PTEN Is a Target of Chromosome 10q Loss in Anaplastic Oligodendrogliomas and PTEN Alterations Are Associated with Poor Prognosis
2001; Elsevier BV; Volume: 159; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)61702-6
ISSN1525-2191
AutoresHikaru Sasaki, Magdalena C. Ƶlatescu, Rebecca A. Betensky, Yasushi Ino, J. Gregory Cairncross, David N. Louis,
Tópico(s)Glioma Diagnosis and Treatment
ResumoAllelic loss of 10q is a common genetic event in malignant gliomas, with three 10q tumor suppressor genes, ERCC6, PTEN, and DMBT1, putatively implicated in the most common type of malignant glioma, glioblastoma. Anaplastic oligodendroglioma, another type of malignant glioma, provides a unique opportunity to study the relevance of particular genetic alterations to chemosensitivity and survival. We therefore analyzed these three genes in 72 anaplastic oligodendrogliomas. Deletion mapping demonstrated 10q loss in 14 of 67 informative cases, with the PTEN and DMBT1 regions involved in all deletions but with the ERCC6 locus spared in two cases. Seven tumors had PTEN gene alterations; two had homozygous DMBT1 deletions, but at least one reflected unmasking of a germline DMBT1 deletion. No mutations were found in ERCC6 exon 2. Chemotherapeutic response occurred in two of the seven tumors with PTEN alterations, but with unexpected short survival times. PTEN gene alterations were not associated with poor therapeutic response in multivariate analysis, but were independently predictive of poor prognosis even after multivariate adjustment for both 10q and 1p loss. In anaplastic oligodendroglioma, therefore, PTEN is a target of 10q loss, and PTEN alterations are associated with poor prognosis, even in chemosensitive cases. Allelic loss of 10q is a common genetic event in malignant gliomas, with three 10q tumor suppressor genes, ERCC6, PTEN, and DMBT1, putatively implicated in the most common type of malignant glioma, glioblastoma. Anaplastic oligodendroglioma, another type of malignant glioma, provides a unique opportunity to study the relevance of particular genetic alterations to chemosensitivity and survival. We therefore analyzed these three genes in 72 anaplastic oligodendrogliomas. Deletion mapping demonstrated 10q loss in 14 of 67 informative cases, with the PTEN and DMBT1 regions involved in all deletions but with the ERCC6 locus spared in two cases. Seven tumors had PTEN gene alterations; two had homozygous DMBT1 deletions, but at least one reflected unmasking of a germline DMBT1 deletion. No mutations were found in ERCC6 exon 2. Chemotherapeutic response occurred in two of the seven tumors with PTEN alterations, but with unexpected short survival times. PTEN gene alterations were not associated with poor therapeutic response in multivariate analysis, but were independently predictive of poor prognosis even after multivariate adjustment for both 10q and 1p loss. In anaplastic oligodendroglioma, therefore, PTEN is a target of 10q loss, and PTEN alterations are associated with poor prognosis, even in chemosensitive cases. Oligodendrogliomas are one of the major histological subtypes of glioma, comprising up to 25% of adult gliomas.1Coons SW Johnson PC Scheithauer BW Yates AJ Pearl DK Improving diagnostic accuracy and interobserver concordance in the classification and grading of primary gliomas.Cancer. 1997; 79: 1381-1393Crossref PubMed Scopus (483) Google Scholar In contrast to other glioma subtypes, such as astrocytomas, many oligodendrogliomas are dramatically sensitive to chemotherapy, in particular to combined chemotherapy with procarbazine, lomustine (CCNU), and vincristine (PCV).2Cairncross JG Macdonald DR Successful chemotherapy for recurrent malignant oligodendroglioma.Ann Neurol. 