MGMT promoter = hypermethylation is a frequent, early, and consistent event in = astrocytoma progression, and not correlated with TP53 mutation

Laboratory Investigation - Human/Animal Tissue

MGMT promoter = hypermethylation is a frequent, early, and consistent event in = astrocytoma=20 progression, and not correlated with TP53 mutation

Floris H. Groenendijk^{1, 2},=20 Walter Taal³, = Hendrikus J. Dubbink¹,=20 Cathleen R. Haarloo¹,=20 Mathilde C. Kouwenhoven^{1, 3}, = Martin J. van=20 den Bent³, Johan M. Kros¹ and=20 Winand N. M. Dinjens¹

(1)

Department of Pathology, Josephine = Nefkens=20 Institute, Be320a, Erasmus MC, University Medical Center = Rotterdam, P.O.=20 Box 2040, 3000 CA Rotterdam, The=20 Netherlands

(2)

Faculty of Health, Medicine and Life = Sciences,=20 University of Maastricht, P.O. Box 616, 6200 MD Maastricht, = The=20 Netherlands

(3)

Department of = Neuro-Oncology/Neurology, Daniel=20 den Hoed Cancer Center, Erasmus MC, University Medical Center, = P.O. Box=20 2040, 3000 CA Rotterdam, The = Netherlands

Winand N. M. = ;Dinjens
Email:=20 w.dinjens@erasmusmc.nl

Received:=20 23 December 2009 Accepted:=20 16 June 2010 Published online:=20 1 July 2010

Abstract

Hypermethylation of the MGMT gene promoter = and mutation=20 of the TP53 tumor-suppressor gene are frequently present in = diffuse=20 astrocytomas. However, there is only anecdotal information about = MGMT=20 methylation status and TP53 mutations during progression of = low-grade=20 diffuse astrocytoma (AII) to anaplastic astrocytoma (AIII) and secondary = glioblastoma (sGB). In this study biopsy specimens from 51 patients with = astrocytic tumors with radiologically proved progression from low to = high-grade=20 malignancy were investigated for the presence and consistency of = MGMT=20 promoter hypermethylation and TP53 mutations. For 27 patients = biopsy=20 samples both of primary tumors and their recurrences were available. For = the=20 other 24 patients histology of either the low-grade lesion or the = high-grade=20 recurrence was available. It was found that MGMT promoter=20 hypermethylation and TP53 mutations are both frequent and early = events in=20 the progression of astrocytomas and that their status is consistent over = time.=20 No correlation was found between MGMT methylation status and the = presence=20 of TP53 mutations. In addition, no correlation was found between=20 MGMT promoter hypermethylation and the type of TP53 = mutations.=20 These results argue against the putative TP53 G:C>A:T = transition=20 mutations suggested to occur preferentially in MGMT = hypermethylated=20 tumors.

Keywords Diffuse astrocytoma = WHO grade=20 II - Anaplastic astrocytoma WHO grade III - = TP53=20 mutation - MGMT promoter=20 hypermethylation - G:C>A:T transition mutations

Johan M. Kros and Winand = N. M. Dinjens=20 contributed equally to this study.

Introduction

Low-grade diffuse astrocytomas (WHO grade II; AII) are = slowly=20 growing tumors with a peak incidence in young adults. They diffusely = infiltrate=20 the normal brain and have an intrinsic tendency to progress to greater=20 malignancy, i.e. anaplastic astrocytoma (WHO grade III; AIII) and = secondary=20 glioblastoma (WHO grade IV; sGB). Currently available treatment = strategies, for=20 example tumor resection, radiotherapy, and/or chemotherapy are only = partially=20 effective and most patients develop recurrent or progressive disease. = For=20 development of new treatment strategies, identification of genetic = alterations=20 associated with pathogenesis, progression, and treatment response of = these=20 slow-growing tumors is necessary.

The tumor-suppressor gene TP53 on chromosome = 17p13.1=20 encodes a key transcription factor involved in several cellular = mechanisms=20 including growth arrest, DNA repair, and induction of apoptosis. = Mutations in=20 the TP53 gene have been reported as early and frequent events in = sGB and=20 their precursor lesions [1,=20 2]=20 whereas de-novo primary glioblastomas reportedly lack TP53 = mutations or=20 acquire them late in tumorigenesis [3,=20 4].=20 However, the presence of TP53 mutations in the progression of AII = to AIII=20 and sGB has mostly been determined in investigations of series of = astrocytic=20 tumors with different grades of malignancy without following individual = tumors=20 for their tumorigenesis [5,=20 6].=20 In some but not all studies on AII, the TP53 mutation was found = to be an=20 independent unfavorable predictor of survival and/or malignant = transformation=20 [3,=20 7=969].=20 Other genetic aberrations in the TP53 pathway, for example = MDM2=20 amplification and p14 ^ARFdeletion or = hypermethylation=20 are also frequently observed in low-grade diffuse astrocytomas [7,=20 10,=20 11].=20

Over the last decade, epigenetic silencing of the=20 O⁶-methylguanine-methyltransferase (MGMT) gene by = promoter=20 hypermethylation has been found to be associated with chemosensitivity = of a=20 variety of tumor types, including gliomas [12=9616].=20 The MGMT gene is located on chromosome band 10q26 and encodes for = O⁶-alkylguanine-DNA-alkyltransferase (AGT), a repair enzyme = that=20 removes promutagenic alkylating DNA adducts from the O⁶ = position of=20 guanine, an important target of alkylating and methylating agents. Lack = of=20 MGMT repair capacity contributes to the genesis and progression = of human=20 cancers, because it leads to the accumulation of DNA mutations and = chromosomal=20 instability [12,=20 14,=20 17].=20 During chemotherapy with alkylating and methylating agents the primary = cell=20 mechanism inducing chemoresistence is the demethylating activity of AGT, = which=20 removes the alkyl adducts. The absence of MGMT activity can = therefore be=20 used as a predictor of response for patients treated with alkylating=20 chemotherapeutic agents. High-grade gliomas, in particular = glioblastomas, with=20 hypermethylated MGMT promoters proved to be more sensitive to=20 chemotherapeutic agents, including temozolomide (TMZ), resulting in an = overall=20 survival benefit for these patients [13,=20 15,=20 18=9620].=20

Apart from the predictive value, the MGMT = promoter=20 hypermethylation status has been associated with shorter = progression-free=20 survival in AII [21,=20 22].=20 In several reports an association between MGMT promoter = hypermethylation=20 and an increased frequency of TP53 mutations, with predominance = of G>A=20 or C>T transitions (G:C>A:T transition mutations), especially at = CpG=20 sites, has been mentioned [11,=20 21,=20 23=9625].=20 Further, in a recent population-based study of glioblastomas a higher = frequency=20 of TP53 G:C>A:T transition mutations in tumors with = MGMT=20 promoter hypermethylation (25%) than in glioblastomas without = MGMT=20 methylation (16%, P =3D 0.0385) was reported [26].=20 However, information about MGMT promoter hypermethylation or = TP53=20 mutation during astrocytoma progression is sparse [11,=20 23].=20 The objective of this study was to investigate the association of = MGMT=20 promoter methylation status and TP53 mutations and to determine = the=20 consistency of these molecular characteristics over time, in a series of = diffuse=20 astrocytomas and their recurrences.

