Multiplex Ligation-Dependent Probe Amplification
2006; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2006.060012
ISSN1943-7811
AutoresJudith W. M. Jeuken, S.J.B. Cornelissen, Sandra Boots‐Sprenger, Sabine Gijsen, Pieter Wesseling,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoGenetic aberrations in tumors are predictive for chemosensitivity and survival. A test is needed that allows simultaneous detection of multiple changes and that is widely applicable in a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semiquantitative polymerase chain reaction-based assay. To assess the applicability of MLPA, we performed MLPA analysis to detect relevant genetic markers in a spectrum of 88 gliomas. The vast majority of these tumors (n = 79) were previously characterized by comparative genomic hybridization. With MLPA kit P088 (78 cases), complete and partial loss of 1p and 19q were reliably identified, even in samples containing only 50% tumor DNA. Distinct 1p deletions exist with different clinically prognostic consequences, and in contrast to the commonly used diagnostic strategies (loss of heterozygosity or fluorescent in situ hybridization 1p36), P088 allows detection of such distinct 1p losses. Combining P088 with P105 will further increase the accurate prediction of clinical behavior because this kit identified markers (EGFR, PTEN, and CDKN2A) of high-grade malignancy in 41 cases analyzed. We conclude that MLPA is a reliable diagnostic tool for simultaneous identification of different region-specific genetic aberrations of tumors. Genetic aberrations in tumors are predictive for chemosensitivity and survival. A test is needed that allows simultaneous detection of multiple changes and that is widely applicable in a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semiquantitative polymerase chain reaction-based assay. To assess the applicability of MLPA, we performed MLPA analysis to detect relevant genetic markers in a spectrum of 88 gliomas. The vast majority of these tumors (n = 79) were previously characterized by comparative genomic hybridization. With MLPA kit P088 (78 cases), complete and partial loss of 1p and 19q were reliably identified, even in samples containing only 50% tumor DNA. Distinct 1p deletions exist with different clinically prognostic consequences, and in contrast to the commonly used diagnostic strategies (loss of heterozygosity or fluorescent in situ hybridization 1p36), P088 allows detection of such distinct 1p losses. Combining P088 with P105 will further increase the accurate prediction of clinical behavior because this kit identified markers (EGFR, PTEN, and CDKN2A) of high-grade malignancy in 41 cases analyzed. We conclude that MLPA is a reliable diagnostic tool for simultaneous identification of different region-specific genetic aberrations of tumors. The majority of gliomas can be classified as astrocytic (As) or oligodendroglial tumors (OTs), the latter including pure oligodendroglial (Os) or mixed oligo-astrocytic (OAs) tumors. An accurate distinction between OTs and As is important because of prognostic and therapeutic implications.1Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein D 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 (1381) Google Scholar2Schmid JS Perry A Borell TJ Lee HK O'Fallon J Hosek SM Kimmel D Yates A Burger PC Scheithauer BW Jenkins RB Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas.J Clin Oncol. 2000; 18: 636-645PubMed Google Scholar3Cairncross JG Aggressive oligodendroglioma: a chemosensitive tumor.Recent Results Cancer Res. 1994; 135: 127-133Crossref PubMed Scopus (22) Google Scholar Unfortunately, unequivocal histopathological criteria are lacking and differences in clinical behavior within a specific histopathological group have been reported (eg, two thirds of the anaplastic Os respond to PCV chemotherapy [procarbazine, lomustine (CCNU), and vincristine]).1Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein D 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 (1381) Google Scholar,3Cairncross JG Aggressive oligodendroglioma: a chemosensitive tumor.Recent Results Cancer Res. 1994; 135: 127-133Crossref PubMed Scopus (22) Google Scholar,4Kyritsis AP Yung WKA Bruner J Gealson M Levin VA The treatment of anaplastic oligodendrogliomas and mixed gliomas.Neurosurgery. 