1988; 23: 360-364Crossref PubMed Scopus (326) Google Scholar However, approximately one third of anaplastic oligodendrogliomas are resistant to chemotherapy, and some tumors that respond nonetheless show rapid regrowth with short patient survival.3Cairncross G Macdonald D Ludwin S Lee D Cascino T Buckner J Fulton D Dropcho E Stewart D Schold CJ Chemotherapy for anaplastic oligodendroglioma. National Cancer Institute of Canada Clinical Trials Group.J Clin Oncol. 1994; 12: 2013-2021PubMed Google Scholar Anaplastic oligodendrogliomas thereby provide a unique opportunity to investigate the relevance of particular genetic alterations to chemosensitivity and survival. Recently, allelic losses of chromosomes 1p and 19q have been shown to correlate with increased chemosensitivity and better prognosis in patients with these tumors.4Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein DM Hammond RR Silver JS Stark PC Macdonald DR Ino Y Ramsay DA Louis DN Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas.J Natl Cancer Inst. 1998; 90: 1473-1479Crossref PubMed Scopus (1389) Google ScholarAllelic loss of chromosome 10q is one of the most frequent genetic alterations in gliomas, and has been reported in 74 to 87% of glioblastomas,5Wang SI Puc J Li J Bruce JN Cairns P Sidransky D Parsons R Somatic mutations of PTEN in glioblastoma multiforme.Cancer Res. 1997; 57: 4183-4186PubMed Google Scholar, 6Rasheed BK Stenzel TT Mclendon RE Parsons R Friedman AH Friedman HS Bigner DD Bigner SH PTEN gene mutations are seen in high-grade but not in low-grade gliomas.Cancer Res. 1997; 57: 4187-4190PubMed Google Scholar, 7Schmidt EE Ichimura K Goike HM Moshref A Liu L Collins VP Mutational profile of the PTEN gene in primary human astrocytic tumors and cultivated xenografts.J Neuropathol Exp Neurol. 1999; 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90: 1473-1479Crossref PubMed Scopus (1389) Google Scholar, 9Lin H Bondy ML Langford LA Hess KR Delclos GL Wu X Chan W Pershouse MA Yung WK Steck PA Allelic deletion analyses of MMAC/PTEN and DMBT1 loci in gliomas: relationship to prognostic significance.Clin Cancer Res. 1998; 4: 2447-2454PubMed Google Scholar, 10Bigner SH Matthews MR Rasheed BK Wiltshire RN Friedman HS Friedman AH Stenzel TT Dawes DM Mclendon RE Bigner DD Molecular genetic aspects of oligodendrogliomas including analysis by comparative genomic hybridization.Am J Pathol. 1999; 155: 375-386Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Although chromosome 10q loss in malignant gliomas usually involves all or most of the long arm, the 10q25-26 region has been suggested as the primary tumor suppressor candidate region.8Ichimura K Schmidt EE Miyakawa A Goike HM Collins VP Distinct patterns of deletion on 10p and 10q suggest involvement of multiple tumor suppressor genes in the development of astrocytic gliomas of different malignancy grades.Genes Chromosom Cancer. 1998; 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58: 1170-1183Crossref PubMed Scopus (73) Google Scholar, 17Zhou XP Li YJ Hoang-Xuan K Laurent-Puig P Mokhtari K Longy M Sanson M Delattre JY Thomas G Hamelin R Mutational analysis of the PTEN gene in gliomas: molecular and pathological correlations.Int J Cancer. 1999; 84: 150-154Crossref PubMed Scopus (117) Google Scholar PTEN negatively regulates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, and thereby affects control of cell cycle and cell survival.18Cantley L Neel BG New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/Akt pathway.Proc Natl Acad Sci USA. 1999; 96: 4240-4245Crossref PubMed Scopus (1740) Google Scholar, 19Di Cristofano A Pandolfi PP The multiple roles of PTEN in tumor suppression.Cell. 2000; 100: 387-390Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar Ectopic expression of wild-type PTEN in PTEN-mutant gliomas markedly sensitizes these cells to irradiation, but not to five chemotherapeutic drugs,20Wick W Furnari FB Naumann U Cavenee WK Weller M PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis.Oncogene. 