Materials and methods

Patients and tumor samples

Archival routine formalin-fixed and paraffin-embedded = (FFPE)=20 tumor tissues were collected from 51 patients diagnosed with:

(1)	an AII at the time of the first operation;=20 or
(2)	a histopathology of high-grade astrocytic = tumor after a=20 wait and see period of at least 1.5 years and a lesion on MR = scan=20 without contrast enhancement (clinically regarded as an original = low-grade=20 astrocytic tumor).

Glioma recurrences were diagnosed by contrast = enhancement on=20 MR scans, with or without clinical symptoms, and/or histopathology of = tissue=20 biopsies. For 27 patients tumor tissue was available both from the first = operation and from the operation for the recurrence. For 17 patients = only tumor=20 tissue from the first operation was available and for seven patients = only tissue=20 from the second surgery was available (Table 1).=20 Hematoxylin and eosin (H&E)-stained tissue sections from all tumors = were=20 reviewed by an independent neuropathologist (JMK) in accordance with the = latest=20 WHO guidelines for typing and grading [27].

Table 1 Results from=20 histopathological review and molecular analysis

Case no.	Sex	Age	P/R	Interval (months)	Intervening treatment	Histology	p53 expression % of cells	Mutation analysis of TP53	LOH of the TP53 locus ^b =	MGMT status
Primary only (n =3D 17) =
1	M	39	P			AII	80	Exon 6, c.13419A>G, p.Tyr220Cys	Yes	Hypermethylated
2	F	29	P			AII	60	Exon 4, c.12243_12260dupTACGGTTTCCGTCTGGGC,=20 p.Tyr107_Gly112dup	No	Hypermethylated
3	F	39	P			AIII	60	Exon 4, c.12253G>T, p.ArgR110Leu/Exon 8, = c.14486C>T,=20 p.Arg273Cys	No	Hypermethylated
4	M	38	P			AII	60	Exon 5, c.13107T>G, p.Val143Gly	Yes	Hypermethylated
5	F	40	P			AII	30	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
6	M	38	P			AII	40	Exon 8, c.14496C>A, p.Ala276Asp	Yes	Hypermethylated
7	M	47	P			AII	30	Exon 8, c.14470_14475dupAACAGC, = p.Asn268_Ser269dup	No	Hypermethylated
8	M	51	P			AII	Neg.	Exon 5, c.13229delG, p.Asp184 fs	No	Hypermethylated
9	F	33	P			AII	30	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
10	M	32	P			AIII	20	No data	Yes	Hypermethylated
11	F	34	P			AII	70	No data	Yes	No data
12	M	36	P			AII	20	Exon 8, c.14487G>A, p.Arg273His	No	Hypermethylated
13	F	59	P			AII	60	Wt	Yes	Unmethylated
14	M	25	P			AII	>10	Exon 5, c.13203G>A, p.Arg175His/Exon 8, = c.14513C>T,=20 p.Arg282Trp	No	Hypermethylated
15	M	38	P			AIII	80	Exon 6, c.13329C>G, p.Pro190Arg	Yes	Hypermethylated
16	M	43	P			AII	80	Wt	Yes	Hypermethylated
17	M	39	P			AII	50	Exon 7, c.14061G>C, p.Gly245Ala	Yes	Unmethylated
Primary and recurrence = (n =3D 27)=20
18^a	M	35	P	4	RT	AIII	30	Exon 5, c.13107T>G, p.Val143Gly	Yes (reverse loss)	Hypermethylated
R	GBM	40	Wt	Unmethylated
19	F	49	P	8	RT	AIII	40	Exon 4, c.12253G>C, p.ArgR110Pro/Exon 5, = c.13152G>A,=20 p.Arg158His	No	Hypermethylated
R	GBM	30	Exon 4, c.12253G>C, p.ArgR110Pro/Exon 5, = c.13152G>A,=20 p.Arg158His	No	Hypermethylated
20	M	41	P	12	RT	GBM	>80	Exon 5, c.13152G>C, p.Arg158Pro	No	Unmethylated
R	GBM	>80	Exon 5, c.13152G>C, p.Arg158Pro	Yes	Unmethylated
21	F	48	P	12	RT + PCV	AII	<10	Wt	No	Hypermethylated
R	GBM	80	Wt	No	Hypermethylated
22	M	34	P	13	RT + PCV	AII	20	Exon 5, c.13149T>G, p.Val157Gly	Yes	Unmethylated
R	AII	70	Exon 5, c.13149T>G, p.Val157Gly	Yes	Unmethylated
23	F	56	P	17	RT	GBM	>80	Exon 5, c.13203G>A, p.Arg175His	No	Unmethylated
R	GBM	>80	Exon 5, c.13203G>A, p.Arg175His	No	Unmethylated
24	F	36	P	18	RT	AII	60	No data	Yes	No data
R	AII	60	No data	Yes	No data
25	M	54	P	23	RT	AIII	>80	Exon 5, c.13203G>A, p.Arg175His	Yes	Hypermethylated
R	GBM	>90	Exon 5, c.13203G>A, p.Arg175His	Yes	Hypermethylated
26	F	31	P	23	RT	AII	>80	Wt	Yes	Hypermethylated
R	GBM	>80	Wt	Yes	Hypermethylated
27	F	55	P	25	RT	AII	<10	Wt	No	Unmethylated
R	AII	<10	Wt	No	Unmethylated
28	M	44	P	25	RT	AII	>10	Exon 7, c.14057G>T, p.Gly244Cys	Yes	Unmethylated
R	AIII	>80	Exon 7, c.14057G>T, p.Gly244Cys	Yes	Unmethylated
29	M	31	P	26	RT + PCV	AII	20	Exon 5, c.13107T>G, p.Val143Gly	Yes	Hypermethylated
R	OIII	>90	Exon 5, c.13107T>G, p.Val143Gly	Yes	Hypermethylated
30	F	34	P	37	RT	OAIII ^c	70	Exon 6, c.13419A>G, p.Tyr220Cys/Exon 8, = c.14487G>A,=20 p.Arg273His	No	Unmethylated
R	OAII ^c	60	Exon 6, c.13419A>G, p.Tyr220Cys/Exon 8, = c.14487G>A,=20 p.Arg273His	No	Unmethylated
31	M	28	P	37	RT	AII	30	Exon 6, c.13419A>G, p.Tyr220Cys	Yes	No data
R	AIII	80	Exon 6, c.13419A>G, p.Tyr220Cys	Yes	Unmethylated
32	M	55	P	40	RT + PCV	AII	Neg.	Exon 5, c.13157_13161delATGGC, = p.Met160 fs	Yes	Unmethylated
R	GBM	Neg.	Exon 5, c.13157_13161delATGGC, = p.Met160 fs	Yes	Unmethylated
33	M	36	P	43	RT	AII	30	Exon 8, c.14487G>A, p.Arg273His	Yes	Hypermethylated
R	AIII	>80	Exon 8, c.14487G>A, p.Arg273His	Yes	Unmethylated
34^a	F	29	P	43	RT	AII	20	Exon 7, c.14063A>G, p.Met246Val	Yes	Unmethylated
R	GBM	60	Exon 8, c.14483G>T, p.Val272Leu	Yes	Hypermethylated
35	F	34	P	47	RT	AII	30	Exon 5, c.13197T>A, p.Val173Glu/Exon 7, = c.14070G>A,=20 p.Arg248Gln	No	Hypermethylated
R	GBM	>80	Exon 5, c.13197T>A, p.Val173Glu/Exon 7, = c.14070G>A,=20 p.Arg248Gln	No	Hypermethylated
36^a	F	33	P	56	RT	AII	30	Wt	Yes (reverse loss)	No data
R	GBM	>80	Exon 6, c.13419A>G, p.Tyr220Cys	Unmethylated
37	F	39	P	62	RT	AIII	60	Exon 5, c.13056A>G, p.Tyr126Cys	No	Hypermethylated
R	OAIII	60	Exon 5, c.13056A>G, p.Tyr126Cys	No	Hypermethylated
38	M	39	P	73	RT	AII	>10	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
R	OAIII	80	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Hypermethylated
39	M	41	P	74	RT	AII	50	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
R	AII	80	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
40	M	55	P	85	RT	AII	<10	Wt	No	Hypermethylated
R	GBM	80	Wt	Yes	Hypermethylated
41	F	37	P	95	RT	AII	60	Wt	Yes	Hypermethylated
R	GBM	80	Wt	Yes	No data
42	F	38	P	96	RT	AII	80	Exon 6, c.13418T>C, p.Tyr220His	Yes	Hypermethylated
R	AII	>10	Exon 6, c.13418T>C, p.Tyr220His	Yes	Hypermethylated
43	M	46	P	146	RT	AII	30	Wt	No	Unmethylated
R	AII	30	Exon 9, c.14719_14735dupAAGAAACCACTGG,=20 p.Glu326 fs	Yes	Unmethylated
44^a	M	37	P	160	RT	AII	80	Exon 9, c.14686C>T, p.Pro309Ser	Yes (reverse loss)	Unmethylated
R	AII	60	Exon 9, c.14710C>T, p.Gln317Stop	Unmethylated
Recurrence only (n =3D 7) =
45	M	43	R			AII	<10	Wt	No	Unmethylated
46	M	43	R			AII	20	Wt	No	Hypermethylated
47	M	27	R			GBM	>90	Exon 5, c.13203G>A, p.Arg175His	Yes	Hypermethylated
48	M	32	R			AIII	70	Exon 8, c.14486C>T, p.Arg273Cys	Yes	Unmethylated
49	M	49	R			GBM	>80	Exon 8, c.14486C>T, p.Arg273Cys	No	Hypermethylated
50	F	44	R			AII	20	Wt	No	No data
51	F	37	R			GBM	>10	Exon 5, c.13203G>A, p.Arg175His	Yes	No data