1993; 32: 365-371Crossref PubMed Scopus (100) Google Scholar Fortunately, loss of 1p and 19q have been identified as diagnostic molecular markers in gliomas predicting response to chemotherapy and long survival.1Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein D 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 (1381) Google Scholar,5Nutt CL Mani DR Betensky RA Tamayo P Cairncross JG Ladd C Pohl U Hartmann C McLaughlin ME Batchelor TT Black PM von Deimling A Pomeroy SL Golub TR Louis DN Gene expression-based classification of malignant gliomas correlates better with survival than histological classification.Cancer Res. 2003; 63: 1602-1607PubMed Google Scholar6Smith JS Perry A Borell TJ Lee HK Fallon JO Hosek SM Kimmel D Yates A Burger PC Scheithauer BW Jenkins RB Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, mixed oligoastrocytomas.J Clin Oncol. 2000; 18: 636-645Crossref PubMed Google Scholar7van den Bent MJ Looijenga LHJ Langberg K Dinjens W Gravenland W Uytdewilligen L Sillevis Smitt PA Jenkins RB Kros JM Chromosomal anomalies in oligodendroglial tumors are correlated with clinical features.Cancer. 2003; 97: 1276-1284Crossref PubMed Scopus (170) Google Scholar8Iuchi T Namba H Iwadate Y Shishikura T Kageyama H Nakamura Y Ohira M Yamaura A Osato K Sakiyama S Nakagawara A Identification of the small interstitial deletion at chromosome band 1p34–p35 and its association with poor outcome in oligodendroglial tumors.Gen Chromosom Cancer. 2002; 35: 170-175Crossref PubMed Scopus (39) Google Scholar9Bauman GS Ino Y Ueki K Zlatescu MC Fisher BJ Macdonald DR Stitt L Louis DN Cairncross JG Allelic loss of chromosome 1p and radiotherapy plus chemotherapy in patients with oligodendrogliomas.Int J Radiat Oncol Biol Phys. 2000; 48: 825-830Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar10Ino Y Betensky RA Zlatescu MC Sasaki H Macdonald DR Stemmer-Rachamimov AO Ramsay DA Cairncross JG Louis DN Molecular subtypes of anaplastic oligodendroglioma: implications for patient management at diagnosis.Clin Cancer Res. 2001; 7: 839-845PubMed Google Scholar Identification of molecular alterations involved in the malignant progression of gliomas such as EGFR amplification and PTEN and CDKN2A loss next to loss of heterozygosity (LOH) 1p36 was reported to predict a less favorable prognosis and a less durable response to chemotherapy.10Ino Y Betensky RA Zlatescu MC Sasaki H Macdonald DR Stemmer-Rachamimov AO Ramsay DA Cairncross JG Louis DN Molecular subtypes of anaplastic oligodendroglioma: implications for patient management at diagnosis.Clin Cancer Res. 2001; 7: 839-845PubMed Google Scholar11Maintz D Fiedler K Koopmann J Rollbrocker B Nechev S Lenartz D Stangl AP Louis DN Schramm J Wiestler OD von Deimling A Molecular genetic evidence for subtypes of oligoastrocytomas.J Neuropathol Exp Neurol. 1997; 56: 1098-1104Crossref PubMed Scopus (226) Google Scholar12Wolter M Reifenberger J Blaschke B Ichimura K Schmidt EE Collins VP Reifenberger G Oligodendroglial tumors frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes.J Neuropathol Exp Neurol. 2001; 60: 1170-1180PubMed Google Scholar13Watanabe T Nakamura M Kros JM Burkhard C Yonekawa Y Kleihues P Ohgaki H Phenotype versus genotype correlation in oligodendrogliomas and low-grade diffuse astrocytomas.Acta Neuropathol (Berl). 