1999; 18: 3936-3943Crossref PubMed Scopus (103) Google Scholar raising the possibility that PTEN status may relate to therapeutic sensitivity in malignant gliomas. However, mutations only rarely occur in other types of tumors that lose 10q, such as malignant meningioma and pancreatic cancer, suggesting the presence of other 10q tumor suppressors.21Bostrom J Cobbers JM Wolter M Tabatabai G Weber RG Lichter P Collins VP Reifenberger G Mutation of the PTEN (MMAC1) tumor suppressor gene in a subset of glioblastomas but not in meningiomas with loss of chromosome arm 10q.Cancer Res. 1998; 58: 29-33PubMed Google Scholar, 22Okami K Wu L Riggins G Cairns P Goggins M Evron E Halachmi N Ahrendt SA Reed AL Hilgers W Kern SE Koch WM Sidransky D Jen J Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.Cancer Res. 1998; 58: 509-511PubMed Google ScholarThe DMBT1 gene at 10q25.3-26.1 encodes a secreted or membrane-linked protein, which seems to participate in epithelial differentiation and in immune regulation.23Mollenhauer J Herbertz S Holmskov U Tolnay M Krebs I Merlo A Schroder HD Maier D Breitling F Wiemann S Grone HJ Poustka A DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.Cancer Res. 2000; 60: 1704-1710PubMed Google ScholarDMBT1 has been proposed as a candidate tumor suppressor gene for glioblastoma, medulloblastoma, lung cancer, and gastrointestinal cancers based on homozygous deletions and lack of expression in these tumors.24Mollenhauer J Wiemann S Scheurlen W Korn B Hayashi Y Wilgenbus KK von Deimling A Poustka A DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.Nat Genet. 1997; 17: 32-39Crossref PubMed Scopus (411) Google Scholar, 25Somerville RP Shoshan Y Eng C Barnett G Miller D Cowell JK Molecular analysis of two putative tumour suppressor genes, PTEN and DMBT, which have been implicated in glioblastoma multiforme disease progression.Oncogene. 1998; 17: 1755-1757Crossref PubMed Scopus (66) Google Scholar, 26Wu W Kemp BL Proctor ML Gazdar AF Minna JD Hong WK Mao L Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer.Cancer Res. 1999; 59: 1846-1851PubMed Google Scholar, 27Mori M Shiraishi T Tanaka S Yamagata M Mafune K Tanaka Y Ueo H Barnard GF Sugimachi K Lack of DMBT1 expression in oesophageal, gastric and colon cancers.Br J Cancer. 1999; 79: 211-213Crossref PubMed Scopus (96) Google Scholar DMBT1, however, has a repetitive genomic structure, including 14 scavenger receptor cysteine-rich domains, and is therefore potentially susceptible to chromosomal instability.28Mollenhauer J Holmskov U Wiemann S Krebs I Herbertz S Madsen J Kioschis P Coy JF Poustka A The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.Oncogene. 1999; 18: 6233-6240Crossref PubMed Scopus (74) Google Scholar Indeed, a subset of normal individuals harbor hemizygous DMBT1 deletions, indicating that homozygous deletions in tumors may be a result of pre-existing constitutional deletions uncovered by allelic loss.23Mollenhauer J Herbertz S Holmskov U Tolnay M Krebs I Merlo A Schroder HD Maier D Breitling F Wiemann S Grone HJ Poustka A DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.Cancer Res. 2000; 60: 1704-1710PubMed Google ScholarFinally, the most centromeric 10q candidate, the ERCC6 gene at 10q11.2-21.2, is responsible for complementation group B of Cockayne syndrome, an autosomal recessive disorder characterized by postnatal growth failure, mental retardation, and cutaneous photosensitivity.29Troelstra C Landsvater RM Wiegant J van der Ploeg M Viel G Buys CH Hoeijmakers JH Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11–q21.Genomics. 