Summary of results from 51 patients diagnosed with a = recurrent=20 astrocytic tumor with a clinical history of primary diffuse astrocytoma = (WHO=20 grade II)

For 27 patients tumor specimens from primary and = recurrent=20 tumor were available; for 17 patients only biopsy material from the = first=20 operation was available; and for seven patients only material from the = second=20 surgery was available. After molecular investigation of the 27 cases = with paired=20 primary tumors and recurrences (cases 18=9644), four cases with = different=20 TP53 mutations and/or LOH pattern were considered as two, = clonally=20 independent, entities (cases 18, 34, 36, and 44)

P, primary; R, recurrence; RT, radiotherapy; PCV,=20 procarbazine, lomustine, and vincristine; AII, low-grade astrocytoma WHO = grade=20 II; AIII, anaplastic astrocytoma WHO grade III; GBM, glioblastoma = multiforme;=20 OIII, anaplastic oligodendroglioma; OAII, oligo-astrocytoma; OAIII, = anaplastic=20 oligo-astrocytoma; Wt, wild-type

^aCases with two clonally unrelated = tumors, on the=20 basis of the molecular results

^bPaired tumors both yes: identical LOH = pattern=20

^cThe histology of the recurrence seemed = to be=20 indicative of lower malignancy grade than the primary tumor; this is = attributed=20 to sampling error in the recurrence (case 30) =

All patients received radiation therapy after surgery = for the=20 primary tumor. Four of the 27 patients (Table 1;=20 cases 21, 22, 29, and 32) were treated with alkylating chemotherapy=20 (procarbazine, lomustine, and vincristine) between the operations for = the=20 primary and recurrent tumors. All patients were treated with = temozolomide after=20 the operation for the recurrent tumor. By the end of this study all = patients had=20 died. The median follow-up time was 65.5 months.

The archival tissue samples were used in accordance = with the=20 code of conduct for appropriate secondary use of tissue: =93Proper = Secondary Use=20 of Human Tissue=94 established by the Dutch Federation of Medical = Scientific=20 Societies (http://www.federa.org/).=20

DNA extraction

H&E-stained sections from the available FFPE = tissue blocks=20 were used by the neuropathologist (JMK) to select parts for DNA = extraction. DNA=20 was isolated by standard procedures from selected tissue fragments = containing a=20 high percentage tumor cells and, when possible, from fragments of normal = tissue=20 [28].=20

Methylation-specific multiplex ligation-dependent = probe=20 amplification (MS-MLPA) for MGMT

The MS-MLPA probe mix used (ME011; MRC Holland, = Amsterdam, The=20 Netherlands) includes 21 probe sequences containing an HhaI = recognition=20 site, which yields information about the methylation status of the = target=20 sequences. In addition, eight control probe sequences not affected by = the=20 methylation-sensitive restriction enzyme HhaI digestion are used. = The=20 probes containing an HhaI recognition site should only generate a = signal=20 if the DNA target is methylated and cannot be digested. The probe mix = was=20 developed to detect CpG island methylation of six mismatch repair genes = and=20 includes three specific probes for semiquantitative hypermethylation = detection=20 of the MGMT promoter region. This MS-MLPA assay for MGMT = promoter=20 hypermethylation of gliomas was performed as described by Jeuken et al., = with=20 minor modifications [29].=20 For fragment analysis, PCR products were separated by capillary gel=20 electrophoresis (ABI Prism 3130xl; Applied Biosystems, Foster City, CA, = USA) and=20 quantified by use of GeneMarker software version 1.7 (SoftGenetics, = State=20 College, PA, USA).