2002; 103: 267-275Crossref PubMed Scopus (134) Google Scholar14von Deimling A Fimmers R Schmidt MC Bender B Fassbender F Nagel J Jahnke R Kaskel P Duerr E Koopmann J Maintz D Steinbeck S Wick W Platten M Muller DJ Przkora R Waha A Blumcke B Wellenreuther R Meyer-Puttlitz B Schmidt O Mollenhauer J Poustka A Stangl AP Lenartz D von Ammon K Henson JW Schramm J Louis DN Wiestler OD Comprehensive allelotype and genetic analysis of 466 human nervous system tumors.J Neuropathol Exp Neurol. 2000; 56: 544-558Google Scholar15Reifenberger G Kros JM Burger PC Louis DN Collins VP Kleihues P Cavenee WK Oligodendroglial tumors and mixed gliomas. WHO classification. Tumours of the Nervous System. International Agency for Research on Cancer (IARC) Press, Lyon1999: 55-70Google Scholar A combined molecular diagnostic approach would therefore be of clinical relevance. The most commonly applied techniques, LOH,10Ino Y Betensky RA Zlatescu MC Sasaki H Macdonald DR Stemmer-Rachamimov AO Ramsay DA Cairncross JG Louis DN Molecular subtypes of anaplastic oligodendroglioma: implications for patient management at diagnosis.Clin Cancer Res. 2001; 7: 839-845PubMed Google Scholar,16Reifenberger G Louis DN Oligodendroglioma: toward molecular definitions in diagnostic neuro-oncology.J Neuropathol Exp Neurol. 2003; 62: 111-126Crossref PubMed Scopus (272) Google Scholar fluorescent in situ hybridization (FISH),17Smith JS Alderete BE Minn Y Borell T Perry A Mohapatra G Smith SM Kimmel D Fallon JO Tates A Feuerstein BG Burger PC Scheithauer BW Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype.Oncogene. 1999; 18: 4144-4152Crossref PubMed Scopus (334) Google Scholar,18Hatanpaa KJ Burger PC Eshleman JR Murphy KM Berg KD Molecular diagnosis of oligodendroglioma in paraffin sections.Lab Invest. 2003; 83: 419-428Crossref PubMed Scopus (57) Google Scholar or quantitative microsatellite analysis,19Ginzinger DG Godfrey TE Nigro J Moore DH Suzuki S Pallavicini MG Gray JW Jensen RH Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis.Cancer Res. 2000; 60: 5405-5409PubMed Google Scholar,20Nigro JM Takahashi MA Ginzinger DG Law M Passe S Jenkins RB Aldape KD Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative polymerase chain reaction assay.Am J Pathol. 2001; 158: 1253-1262Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar specifically analyze the chromosomal region 1p36 to identify OTs with a favorable prognosis and therapy response. The clinical value of such tests has been clearly proven. However, not all chemosensitive OTs were identified, and vice versa, some of the tumors with a loss of 1p36 proved to be chemoresistant.1Cairncross JG Ueki K Zlatescu MC Lisle DK Finkelstein D 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 (1381) Google Scholar,16Reifenberger G Louis DN Oligodendroglioma: toward molecular definitions in diagnostic neuro-oncology.J Neuropathol Exp Neurol. 2003; 62: 111-126Crossref PubMed Scopus (272) Google Scholar Furthermore, it is becoming increasingly clear that distinct types of 1p deletions (complete versus partial) exist in (oligodendro)gliomas, sometimes with opposite clinical and biological consequences.21Felsberg J Erkwoh A Sabel MC Kirsch L Fimmers R Blaschke B Schlegel U Schramm J Wiestler OD Reifenberger G Oligodendroglial tumors: refinement of candidate regions on chromosome arm 1p and correlation of 1p/19q status with survival.Brain Pathol. 2004; 14: 121-130Crossref PubMed Scopus (149) Google Scholar,22McLendon RE Herndon JE West B Reardon D Wiltshire R Rasheed BK Quinn J Friedman HS Friedman AH Bigner DD Survival analysis of presumptive prognostic markers among oligodendrogliomas.Cancer. 2005; 104: 1693-1699Crossref PubMed Scopus (43) Google Scholar Unfortunately, however, the above-mentioned diagnostic strategies do not discriminate between these distinct types. Until the exact combination of genes on 1p/19q responsible for the favorable clinical behavior of these gliomas have been identified, analysis of multiple regions on 1p and 19q seems preferable, thereby enabling an even more accurate identification of these clinically favorable gliomas. Furthermore, it is noteworthy that techniques identifying copy number changes such as (array) comparative genomic hybridization (CGH) identified 1p and 19q gains in gliomas.23Idbaih A Marie Y Pierron G Brennetot C Hoang-Xuan K Kujas M Mokhtari K Sanson M Lejeune J Aurias A Delattre O Delattre JY Two types of chromosome 1p losses with opposite significance in gliomas.Ann Neurol. 