10p has been found in prostate cancer.…”

Section: Dear Sirmentioning

confidence: 99%

KLF6 is not the major target of chromosome 10p losses in glioblastomas

Montanini

Bissola

Finocchiaro

2004

Intl Journal of Cancer

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Dear Sir,Complete or partial deletions of chromosome 10 are critical events during the malignant progression of gliomas. 1 They are present in the large majority of patients with glioblastoma multiforme (GBM), the most malignant glioma. Recent evidence suggests that complete losses of one copy of chromosome 10 are more frequent in primary GBM and partial losses in secondary GBM. 2 More often deletions affect the long arm of chromosome 10 but in a large fraction of patients they are also present on the short arm. 3 A comparable frequency of deletions on 10p has been found in prostate cancer. 4 KLF6, a gene that is part of the Kruppel family of transcription factors, 5 is located on 10p and according to several reports is mutated in about 50% of these patients 6 (a recent report, however, does not confirm this finding 7 ).A recent report by Jeng and Hsu describes the presence of KLF6 mutations in astrocytic gliomas and concludes that "mutations of the KLF6 gene play a role in the pathogenesis of astrocytic gliomas." 8 We have investigated the loss of heterozygosity in the chromosomal region flanking KLF6 and looked for mutation of KLF6 in 34 patients with GBM and in 2 GBM cell lines. LOH studies were done using microsatellite markers KLF6-M1 and KLF6-M2 and methods described previously. 9 LOH with primer M1 (telomeric) was present in 16/34 cases (47%). LOH with primer M2 (centromeric) was found in 8/34 (24%). Overall at least one loss was present in 62% of the cases but only in 3 tumors allelic imbalance was present on both the markers flanking KLF6.We have analyzed the coding sequence of KLF6 in these 34 patients. The 4 exons were amplified using PCR primers and temperatures outlined in Table I.The sequence was found identical to normal in all patients, with one exception. In Patient 19, showing LOH on M1, exons 1, 3 and 4 could be amplified by PCR whereas exon 2 failed to be amplified. Using PCR primers on exons 1 and 4 we could amplify more than 5 kb of sequence of KLF6 in 2 independent DNAs but not in tumor DNA of Patient 19, suggesting the presence of a translocation interrupting the coding sequence of KLF6 in exon 2. In Patient 8, the sequence of lymphocyte DNA showed heterozygosity on codon 201 (CGG/CGA) and loss of heterozygosity on tumor DNA (CGA). Both codons however encode the same arginine residue (reverse sequences are shown in the Fig. 1).To test whether reduced expression of KLF6 was present in some patients, we analyzed by real-time PCR the expression of KLF6 in 8 patients, in 2 established cell lines deriving from malignant gliomas (U87 and U373) and in normal brain. RNA was extract with Eurozol (EuroClone, Wetherby, UK) and 1 g of RNA was reverse-transcribed with Expand Reverse Transcriptase (Roche Diagnostics, Milan, Italy). Real-time PCR was carried out using GeneAmp 5700 and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and primers of KLF6 and GAPDH (Assay on Demand cat no. Hs00154550 and Hs99999905, respectively), all from Applied Biosystems. PCR conditions were 2 min ...

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Section: Dear Sirmentioning

confidence: 99%

KLF6 is not the major target of chromosome 10p losses in glioblastomas

Montanini

Bissola

Finocchiaro

2004

Intl Journal of Cancer

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“…DNA was extracted from paraffin sections, as described previously, to be used for microsatellite analysis. 3,6 Control genomic DNA was extracted from separate tissue blocks not involved with the tumor.…”

Section: Tumor Samples and Dna Extractionmentioning

confidence: 99%

“…Another type of glioblastoma, the so-called secondary glioblastoma, progressively develops more slowly from low-grade diffuse (WHO grade II) or anaplastic astrocytoma (WHO grade III). 1 Recent genetic analyses have shown that these glioblastoma subtypes develop through different genetic pathways; 2-4 primary glioblastomas occur in older patients characterized by EGFR amplification/overexpression, PTEN mutation, homozygous p16 INK4a deletion, and loss of heterozygosity (LOH) throughout chromosome 10, 3,5,6 while secondary glioblastomas develop in younger patients and typically show frequent p53 mutations, LOH preferentially on chromosomes 10q and 19q, 3,[5][6][7] and promoter methylation of the RB1 gene. 8 LOH on 22q seems to be of interest, since this allelic loss is a common event in gliomas, occurring with a frequency varying between 11 and 39%.…”

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Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Nakamura

Ishida

Shimada

et al. 2005

Laboratory Investigation

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Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3-13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3-13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.

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“…Recent studies have shown that these glioblastoma subtypes develop through different genetic pathways Kleihues and Ohgaki, 1999;Lang et al, 1994;Tohma et al, 1998;von Deimling et al, 1992;Watanabe et al, 1996). Primary glioblastomas are characterized by EGFR amplification/overexpression, PTEN mutations, p16 INK4a homozygous deletion, and loss of heterozygosity (LOH) on chromosomes 10p and 10q Fujisawa et al, 2000;Kleihues and Ohgaki, 1999;Tohma et al, 1998;Watanabe et al, 1996), whereas secondary glioblastomas contain frequent p53 mutations and show LOH preferentially on chromosomes 19q and 10q (Fujisawa et al, 2000;Kleihues and Ohgaki, 1999;Nakamura et al, 2000b;Watanabe et al, 1996Watanabe et al, , 1997.…”

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Promoter Hypermethylation of the RB1 Gene in Glioblastomas

Nakamura

Yonekawa

Kleihues

et al. 2001

Laboratory Investigation

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SUMMARY:Loss of expression of the retinoblastoma gene (RB1) has been shown to occur in up to 25% of glioblastomas (WHO Grade IV). To elucidate the underlying mechanism, we assessed RB1 promoter hypermethylation using methylation-specific polymerase chain reaction and RB1 expression by immunohistochemistry in 35 primary (de novo) glioblastomas and in 21 secondary glioblastomas that had progressed from low-grade diffuse astrocytoma (WHO Grade II) or anaplastic astrocytoma (WHO Grade III). Promoter hypermethylation was significantly more frequent in secondary (9 of 21, 43%) than in primary glioblastomas (5 of 35, 14%; p ϭ 0.0258). There was a clear correlation between loss of RB1 expression and promoter hypermethylation. In the majority of glioblastomas with loss of RB1 expression, there was promoter hypermethylation (11 of 13, 85%), whereas 93% of tumors with RB1 expression had a normal RB1 gene status (p Ͻ 0.0001). In three glioblastomas, areas with and without RB1 expression were microdissected; promoter hypermethylation was detected only in areas lacking RB1 expression. In patients with multiple biopsies, methylation of the RB1 promoter was not detectable in the less malignant precursor lesions, ie, low-grade diffuse and anaplastic astrocytoma. These results indicate that promoter hypermethylation is a late event during astrocytoma progression and is the major mechanism underlying loss of RB1 expression in glioblastomas. (Lab Invest 2001, 81:77-82).

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Loss of Heterozygosity on Chromosome 10 Is More Extensive in Primary (De Novo) Than in Secondary Glioblastomas

Cited by 151 publications

References 40 publications

KLF6 is not the major target of chromosome 10p losses in glioblastomas

KLF6 is not the major target of chromosome 10p losses in glioblastomas

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Promoter Hypermethylation of the RB1 Gene in Glioblastomas

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