MS-MLPA data analysis

The MS-MLPA results were normalized by dividing the = peak height=20 for each MGMT probe signal by the mean peak height for the eight = control=20 fragments within the same sample. To estimate the fraction of methylated = MGMT promoter DNA, normalized values of each MGMT probe of = digested DNA samples were divided by normalized values of corresponding=20 undigested DNAs. Methylation analyses were performed in duplicate and = the=20 average ratios of both experiments for each probe were calculated. For = the=20 promoter hypermethylation detection of MGMT, we used the = MGMT=20 ₂probe from the probe mix only. This probe is located = within=20 the widely used CpG island region for MGMT promoter = hypermethylation=20 detection by methylation-specific PCR (MS-PCR), and hypermethylation of = this=20 region has been found to be an independent predictor of the response of = gliomas=20 to TMZ [13,=20 15,=20 19,=20 20].=20

Preliminary comparison of the widely used MS-PCR and = the=20 MGMT ₂probe analyses demonstrated concordant = results=20 (not shown). Also, in our hands, the MGMT _{2 =}probe also=20 resulted in better assay reproducibility (Spearman=92s rho 0.658,=20 P < 0.001) than the two other MGMT probes=20 (MGMT ₁probe Spearman=92s rho 0.448;=20 P =3D 0.032 and MGMT ₃probe = Spearman=92s=20 rho 0.431; P =3D 0.040). Cell lines were used as = positive (TE-4,=20 OEC33, FLO-1, SW620) or negative (TE-1, TE-5, TE-13, TE-14, SK-GT-2, = BE-3,=20 ESO51) controls for evaluation of MGMT promoter methylation. When = the=20 MGMT digested/MGMT undigested ratio of probe MGMT=20 ₂is below 0.30 the MGMT promoter fragment is = regarded=20 as non-methylated, as described previously for MS-MLPA assays [30,=20 31].=20

Mutation analysis of TP53

Tumor samples from each patient were screened for = TP53=20 mutations in exons 4=969, including intron=96exon boundaries, by = polymerase chain=20 reaction (PCR) followed by bi-directional direct DNA sequencing using = the=20 M13-tailed primer method. Intronic primer sets for PCR amplification are = shown=20 in Table 2A.=20 PCR products were generated in a 15-μl reaction mixture containing = 1.0 μl=20 DNA solution, 10 μmol of each primer, 25 mM = MgCl₂,=20 10 mM dNTPs,=20 and 1U Taq polymerase (Promega, Madison, WI, USA). The PCR reaction was=20 performed using a thermocycler (Biometra, G=F6ttingen, Germany) with an = initial=20 denaturing step (95=B0C) for 3 min, followed by 35 cycles = consisting of=20 denaturing (95=B0C) for 30 s, annealing (60=B0C) for 45 s, and = extension=20 (72=B0C) for 45 s. After the final cycle, an extension period of = 10 min=20 at 72=B0C was performed. The PCR products (5 μl) were digested = with 1 μl=20 exonuclease I (ExoSAP-IT; USB, Cleveland, OH, USA) at 37=B0C for = 30 min=20 followed by inactivation of the enzyme at 80=B0C for 15 min. The = PCR products=20 were sequenced on an ABI Prism 3130xl genetic analyzer using the ABI = Prism=20 BigDye Terminator v3.1 kit (both from Applied Biosystems). = Samples were=20 analyzed by use of Mutation Surveyor software package version 3.2 = (SoftGenetics,=20 State College, PA, USA) and compared with the public sequence in GenBank = (NM_000546).

Table 2 Primer sets=20 for TP53 mutation analysis and LOH analysis of the TP53 = Locus=20

Exon	Size (bp)^a	Forward primer	Reverse primer
A Nucleotide sequences of primers used = for=20 TP53 mutation analysis
Exon 4A	300	5′-CTGGTAAGGACAAGGGTTGG-3′	5′-GATGACAGGGGCCAGGAG-3′
Exon 4B	249	5′-AGATGAAGCTCCCAGAATGC-3′	5′-GATACGGCCAGGCATTGAAG-3′
Exon 5A	222	5′-TGCTGCCGTCTTCCAGTTGC-3′	5′-CTCACAACCTCCGTCATGTG-3′
Exon 5B	197	5′-CAGCTGTGGGTTGATTCCAC-3′	5′-TGAGGAATCAGAGGCCTG-3′
Exon 6	263	5′-TCAGATAGCGATGGTGAGCA-3′	5′-GCCACTGACAACCACCCTTA-3′
Exon 7	215	5′-CGCACTGGCCTCATCTTG-3′	5′-AGGGGTCAGAGGCAAGCAGA-3′
Exon 8	248	5′-GGGACAGGTAGGACCTGATTT-3′	5′-GCATAACTGCACCCTTGGTC-3′
Exon 9	232	5′-GGAGACCAAGGGTGCAGTTA-3′	5′-CCCCAATTGCAGGTAAAACA-3′
B Nucleotide sequences of primers used = for LOH=20 analysis of the TP53 Locus
D17S520	142	5′-GGAGAAAGTGATACAAGGGA-3′	5′-TAGTTAGATTAATACCCACC-3′
D17S786	167	5′-TACAGGGATAGGTAGCCGAG-3′	5′-GCTGTGAGTCCCAAAAGAGG-3′
D17S1353	122	5′-ATTCCCACTGCCACTCCTTG-3′	5′-CAGCTGAGGGATACTATTCAG-3′
D17S1566	189	5′-AAAGATCCTTATTGCCACTTTACTG-3′	5′-CTCTTACCTTGCTGGTGAGATTG-3′
D17S1866	175	5′-TGGATTCTGTAGTCCCAGG-3′	5′-GGTTCAAAGACAACTCCCC-3′

^aAmplicon sizes based on the UCSC Genome = Browser=20 (http://genome.ucsc.edu/)=20

Loss of heterozygosity (LOH) analysis of the = TP53=20 locus (17p13)

Five highly polymorphic microsatellite markers located = on=20 chromosome 17p13 (D17S1353, D17S1866, D17S1566, D17S786, and D17S520) = were used=20 for LOH analysis. Microsatellites were amplified with = fluorescence-labeled=20 forward and unlabeled reverse primers (Table 2B).=20 Because normal DNA from non-neoplastic tissues was not available for = most of the=20 tumor samples, allelic losses were determined by analysis of the allelic = patterns and by integration of data from multiple highly polymorphic = markers, as=20 described elsewhere [32].=20 LOH analysis was performed using the ABI Prism 3130xl Genetic Analyzer = (Applied=20 Biosystems) and the GeneMarker software package version 1.7 = (SoftGenetics).=20