1992; 12: 745-749Crossref PubMed Scopus (31) Google Scholar ERCC6 is involved in a subpathway of nucleotide excision repair (transcription-coupled repair) for preferential repair of damage to the transcribed strand of active genes.30Troelstra C van Gool A de Wit J Vermeulen W Bootsma D Hoeijmakers JH ERCC6, a member of a subfamily of putative helicase, is involved in Cockayne's syndrome and preferential repair of active genes.Cell. 1992; 71: 939-953Abstract Full Text PDF PubMed Scopus (617) Google Scholar, 31Selby CP Sancar A Human transcription-repair coupling factor CSB/ERCC6 is a DNA-stimulated ATPase but is not a helicase and does not disrupt the ternary transcription complex of stalled RNA polymerase II.J Biol Chem. 1997; 272: 1885-1890Crossref PubMed Scopus (215) Google Scholar Of note, mutations in exon 2 of the ERCC6 gene have been noted in 17.5% of high-grade gliomas.32Jaeckle KA Zhang L Lee PSY Connor T Ali-Osman F ERCC6 mutations in human malignant astrocytomas.Neuro-Oncology. 1999; 1: 317Google ScholarTo date, no extensive analysis of these 10q candidate glioma suppressor genes has been reported for anaplastic oligodendrogliomas, which undergo molecular alterations that are often distinct from astrocytic malignant gliomas such as glioblastoma.33Louis DN Gusella JF A tiger behind many doors: multiple genetic pathways to malignant gliomas.Trends Genet. 1995; 11: 412-415Abstract Full Text PDF PubMed Scopus (191) Google Scholar Moreover, because our previous studies suggested that chromosome 10q loss may denote tumors that respond less often to chemotherapy,4Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein DM Hammond RR Silver JS Stark PC Macdonald DR Ino Y Ramsay DA Louis DN Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas.J Natl Cancer Inst. 1998; 90: 1473-1479Crossref PubMed Scopus (1389) Google Scholar the identification of a 10q anaplastic oligodendroglioma gene could provide biological information relevant to understanding either chemoresistance or overall tumor behavior. We therefore analyzed the PTEN, DMBT1, and ERCC6 genes as well as the regional pattern of 10q loss in a large series of anaplastic oligodendrogliomas, and addressed the relevance of the genetic alterations to pathogenesis, chemosensitivity, and prognosis.Materials and MethodsTissues and Clinical ParametersSeventy-two anaplastic (grade III) oligodendrogliomas were classified and graded according to World Health Organization criteria34Reifenberger G Kros JM Burger PC Louis DN Collins VP Oligodendroglial tumors and mixed gliomas.in: Kleihues P Cavenee WK World Health Organization Classification of Tumours of the Central Nervous System. IARC/WHO, Lyon2000: 55-70Google Scholar by at least two neuropathologists, and cases with definite astrocytic components were excluded. Of the 72 patients, 47 were newly diagnosed patients who underwent chemotherapy as an integral part of initial treatment strategy; 45 patients received the PCV regimen of procarbazine, lomustine (CCNU), and vincristine, one received carmustine (BCNU) and one received temozolamide. Thirty-seven of these 47 patients received radiation therapy after completing a chemotherapy program or at the time of tumor recurrence after chemotherapy. Twenty-five of the 72 patients were treated with chemotherapy at recurrence after initial treatment with radiation therapy. Neuroradiological responses to chemotherapy were noted in 24 of the 36 evaluable (ie, with neuroradiologically assessable residual disease after surgery) tumors (67%) treated with chemotherapy as an initial treatment regimen, and in 23 of the 24 evaluable tumors (96%) treated with chemotherapy at recurrence. These investigations have been approved by the Massachusetts General Hospital Subcommittee on Human Studies and the Review Board for Health Science Research Involving Human Subjects at the University of Western Ontario. Tumor DNA was extracted from formalin-fixed, paraffin-embedded sections.35Louis DN von Deimling A Seizinger BR A (CA)n dinucleotide repeat assay for evaluating loss of allelic heterozygosity in small and archival human brain tumor specimens.Am J Pathol. 1992; 141: 777-782PubMed Google Scholar Constitutional DNA was extracted from blood leukocytes or from formalin-fixed, paraffin-embedded sections of adjacent, uninvolved brain or other tissues.Analysis of Allelic Loss of Chromosome 10qAllelic loss of chromosome 10q was assessed at 14 polymorphic loci, with particular emphasis on the ERCC6, PTEN, and DMBT1 regions: at D10S196, D10S109, D10S1687, D10S608, D10S215, D10S2491 (∼20 kb from the 5′ end of the PTEN gene36Cairns P Okami K Halachmi S Halachmi N Esteller M Herman JG Jen J Isaacs WB Bova GS Sidransky D Frequent inactivation of PTEN/MMAC1 in primary prostate cancer.Cancer Res. 1997; 57: 4997-5000PubMed Google Scholar), D10S583, D10S185, D10S88, D10S187, D10S587 (near DMBT124Mollenhauer J Wiemann S Scheurlen W Korn B Hayashi Y Wilgenbus KK von Deimling A Poustka A DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.Nat Genet. 1997; 17: 32-39Crossref PubMed Scopus (411) Google Scholar), D10S1723, and D10S169 (from centromeric to telomeric, Figure 1) using microsatellite analysis;35Louis DN von Deimling A Seizinger BR A (CA)n dinucleotide repeat assay for evaluating loss of allelic heterozygosity in small and archival human brain tumor specimens.Am J Pathol. 1992; 141: 777-782PubMed Google Scholar and at the ERCC6 locus with a single nucleotide polymorphism in exon 2 (135G/C, Leu45Leu) using single-strand conformation polymorphism (SSCP) analysis (see below).SSCP AnalysisThe entire coding sequence and intron/exon borders of PTEN were screened for mutations using SSCP.37Ueki K Rubio MP Ramesh V Correa KM Rutter JL von Deimling A Buckler AJ Gusella JF Louis DN MTS1/CDKN2 gene mutations are rare in primary human astrocytomas with allelic loss of chromosome 9p.Hum Mol Genet. 1994; 3: 1841-1845Crossref PubMed Scopus (105) Google Scholar, 38Duerr EM Rollbrocker B Hayashi Y Peters N Meyer-Puttlitz B Louis DN Schramm J Wiestler OD Parsons R Eng C von Deimling A PTEN mutations in gliomas and glioneuronal tumors.Oncogene. 1998; 16: 2259-2264Crossref PubMed Scopus (310) Google Scholar Forward primers to amplify exon 1 and exon 3 were redesigned to avoid primer overlap and to allow amplification of DNA extracted from archival materials (ex 1f: 5′-CATCCTGCAGAAGAAGCCCC-3′, 182 bp; ex 3f: 5′-TTGTTAATGGTGGCTTTTTG-3′, 169 bp). In tumors with 10q loss, the 5′ UTR 252 bp upstream of the open reading frame was also examined.38Duerr EM Rollbrocker B Hayashi Y Peters N Meyer-Puttlitz B Louis DN Schramm J Wiestler OD Parsons R Eng C von Deimling A PTEN mutations in gliomas and glioneuronal tumors.Oncogene. 1998; 16: 2259-2264Crossref PubMed Scopus (310) Google Scholar The primers are intronic (except for exon 8-2) and therefore do not amplify the processed PTEN pseudogene.39Teng DH Hu R Lin H Davis T Iliev D Frye C Swedlund B Hansen KL Vinson VL Gumpper KL Ellis L El-Naggar A Frazier M Jasser S Langford LA Lee J Mills GB Pershouse MA Pollack RE Tornos C Troncoso P Yung WKA Fujii G Berson A Bookstein R Bolen JB Tavtigian SV Steck PA MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.Cancer Res. 1997; 57: 5221-5225PubMed Google Scholar, 40Dahia PL FitzGerald MG Zhang X Marsh DJ Zheng Z Pietsch T von Deimling A Haluska FG Haber DA Eng C A highly conserved processed PTEN pseudogene is located on chromosome band 9p21.Oncogene. 