2005; 58: 483-487Crossref PubMed Scopus (148) Google Scholar24Jeuken JWM Boots-Sprenger SHE Wesseling P Zhang W Fuller GN Chromosomal imbalances in oligodendroglial tumors as detected by comparative genomic hybridization (CGH). Genomic and Molecular Neuro-Oncology. Jones and Bartlett Publishers, Boston2004: 185-198Google Scholar25Jeuken JWM Sprenger SHE Wesseling P Macville MVE von Deimling A Teepen HLJM van Overbeeke JJ Boerman RH Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.J Neuropathol Exp Neurol. 1999; 58: 606-612Crossref PubMed Scopus (90) Google Scholar26Jeuken JWM Sprenger SHE Boerman RH von Deimling A Teepen HLJM van Overbeeke JJ Wesseling P Subtyping of oligo-astrocytic tumours by comparative genomic hybridisation.J Pathol. 2001; 194: 81-87Crossref PubMed Scopus (55) Google Scholar27Jeuken JWM Sprenger SHE Vermeer H Kappelle AC Boerman RH Wesseling P Chromosomal imbalances in primary oligodendroglial tumors and their recurrences; clues for malignant progression as detected by CGH.J Neurosurg. 2002; 96: 559-564Crossref PubMed Scopus (42) Google Scholar These gains could be easily misinterpreted as loss of heterozygosity using microsatellite approach.23Idbaih A Marie Y Pierron G Brennetot C Hoang-Xuan K Kujas M Mokhtari K Sanson M Lejeune J Aurias A Delattre O Delattre JY Two types of chromosome 1p losses with opposite significance in gliomas.Ann Neurol. 2005; 58: 483-487Crossref PubMed Scopus (148) Google Scholar24Jeuken JWM Boots-Sprenger SHE Wesseling P Zhang W Fuller GN Chromosomal imbalances in oligodendroglial tumors as detected by comparative genomic hybridization (CGH). Genomic and Molecular Neuro-Oncology. Jones and Bartlett Publishers, Boston2004: 185-198Google Scholar25Jeuken JWM Sprenger SHE Wesseling P Macville MVE von Deimling A Teepen HLJM van Overbeeke JJ Boerman RH Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.J Neuropathol Exp Neurol. 1999; 58: 606-612Crossref PubMed Scopus (90) Google Scholar26Jeuken JWM Sprenger SHE Boerman RH von Deimling A Teepen HLJM van Overbeeke JJ Wesseling P Subtyping of oligo-astrocytic tumours by comparative genomic hybridisation.J Pathol. 2001; 194: 81-87Crossref PubMed Scopus (55) Google Scholar27Jeuken JWM Sprenger SHE Vermeer H Kappelle AC Boerman RH Wesseling P Chromosomal imbalances in primary oligodendroglial tumors and their recurrences; clues for malignant progression as detected by CGH.J Neurosurg. 2002; 96: 559-564Crossref PubMed Scopus (42) Google Scholar Next to analysis of multiple 1p/19q loci, analysis of additional genes, for example those reported to be involved in malignant progression, will prove to be of additional diagnostic value. Unfortunately, with the above-mentioned techniques only one or a few loci can be analyzed per experiment. Array CGH or, at a lower resolution, conventional CGH provides an overview of copy number changes through the entire tumor genome.28Jeuken JWM Sprenger SHE Wesseling P Comparative genomic hybridization: practical guidelines.Diagn Mol Pathol. 2002; 11: 193-203Crossref PubMed Scopus (28) Google Scholar29Cowell JK Barnett GH Nowak NJ Characterization of the 1p/19q chromosomal loss in oligodendrogliomas using comparative genomic hybridization arrays (CGHa).J Neuropathol Exp Neurol. 2004; 63: 151-158PubMed Google Scholar30Kitange G Misra A Law M Passe S Kollmeyer TM Maurer M Ballman K Feuerstein BG Jenkins RB Chromosomal imbalances detected by array comparative genomic hybridization in human oligodendrogliomas and mixed oligoastrocytomas.Gene Chromosom Cancer. 