Immunohistochemistry (IHC) for p53

IHC for p53 was performed on 4-μm sections of = routine FFPE=20 tissues. Briefly, sections were deparaffinized in xylene and hydrated = through=20 descending ethanol gradients. Endogenous peroxidase was inactivated by = treatment=20 with 3% H₂O₂ in phosphate-buffered saline (PBS). = Antigen=20 retrieval was accomplished by boiling the sections for 15 min in=20 10 mmol/l Tris=96EDTA, pH 9.0 in a microwave oven. The = sections were=20 incubated for 1 h at room temperature with mouse monoclonal = antibody DO-7=20 against p53 protein (Dako, Glostrup, Denmark) at 1:50 dilution. After = rinsing in=20 PBS, immunoreactivity was visualized using the Envision kit (Dako). The = sections=20 were subsequently counterstained with Mayer hematoxylin. Although the = DO-7=20 antibody binds to both normal and mutant p53 protein, in general, normal = levels=20 of wild-type p53 protein are too low to be detected by = immunohistochemistry. In=20 each section, the number of clearly positive cells out of 1000 cells was = counted=20 and >10% was regarded as positive [33].=20

Statistical analysis

Correlation coefficients were calculated by use of = Student=92s=20 t-test and Fisher=92s exact test. Data were regarded as = statistically=20 significant at P < 0.05.

Results

The results from histopathological review and = molecular analysis=20 are summarized in Table 1.=20 After molecular investigation of the 27 cases of paired primary tumors = and=20 recurrences (Table 1,=20 cases 18=9644) four cases with different TP53 mutations and/or = reverse LOH=20 patterns were regarded as two clonally independent entities (cases 18, = 34, 36,=20 and 44). The strongest indication of clonally independent entities is = the=20 finding of specific molecular aberrations in the primary tumors which = are not=20 found in the recurrences. In case 43 neither a TP53 mutation nor = LOH was=20 found in the primary tumor whereas in the recurrence a TP53 = mutation and=20 LOH were detected. This could indicate clonally independent tumors. = However,=20 because no molecular aberrations were detected in the primary tumor and = because=20 of the long period of time between occurrence of the primary lesion and=20 recurrence (146 months) we regard case 43 as more likely to be = clonally=20 related tumors with generation and clonal selection of the aberrations = during=20 the long time interval between both tumors.

Patients and tumor histology

According to central review, 34 of 44 (77%) primary = tumors were=20 diffuse astrocytomas of WHO grade II (AII); 6 of 44 (14%) were = anaplastic=20 astrocytomas (AIII), and 3 of 44 (7%) were glioblastoma (GB). One = primary tumor=20 was diagnosed as an anaplastic mixed oligoastrocytoma (OAIII). Of the = recurrent=20 tumors 10 of 34 (29%) were still AII; 5 of 34 (15%) had progressed to = AIII; and=20 15 of 34 (44%) became secondary glioblastomas (sGBs). Two recurrent = tumors were=20 diagnosed as anaplastic mixed oligoastrocytomas (OAIII), one as = anaplastic=20 oligodendroglioma (OIII), and one as low-grade oligoastrocytoma (OAII). = In all=20 tumors with an oligodendroglial component found by central review, loss = of=20 chromosome 1p and/or 19q was excluded in these cases (results not = shown).

Of the 27 cases of which both the primary and = recurrent tumor=20 were investigated, 23 cases were regarded as having clonally related = tumors and=20 four cases clonally unrelated tumors, on the basis of molecular results. = Thirteen of 23 (57%) clonally related tumor pairs showed histological=20 progression of malignancy grade. In nine cases (39%) the tumor histology = had not=20 progressed in malignancy grade. In one case the histology of the = recurrence=20 seemed to indicate lower malignancy grade than the primary tumor; this = was=20 attributed to sampling error in the recurrence (Table 1,=20 case 30). For the 23 paired, clonally related histological samples the = mean=20 interval between surgery for the primary tumor and recurrence was=20 45.1 =B1 35.2 months (range 8=96146 months). The = mean interval=20 between surgery for primary and recurrent tumors of those cases in which = the=20 histology of the first biopsy had not progressed in malignancy grade was = 50.0 months; for the tumors in which histological progression had = occurred=20 the mean interval was 41.3 months (Student=92s t-test,=20 P =3D 0.57).

Hypermethylation analysis of the MGMT = promoter

Seventy-eight tumor samples were available for = MGMT=20 promoter hypermethylation and TP53 mutation analysis (viz.: 21 = samples=20 from patients for whom only the first specimens were available; 54 = samples from=20 27 patients for whom both the first and second surgery tissue samples = were=20 available; and three samples from patients for whom only the second = tumor=20 specimens were available). Results showing MGMT promoter = hypermethylation=20 by MS-MLPA were obtained for 70 of 78 (90%) of the (routine FFPE) tumor = tissues=20 (Table 1).=20 MGMT promoter hypermethylation was identified in 39 of these 70 = (56%)=20 tumor samples. There was no relationship between tumor grade (viz., AII, = AIII or=20 sGB) and methylation status (53% vs. 64% vs. 56% hypermethylated, = respectively).=20 For 20 of 23 clonally related tumor pairs MGMT promoter = hypermethylation=20 results were obtained from both tumors. In 18 of 20 cases (90%), primary = and=20 recurrent tumors had a similar MGMT promoter methylation status = (nine=20 tumor pairs hypermethylated; nine tumor pairs not methylated). For two = pairs of=20 related tumors a change of MGMT methylation status between the = primary=20 and the recurrent tumors was found. There was no relationship between = change in=20 MGMT methylation status and intervening treatment with alkylating = chemotherapy. Of the four cases with clonally independent tumors, one = had=20 different MGMT hypermethylation status, two had identical = MGMT=20 hypermethylation status, and for one case no data could be obtained from = the=20 second tumor.