1998; 16: 2403-2406Crossref PubMed Scopus (76) Google Scholar Because mobility shifts caused by PTEN mutations could be subtle because of high A-T content,7Schmidt EE Ichimura K Goike HM Moshref A Liu L Collins VP Mutational profile of the PTEN gene in primary human astrocytic tumors and cultivated xenografts.J Neuropathol Exp Neurol. 1999; 58: 1170-1183Crossref PubMed Scopus (73) Google Scholar most polymerase chain reaction (PCR) fragments were examined with more than one gel condition. For SSCP of ERCC6 exon 2, novel primer sets were designed to amplify three overlapping fragments covering 91% of the coding region of exon 2, including all reported mutation sites.32Jaeckle KA Zhang L Lee PSY Connor T Ali-Osman F ERCC6 mutations in human malignant astrocytomas.Neuro-Oncology. 1999; 1: 317Google Scholar ERCC6 products were separated on 8% and/or 10% nondenaturing polyacrylamide gel (AA:BIS 19:1) containing 10% glycerol at room temperature at 6 to 8 W. Tumors with altered PTEN or ERCC6 migration patterns were cycle-sequenced (fmol DNA cycle sequencing system; Promega, Madison, WI) bidirectionally using separately amplified PCR products.Reverse Transcriptase-PCRTwo glioma cell lines (Gli13, Gli46) that have a T insertion in a stretch of 15 T before exon 8 of PTEN were investigated by reverse transcriptase-PCR for expression of an aberrant transcript. Gli46 was heterozygous, whereas Gli13 was homozygous, for the insertion. Total RNA was extracted from these two cell lines as well as from two cell lines (Gli36, Gli49) with wild-type sequence, using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH). Reverse transcription with Superscript II and the oligo (dT) primer (Life Technologies, Inc., Rockville, MD) was followed by 35 cycles of PCR using ex 7-2f and ex 9-2r as primers.38Duerr EM Rollbrocker B Hayashi Y Peters N Meyer-Puttlitz B Louis DN Schramm J Wiestler OD Parsons R Eng C von Deimling A PTEN mutations in gliomas and glioneuronal tumors.Oncogene. 1998; 16: 2259-2264Crossref PubMed Scopus (310) Google Scholar PCR products were separated on 1.2% Tris borate-ethylenediaminetetraacetic acid (TBE)-agarose gel and visualized by ethidium bromide staining.Multiplex PCRHomozygous deletions of the PTEN gene were assayed using a modification of a comparative multiplex PCR approach described previously.38Duerr EM Rollbrocker B Hayashi Y Peters N Meyer-Puttlitz B Louis DN Schramm J Wiestler OD Parsons R Eng C von Deimling A PTEN mutations in gliomas and glioneuronal tumors.Oncogene. 1998; 16: 2259-2264Crossref PubMed Scopus (310) Google Scholar Two sets of oligonucleotide primers were used to amplify a 165-bp fragment of the 3′ part of PTEN exon 5 (ex 5-2f, ex 5-2r), and a 171-bp fragment of intron 7 of the desmin gene (in7f, in7r) as a control. The desmin gene is on chromosome 2q, a site rarely altered in oligodendrogliomas.10Bigner SH Matthews MR Rasheed BK Wiltshire RN Friedman HS Friedman AH Stenzel TT Dawes DM Mclendon RE Bigner DD Molecular genetic aspects of oligodendrogliomas including analysis by comparative genomic hybridization.Am J Pathol. 