2005; 42: 68-77Crossref PubMed Scopus (72) Google Scholar Because such experiments are rather specialized, they are not available in most standard molecular biology laboratories and may therefore be less suitable for a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) is a technique by which up to 45 different sequences can be targeted in a single, semiquantitative polymerase chain reaction (PCR)-based experiment (see Figure 1).31Schouten JP McElgunn CJ Waaijer R Zwijnenburg D Diepvens F Pals G Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2067) Google Scholar The sequences detected can be small (∼60 nucleotides), enabling analysis of fragmented DNA. Furthermore, the MLPA reaction is fast, relatively inexpensive, and easy to perform, and the equipment needed for MLPA analysis is present in most molecular biology laboratories. Two MLPA kits were designed for molecular analysis of (oligodendro)gliomas, one to detect copy number changes on 1p and 19q (kit P088; MRC-Holland, Amsterdam, The Netherlands) and one to detect aberrations of EGFR, TP53, PTEN, CDKN2A, and ERBB2 (kit P105; MRC-Holland). To establish the potential of MLPA in a routine diagnostic setting, we analyzed a spectrum of 88 glial tumors using P088 (n = 78) and P105 (n = 41). The vast majority (79 of 88) of these tumors were previously characterized genetically by conventional CGH. Eighty-eight specimens obtained from glioma patients treated in the Department of Neurosurgery of the Radboud University Nijmegen Medical Centre, The Netherlands, were selected. The use of brain tumor tissue after completing histopathological diagnosis for research purposes was approved by the ethics committee of the Radboud University Nijmegen Medical Centre, and informed consent was given by the patients. Tumors were classified according to the World Health Organization–2000 classification32Kleihues P Cavenee WK Pathology and genetics. Tumours of the nervous system. World Health Organization Classification of Tumours. International Agency for Research on Cancer (IARC) Press, Lyon, France2000Google Scholar and included three pilocytic astrocytomas (A-I), six low-grade diffuse astrocytomas (A-II), one anaplastic astrocytoma (A-III), 24 glioblastomas multiforme (GBM), nine low-grade oligodendrogliomas (O-II), 16 anaplastic oligodendrogliomas (O-III), seven low-grade oligo-astrocytomas (OA-II), 17 anaplastic oligo-astrocytomas (OA-III), three low-grade ependymomas (E-II), and two anaplastic ependymomas (E-III). Most samples used in this study (79 of 88) were previously analyzed by conventional CGH.25Jeuken JWM Sprenger SHE Wesseling P Macville MVE von Deimling A Teepen HLJM van Overbeeke JJ Boerman RH Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.J Neuropathol Exp Neurol. 1999; 58: 606-612Crossref PubMed Scopus (90) Google Scholar26Jeuken JWM Sprenger SHE Boerman RH von Deimling A Teepen HLJM van Overbeeke JJ Wesseling P Subtyping of oligo-astrocytic tumours by comparative genomic hybridisation.J Pathol. 2001; 194: 81-87Crossref PubMed Scopus (55) Google Scholar27Jeuken JWM Sprenger SHE Vermeer H Kappelle AC Boerman RH Wesseling P Chromosomal imbalances in primary oligodendroglial tumors and their recurrences; clues for malignant progression as detected by CGH.J Neurosurg. 2002; 96: 559-564Crossref PubMed Scopus (42) Google Scholar DNA was isolated from snap-frozen tumor tissue with the DNeasy tissue kit, as described by the manufacturer (Qiagen, Venlo, The Netherlands), supplemented with an additional wash step using the supplied wash buffer AW2 before elution. In case of paraffin-embedded tissue, 50-μm paraffin sections were cut and incubated in P-buffer (50 mmol/L Tris-HCl, pH 8.2, 100 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 0.5% Tween 20, 0.5% Nonidet P-40, 20 mmol/L dithiothreitol) at 90°C for 15 minutes, after which a protein digestion was performed using 0.5 mg/ml of proteinase K (Roche Diagnostics GmbH, Mannheim, Germany) at 55°C overnight. Subsequently, DNA was isolated using the DNeasy tissue kit. Furthermore, DNA previously isolated using a salting out procedure25Jeuken JWM Sprenger SHE Wesseling P Macville MVE von Deimling A Teepen HLJM van Overbeeke JJ Boerman RH Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.J Neuropathol Exp Neurol. 