TP53 mutation analysis and p53 expression =

TP53 exon 4=969 DNA sequence data were obtained = from 74 of=20 78 (95%) tumor samples. Four samples had inferior DNA quality resulting = in no=20 reliable sequencing data. TP53 mutation analysis revealed = mutations in 40=20 of the 52 (77%) independent cases. In 35 of these 40 (88%) independent = cases one=20 mutation, and in 5 of 40 (13%) cases two TP53 mutations within = the same=20 tumor sample were detected. TP53 mutations were detected in 28 of = 41=20 (68%) tumors with histological diagnosis of AII, in 10 of 10 (100%) = AIII, and in=20 13 of 18 (72%) GB (Table 1).=20 Mutation of TP53 was found in 17 of 23 (74%) tumor pairs (primary = tumors=20 and their recurrences) regarded as clonally related. In three of these = 17 tumor=20 pairs two different TP53 mutations in the same tumor sample were = detected=20 and these mutations all remained present in the recurrent tumors. Two = tumor=20 pairs had TP53 mutations in the recurrent tumors only, and in one = case=20 TP53 mutation was only found in the primary tumor. In two tumor = pairs the=20 mutation in the primary tumor differed from that in the recurrence. In = five=20 tumor pairs TP53 mutations were not found in either the primary = or=20 recurrent tumor, and in one tumor pair no sequence data were obtained. =

A total of 45 TP53 mutations were detected in = 40 of 55=20 independent tumors. Three mutations were found in TP53 exon 4, = fifteen in=20 exon 5, six in exon 6, four in exon 7, fourteen in exon 8, and three in = exon 9.=20 A not previously reported in-frame duplication of 18 nucleotides (codon = 107=96112)=20 in exon 4 was found in one patient (Table 1,=20 case 2). Of all the TP53 mutations identified in independent = cases, 20 of=20 45 (44%) were G:C>A:T transition mutations; of these, 18 of 20 (90%) = were=20 located at CpG sites. Of the 25 not G:C>A:T transition mutations only = three=20 mutations (12%) were located at CpG sites. The two G:C>A:T transition = mutations that were not located at CpG sites were found in exon 9, the = exon of=20 TP53 without CpG sites.

Positive immunohistochemistry for p53 was associated = with either=20 mutation of the TP53 gene (Fisher=92s exact test,=20 P =3D 0.02) or LOH of the TP53 locus (Fisher=92s = exact test,=20 P =3D 0.02) or both (Fisher=92s exact test,=20 P < 0.001). In five of eight tumor samples without = p53=20 expression neither TP53 mutation nor LOH was found. The remaining = three=20 samples without p53 expression had TP53 frameshift mutations and, = in=20 accordance with this type of mutation, p53 expression was absent. =

LOH analysis of the TP53 locus (17p13) =

All 78 tumor samples in the study were informative for = at least=20 two of five microsatellite markers on chromosome 17p13. In 38 of 48 = (78%) tumors=20 with a single TP53 mutation, LOH of the TP53 locus was = found. In=20 all tumors in which two TP53 mutations were found, no allelic = loss on=20 17p13 was observed. LOH of the TP53 locus was found in five out = of 13=20 (38%) AII without TP53 mutation and in four out of seven (57%) = recurrent=20 tumors without TP53 mutation. The allelic loss pattern was = different=20 between three tumor pairs in which LOH was found in the recurrent tumor = only.=20 Further, reverse allelic loss of the primary and recurrent tumor was = detected in=20 three patients. One of these had a different mutation in each tumor = whereas two=20 patients had a mutation in only one of the tumor pairs.

Correlation between MGMT promoter = hypermethylation=20 and TP53 mutations

There was no significant correlation between = MGMT=20 promoter hypermethylation and TP53 mutation (Fisher=92s exact = test,=20 P =3D 0.77; Table 3).=20 In addition, there was no significant correlation between MGMT = promoter=20 hypermethylation and the presence of G:C>A:T transition mutations in=20 TP53. G:C>A:T transition mutations were found in 10 of 21 = (48%)=20 independent tumors with a hypermethylated MGMT promoter and a = TP53=20 mutation, and in nine of 16 (56%) MGMT promoter unmethylated = tumors with=20 a TP53 mutation (Fisher=92s exact test, = P =3D 0.80;=20 Table 3).

Table 3 Correlation=20 between MGMT promoter hypermethylation status and TP53 = mutation=20 status

	MGMT hypermethylated=20 (n =3D 29)^a	MGMT not hypermethylated = (n =3D 20)=20	P value
TP53 mutated	21	16	0.77
G:C>A:T mutation	10	9	0.80
G>A transition	7	2
At CpG site	7	2
C>T transition	3	7
At CpG site	3	5
TP53 not mutated	7	4

^aCase 10: no data on TP53 mutation = could be=20 obtained

Discussion

In this study we demonstrated that MGMT = promoter=20 hypermethylation and TP53 aberrations are early and frequent = events in=20 the progression of astrocytomas and that both are largely consistent = over time.=20 In addition, we did not find a correlation between MGMT promoter=20 methylation status and TP53 mutations. These results argue = against the=20 putative TP53 G:C>A:T transition mutations which have been = suggested=20 to occur preferentially in MGMT hypermethylated tumors.

MGMT promoter hypermethylation

No data on the MGMT promoter methylation status = during=20 astrocytic tumor progression have yet been reported. In this study, we = showed=20 that MGMT promoter hypermethylation is not limited to high-grade=20 astrocytomas but should be regarded as an early and frequent event in = primary=20 AII and AIII, which remains present during tumor progression. = MGMT=20 promoter hypermethylation was found in 58% of primary AII, consistent = with=20 frequencies reported by Nakamara et al., Watanabe et al., and Nakasu et=20 al. [11,=20 23,=20 34],=20 who demonstrated MGMT promoter hypermethylation by MS-PCR or=20 immunohistochemistry in 48, 63, and 68% of AII, respectively.

Discrepancies between results from = immunohistochemistry and from=20 PCR experiments should be taken into consideration. We found concordance = in=20 MGMT promoter hypermethylation status between primary and = recurrent=20 tumors in 18 of 20 (90%) clonally related tumor pairs. The remaining two = cases=20 demonstrate that MGMT promoter hypermethylation may change during = astrocytoma progression, e.g., by reversibility of the hypermethylation, = clonal=20 selection of methylated or unmethylated subclones, or by loss of the=20 hypermethylated MGMT allele. No relationship between a change in=20 MGMT promoter methylation status and intervening treatment with=20 alkylating chemotherapy was revealed. Recently, Brandes et al. reported = their=20 study on the stability of MGMT methylation status in a series of = 38=20 patients with a glioblastoma and second surgery for recurrence. = MGMT=20 methylation status changed in 37% of the patients and more frequently in = methylated than in unmethylated tumors [35].=20

TP53 mutations

With regard to the analyses of molecular aberrations = in tumors=20 it must be considered that, because of the general procedures used, = technically=20 only aberrations present in most of the cells from which the DNA was = isolated=20 (i.e. clonal aberrations) will be detected. Tumors are composed of = neoplastic=20 and non-neoplastic (normal) cells and it is generally accepted that the=20 neoplastic cells, in contrast with normal cells, harbor clonal molecular = aberrations. DNA isolated from a part of the tumor is composed of the = DNA of the=20 neoplastic cells and the DNA of the normal cells. These facts imply that = when a=20 specific molecular aberration is reliably identified in the DNA sample = isolated=20 from the tumor, this DNA sample is retrieved from a part of the tumor = composed=20 of a high percentage of clonal neoplastic cells. Admixture of a high = percentage=20 of normal cells or the presence of heterogeneous (with regard to the = determined=20 aberration) neoplastic cell populations would have masked the aberration = and=20 prevented its detection.