1999; 155: 375-386Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar PCR was performed in a final volume of 10 μl containing 1 to 10 ng genomic DNA, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl, pH 8.3, 0.2 mmol/L of each dNTP, 1 μmol/L of each primer, 0.5 U Taq polymerase (Fisher Scientific, Pittsburgh, PA). PCR conditions consisted of initial denaturation at 95°C for 5 minutes, 31 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 40 seconds, and a final extension step of 10 minutes at 72°C. PCR products were separated on 4% TBE-agarose gel and visualized by ethidium bromide staining. Quantitation of the bands was performed with the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD), and the PTEN:desmin ratio was calculated. Because DNA extracted from paraffin-embedded tissues tended to show lower PTEN:desmin ratios than DNA extracted from blood, titration assays using serial mixtures of normal DNA and a PTEN homozygously deleted cell line DNA was not applicable. Instead, homozygous deletions were scored by comparison to the ratios of tumors with PTEN mutations accompanied by allelic loss.For homozygous deletions of DMBT1, two multiplex PCR assays were used. Because most reported homozygous deletions (23 of 27) within the DMBT1 gene involved the g14ext/g14 locus in intron 18,24Mollenhauer J Wiemann S Scheurlen W Korn B Hayashi Y Wilgenbus KK von Deimling A Poustka A DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.Nat Genet. 1997; 17: 32-39Crossref PubMed Scopus (411) Google Scholar, 25Somerville RP Shoshan Y Eng C Barnett G Miller D Cowell JK Molecular analysis of two putative tumour suppressor genes, PTEN and DMBT, which have been implicated in glioblastoma multiforme disease progression.Oncogene. 1998; 17: 1755-1757Crossref PubMed Scopus (66) Google Scholar, 26Wu W Kemp BL Proctor ML Gazdar AF Minna JD Hong WK Mao L Expression of DMBT1, a candidate tumor suppressor gene, is frequently lost in lung cancer.Cancer Res. 1999; 59: 1846-1851PubMed Google Scholar, 27Mori M Shiraishi T Tanaka S Yamagata M Mafune K Tanaka Y Ueo H Barnard GF Sugimachi K Lack of DMBT1 expression in oesophageal, gastric and colon cancers.Br J Cancer. 1999; 79: 211-213Crossref PubMed Scopus (96) Google Scholar these two closely positioned STSs were used as target sequences. A 190-bp sequence from chromosome 8 (c12) and a 187-bp sequence of the APEX nuclease gene on chromosome 14q were used as controls for g14ext and g14, respectively. All primer sequences, except the forward primer to amplify STS g14 (5′-ATTAGGGCTGCTGAGCAAAG-3′), have been published.24Mollenhauer J Wiemann S Scheurlen W Korn B Hayashi Y Wilgenbus KK von Deimling A Poustka A DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.Nat Genet. 1997; 17: 32-39Crossref PubMed Scopus (411) Google Scholar, 41Ono Y Tamiya T Ichikawa T Kunishio K Matsumoto K Furuta T Ohmoto T Ueki K Louis DN Malignant astrocytomas with homozygous CDKN2/p16 gene deletions have higher Ki-67 proliferation indices.J Neuropathol Exp Neurol. 1996; 55: 1026-1031Crossref PubMed Scopus (90) Google Scholar PCR was performed for 29 cycles at annealing temperature of 60°C to amplify g14ext/c12, and the annealing temperature was gradually decreased from 63°C to 56°C with 30 cycles in total for amplification of g14/APEX. The products were separated on 3% TBE-agarose gels. Homozygous deletions were scored by comparing the DMBT1:control ratio of the test tumor to that of mixture consisting of 30% constitutional DNA of each case and 70% U343MG cell line DNA, which has a homozygous deletion at the g14ext/g14 locus.Long-Range PCRLong-range PCR was used to evaluate constitutional DMBT1 deletions. The extent of the deletion was initially estimated based on published Southern blot data of a constitutional hemizygous deletion involving the g14ext/g14 locus in a normal individual (G3 configuration),23Mollenhauer J Herbertz S Holmskov U Tolnay M Krebs I Merlo A Schroder HD Maier D Breitling F Wiemann S Grone HJ Poustka A DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer.Cancer Res. 2000; 60: 1704-1710Pu
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