1999; 58: 606-612Crossref PubMed Scopus (90) Google Scholar,33Miller SA Dykes DD Polsky HF A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17628) Google Scholar was purified using the DNeasy tissue kit. P088 contains 15 1p probes, eight 19q probes, and 21 control probes specific to other chromosomes (MRC Holland). During our study a minor change (replacement of two 1p probe by a new 1p and control probe) was introduced by the manufacturer, and lot-nr 0804 was replaced by lot-nr 0305. SALSA P105 (lot-nr 0804) contains nine PTEN probes, five CDKN2A probes, eight TP53 probes, three EGFR probes, two ERBB2 probes, and 15 control probes. All MLPA probe pairs code for unique human single copy DNA sequences and were designed and prepared as described by Schouten and colleagues.31Schouten JP McElgunn CJ Waaijer R Zwijnenburg D Diepvens F Pals G Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2067) Google Scholar Probe sequences and genes detected by the control probes are available on request by the manufacturer (MRC Holland). Probes used to detect imbalances are listed in Figure 2, Figure 3, Figure 4, Figure 5.Figure 3Overview of the distribution of individual probe ratios in control and −1p/−19q-containing samples. The x axis shows probe names in chromosomal order whereas the y axis shows ratios. Probe ratios for the reference DNAs are shown in gray, and ratios in tumors with a complete loss of 1p and 19q as detected by CGH are shown in black. For reference DNA, horizontal lines represent mean probe values; vertical lines represent mean values ± 2 times SD; dot represents individual probe ratio. The increased variation in probe ratios among the −1p/−19q tumors compared to the control samples is caused by the fact that the amount of tumor cells within a tumor sample directly affecting the probe ratios varies among the different tumors. Mean probe ratios and standard deviations were therefore not calculated for tumor DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Overview of the detection of partial 1p and 19q losses using MLPA kit P088. A: Partial deletions as detected by MLPA kit P088. Legends are as described for Figure 2. Partial deletions as detected by CGH are listed on the left. Red and green boxes represent probe ratios 1.2. Additionally, yellow and light green boxes represent regions that, based on the relative low/high ratios and ratios of adjacent probes, were considered to be lost or gained even though the threshold (0.8 and 1.2) was not reached. The total deleted or gained regions as detected by this method are boxed in black. B: An example of comparison of conventional CGH analysis (left) and MLPA analysis using kit P088 (right) both identifying a partial deletion on 1p and 19q (case N182). The partial deletions detected in case N182 by CGH involve 1p11-31 and 19q13.2-qter, whereas MLPA detects a loss on 1p from NRAS (1p13.1) to LPHN2 (1p31.1) and a loss on 19q from ZNF342 (19q13.32) to BC2 (19q13.43); in addition, MLPA analysis shows a partial gain on 19q, and this gain is also indicated by CGH, but here the threshold is not crossed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Results of MLPA analysis of gliomas using kit P105 (for EGFR, CDKN2A, PTEN, TP53, and ERBB2). See also legend in Figure 2. The probe names (gene name and exon analyzed are indicated) are listed; probe CDKN2A prom is located in the promotor region, CDKN2A intA is located 0.5 kb before the start of p14ARF exon 1, CDKN2A intB is located between p16 exon 1 and p14 exon 1, whereas ERBB2 1 