The high percentage of mutations of TP53 (77%) = detected=20 in the astrocytic tumors in this study is in accordance with the highest = frequencies reported in the literature [1,=20 3,=20 6=968].=20 The presence or absence of nuclear p53 expression, determined by=20 immunohistochemistry, was highly significantly associated with the = presence or=20 absence of TP53 aberrations. Significant correlation between p53=20 expression, TP53 mutations, and TP53 locus LOH was shown = in other=20 studies [1=963,=20 8].=20 In 13 cases with high p53 expression and/or LOH of chromosome 17p13 no=20 TP53 mutations were detected. This may be the result of = intratumoral=20 heterogeneity of TP53 gene mutations [36]=20 or TP53 mutations escaping detection by our sequencing method = (especially=20 in cases without detected LOH, probably because of admixture of normal = DNA). Yet=20 another explanation may be that TP53 mutations were located = outside the=20 investigated region of the gene (especially in cases with LOH). In ten = cases a=20 single TP53 mutation was detected without concomitant TP53 = locus=20 LOH. It may well be that in these cases LOH was probably not identified = in the=20 tumor DNA because of too much admixture of normal DNA. In addition, = TP53=20 LOH could be absent because of the presence of a dominant-negative = TP53=20 mutation in the tumors without concomitant loss of the wild type allele. = In five=20 cases two TP53 mutations were found and in all eight tumor = samples from=20 these cases no LOH of the TP53 locus was observed, which is more=20 supportive of monoclonal tumor cell populations with bi-allelic = TP53=20 mutations than of dual clonality.

TP53 mutations are consistent during = astrocytoma=20 progression, because we demonstrate identical TP53 mutation and=20 TP53 locus LOH status in 21 of 27 investigated pairs of primary = tumor and=20 recurrence, indicating clonal relationships between these tumor pairs. = In two=20 cases TP53 aberrations were found in the tumor recurrences only. = It may=20 well be that the mutations were present in the first samples but not = detected=20 because the percentage of the mutation-carrying tumor cells was too low. = The=20 fact that in both primary tumors no TP53 LOH was observed and = only a low=20 percentage of p53 positive cells was seen also indicates the presence of = many=20 normal cells in these tumor specimens. In four of 27 (15%) patients = differences=20 in TP53 mutation and TP53 LOH between the primary tumors = and=20 recurrences were unequivocally found. (One of these cases, case 44, is=20 illustrated in Fig. 1)=20 Allelotyping excluded tissue admixture in these cases. The differences = between=20 the primary and recurrent tumors may well be the result of intratumoral=20 heterogeneity or alternatively, may be indicative of two, clonally = independent,=20 tumors. Intratumoral heterogeneity for TP53 aberrations has been=20 described previously for astrocytic brain tumors [36]=20 and in the setting of systemic metastasis of glioblastomas [37].=20 However, in a large study on 144 biopsies from 67 patients with = recurrent=20 astrocytoma no indication of intratumoral heterogeneity was obtained = [1].=20 In our study all eight tumor samples from the four paired cases reliably = demonstrated TP53 locus LOH with loss of the reverse alleles in = three=20 cases, indicating that the isolated DNA was retrieved from a high = percentage of=20 clonal tumor cells with little admixture of normal cells or tumor cells = of=20 another clone. In addition, in cases 34 and 44 (Table 1)=20 the TP53 mutation identified in the primary tumor was not found = in the=20 recurrence and, vice versa, the mutation detected in the recurrence was = not=20 present in the primary tumor. Because TP53 mutation is an early = event in=20 astrocytoma tumorigenesis the finding of different TP53 mutations = in=20 paired primary tumor and recurrence is supportive for dual clonality. = Although=20 intratumoral heterogeneity cannot be excluded as the cause of different=20 TP53 aberrations in the paired primary tumors and recurrences, we = believe=20 our results are more supportive for the presence of two, clonally = unrelated,=20 entities in these four patients.=20

Fig. 1 Combined=20 analysis of TP53 and MGMT, case 44 primary and second = tumor.=20 a Diffuse astrocytoma (WHO grade II); primary tumor (the = perinuclear=20 halos are interpreted as edema of the neuropilema rather than an=20 oligodendroglial tumor component). b Diffuse astrocytoma (WHO = grade II);=20 second tumor. c, d Immunohistochemistry for p53 showing = many=20 positive (brown) nuclei in both tumor samples. e, f = TP53=20 mutation analysis: different mutations in the primary and secondary = tumors,=20 suggestive of the presence of dual clonality. g Loss of = heterozygosity=20 (LOH) analysis showing loss of the larger allele in the primary tumor=20 (microsatellite marker D17S1866). h LOH analysis showing = loss of=20 the shorter allele in the second tumor (reverse loss, indicative of dual = clonality). i MGMT promoter hypermethylation analysis = (digested=20 sample), both tumors were not hypermethylated for MGMT = (MGMT=20 digested/MGMT undigested ratio of MGMT ₂ probe=20 <0.30)

Thirteen of the 23 clonally related pairs of primary = tumor and=20 recurrence demonstrated histologically confirmed malignant progression = and all=20 cases were consistent with regard to TP53 aberrations (mutation, = LOH and=20 p53 expression). This result is in contrast with a study by Sarkar et = al. who=20 reported increased p53 expression upon malignant progression of = astrocytic=20 tumors. However, in that study only p53 expression was determined and no = TP53 mutation nor LOH analysis was performed. Comparable with our = study,=20 no correlation between TP53 aberration and interval to recurrence = was=20 found [38].=20

Correlation between MGMT promoter = hypermethylation=20 and TP53 mutation

Methylation of the MGMT promoter, and thus = absence of AGT=20 expression in gliomas reportedly correlates with G:C>A:T transition = mutations=20 of TP53, in particular at CpG sites [11,=20 21,=20 23=9625].=20 The mechanism of the preferential occurrence of these mutations may be = related=20 to the cytosine methylation-enhanced formation of adducts at methylated = CpG=20 sites. A 5-methylcytosine adjacent to the 5′ position of an=20 O⁶-methylated guanine strongly hampers repair of this defect = by AGT.=20 Accordingly, inactivation of MGMT gene expression causes = retention of the=20 methylated adducts at the O⁶ position of guanine. This = results in=20 spontaneous or factor-mediated deamination which converts the = 5-methylcytosine=20 into thymine. During DNA replication thymine is incorporated and = subsequently a=20 G:C to A:T transition mutation may originate at that spot. This would = support=20 the relationship between epigenetic inactivation of MGMT and = accumulation=20 of this mutation in TP53 at CpG sites [23,=20 26].=20