and ERBB2 2 represent the 142-bp and 409-bp fragment of ERBB2 2, respectively (exact location not provided). CGH results are provided for chromosomal regions on which the genes are located [7p12 (EGFR), 9p21 (CDKN2A), 10q23.3 (PTEN), 17p13.1 (TP53) and 17q21.1 (ERBB2)]. Abbreviations used for CGH imbalances are as in Figure 2; the following symbols are also used: −, CGH ratio of ∼0.6 suggesting the presence of a homozygous deletion, ++, high copy amplification as indicated by a CGH ratio greater than 1.4. Red and green boxes represent probe ratios less than 0.8 and more than 1.2. Additionally, dark green boxes represent MLPA ratios more than 2.0 indicating high copy number amplifications, whereas pink boxes represent ratios less than 0.4, which may indicate homozygous deletions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) MLPA was performed as described by the manufacturer with minor modifications. Briefly, DNA (250 to 450 ng) was dissolved in 5 μl of TE-buffer (10 mmol/L Tris, pH 8.2, 1 mmol/L ethylenediaminetetraacetic acid, pH 8.0) or Milli-Q water, denatured, and subsequently cooled to 25°C. After adding the probe mix, the sample was denatured, and the probes were allowed to hybridize (16 hours at 60°C). After ligation of both probe pairs and inactivation of ligase, PCR was performed in a volume of 50 μl containing 10 μl of the ligation reaction mixture using the PTC 200 thermal cycler (MJ Research Inc., Waltham, MA) (33 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute with a final extension of 20 minutes at 72°C). We included an additional agarose gel electrophoresis to examine MLPA efficiency (Figure 1). Fragments were separated and quantified by electrophoresis on an ABI 3730 capillary sequencer (Applied Biosystems, Foster City, CA) and Genemapper analysis (Applied Biosystems).34van Dijk MC Rombout PD Boots-Sprenger SH Straatman H Bernsen MR Ruiter DJ Jeuken JW Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.Diagn Mol Pathol. 2005; 14: 9-16Crossref PubMed Scopus (57) Google Scholar In each set of MLPA experiments, in addition to the tumor samples to be analyzed, we included at least four normal control DNA samples for data processing. Reference DNAs were isolated either from blood of healthy volunteers or from normal (nontumorous) paraffin-embedded samples for use in MLPA analysis of tumor DNAs from frozen or paraffin-embedded tissue, respectively.34van Dijk MC Rombout PD Boots-Sprenger SH Straatman H Bernsen MR Ruiter DJ Jeuken JW Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.Diagn Mol Pathol. 2005; 14: 9-16Crossref PubMed Scopus (57) Google Scholar Data analysis was performed in Excel as described by the manufacturer (MRC-Holland). First, the fraction of each peak was calculated by dividing the peak value (peak height or area) of each probe amplification product by the combined value of the control probes within the sample, to compensate for PCR efficiency of the individual samples. Subsequently this relative peak value or so-called probe fraction is divided by the mean probe fraction of this fragment within the included reference DNAs, generating the normalized peak value or the so-called probe ratio. For quality control, five control peaks were included in each reaction: four control fragments generating amplification products of 64, 70, 76, and 82 bp if the DNA quality (amount or purity) is insufficient, and a fifth peak showing an amplification product of 94 bp that is indicative for hybridization and ligation efficiency. For these quality control fragments, the relative peak values are calculated (peak value/average peak value of the control probes within a sample) to identify unreliable experiments (Table 1). Based on our previous experience with MLPA kit P00534van Dijk MC Rombout PD Boots-Sprenger SH Straatman H Bernsen MR Ruiter DJ Jeuken JW Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.Diagn M
Referência(s)