In this study 90% of the G:C>A:T transition = mutations were=20 located at CpG sites. However, we neither found a significant = correlation=20 between MGMT hypermethylation and TP53 mutation, nor a = correlation=20 between MGMT hypermethylation and specific TP53 G:C>A:T = transition mutations. Zawlik et al. found a significantly higher = frequency of=20 TP53 G:C>A:T transition mutations in MGMT promoter=20 hypermethylated glioblastomas, whereas the total frequency of = TP53=20 mutations in MGMT promoter hypermethylated and unmethylated = glioblastomas=20 was similar. Interestingly, no association of MGMT promoter=20 hypermethylation and the accumulation of G:C>A:T transition mutations = in=20 PTEN or other genes was found in that study [26],=20 suggesting specific involvement of the TP53 gene. However, in = colorectal=20 tumorigenesis and gastric cancer inactivation of MGMT by promoter = hypermethylation seemed to be associated with G>A mutations in=20 K-ras [39=9641].=20 In accordance with our findings, Jesien-Lewandowicz et al. recently = reported no=20 correlation between methylation of the MGMT promoter and = G:C>A:T=20 TP53 mutations in a series of 32 primary glioblastomas treated = with=20 radiotherapy and surgery [42].=20 Future studies may reveal whether the specific methylation of = MGMT is=20 representative of a generalized status of genomic methylation and = whether there=20 is a gene-specificity of the G:C>A:T transition mutations. =

Acknowledgements We thank Ludo Uytdewilligen for excellent technical = assistance and=20 Frank van der Panne for assistance with the photographic work.

Open=20 Access =20

This article is distributed under the terms of the = Creative=20 Commons Attribution Noncommercial License which permits any = noncommercial use,=20 distribution, and reproduction in any medium, provided the original = author(s)=20 and source are credited.

References

1.	Watanabe K, Sato K, Biernat W et al (1997) = Incidence=20 and timing of p53 mutations during astrocytoma progression in = patients=20 with multiple biopsies. Clin Cancer Res 3:523=96530

2.	Reifenberger J, Ring GU, Gies U et al (1996) = Analysis=20 of p53 mutation and epidermal growth factor receptor amplification = in=20 recurrent gliomas with malignant progression. J Neuropathol Exp = Neurol=20 55:822=96831

3.	Peraud A, Kreth FW, Wiestler OD et al (2002)=20 Prognostic impact of TP53 mutations and P53 protein overexpression = in=20 supratentorial WHO grade II astrocytomas and oligoastrocytomas. = Clin=20 Cancer Res 8:1117=961124

4.	Ohgaki H, Kleihues P (2007) Genetic pathways = to=20 primary and secondary glioblastoma. Am J Pathol = 170:1445=961453

5.	Ohgaki H, Dessen P, Jourde B et al (2004) = Genetic=20 pathways to glioblastoma: a population-based study. Cancer Res=20 64:6892=966899

6.	Ohgaki H, Kleihues P (2005) Population-based = studies=20 on incidence, survival rates, and genetic alterations in = astrocytic and=20 oligodendroglial gliomas. J Neuropathol Exp Neurol = 64:479=96489

7.	Watanabe T, Katayama Y, Yoshino A et al (2003) = Deregulation of the TP53/p14ARF tumor suppressor pathway in = low-grade=20 diffuse astrocytomas and its influence on clinical course. Clin = Cancer Res=20 9:4884=964890

8.	Stander M, Peraud A, Leroch B et al (2004) = Prognostic=20 impact of TP53 mutation status for adult patients with = supratentorial=20 World Health Organization Grade II astrocytoma or = oligoastrocytoma: a=20 long-term analysis. Cancer 101:1028=961035

9.	Okamoto Y, Di Patre PL, Burkhard C et al = (2004)=20 Population-based study on incidence, survival rates, and genetic=20 alterations of low-grade diffuse astrocytomas and = oligodendrogliomas. Acta=20 Neuropathol 108:49=9656

10.	Ichimura K, Bolin MB, Goike HM et al (2000)=20 Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for = human=20 astrocytic gliomas with G1-S transition control gene = abnormalities. Cancer=20 Res 60:417=96424

11.	Watanabe T, Katayama Y, Yoshino A et al = (2007)=20 Aberrant hypermethylation of p14ARF and O6-methylguanine-DNA=20 methyltransferase genes in astrocytoma progression. Brain Pathol=20 17:5=9610

12.	Esteller M, Hamilton SR, Burger PC et al = (1999)=20 Inactivation of the DNA repair gene O6-methylguanine-DNA = methyltransferase=20 by promoter hypermethylation is a common event in primary human = neoplasia.=20 Cancer Res 59:793=96797

13.	Esteller M, Garcia-Foncillas J, Andion E et = al (2000)=20 Inactivation of the DNA-repair gene MGMT and the clinical response = of=20 gliomas to alkylating agents. N Engl J Med 343:1350=961354

14.	Gerson SL (2004) MGMT: its role in cancer = aetiology=20 and cancer therapeutics. Nat Rev Cancer 4:296=96307

15.	Hegi ME, Diserens AC, Gorlia T et al (2005) = MGMT gene=20 silencing and benefit from temozolomide in glioblastoma. N Engl J = Med=20 352:997=961003

16.	Levin N, Lavon I, Zelikovitsh B et al (2006)=20 Progressive low-grade oligodendrogliomas: response to temozolomide = and=20 correlation between genetic profile and O6-methylguanine DNA=20 methyltransferase protein expression. Cancer 106:1759=961765

17.	Esteller M, Herman JG (2004) Generating = mutations but=20 providing chemosensitivity: the role of O6-methylguanine DNA=20 methyltransferase in human cancer. Oncogene 23:1=968

18.	Friedman HS, McLendon RE, Kerby T et al = (1998) DNA=20 mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis = and=20 response to Temodal in newly diagnosed malignant glioma. J Clin = Oncol=20 16:3851=963857

19.	Hegi ME, Diserens AC, Godard S et al (2004) = Clinical=20 trial substantiates the predictive value of O-6-methylguanine-DNA=20 methyltransferase promoter methylation in glioblastoma patients = treated=20 with temozolomide. Clin Cancer Res 10:1871=961874

20.	Paz MF, Yaya-Tur R, Rojas-Marcos I et al = (2004) CpG=20 island hypermethylation of the DNA repair enzyme methyltransferase = predicts response to temozolomide in primary gliomas. Clin Cancer = Res=20 10:4933=964938

21.	Komine C, Watanabe T, Katayama Y et al (2003) = Promoter hypermethylation of the DNA repair gene = O6-methylguanine-DNA=20 methyltransferase is an independent predictor of shortened = progression=20 free survival in patients with low-grade diffuse astrocytomas. = Brain=20 Pathol 13:176=96184

22.	Everhard S, Kaloshi G, Criniere E et al = (2006) MGMT=20 methylation: a marker of response to temozolomide in low-grade = gliomas.=20 Ann Neurol 60:740=96743

23.	Nakamura M, Watanabe T, Yonekawa Y et al = (2001)=20 Promoter methylation of the DNA repair gene MGMT in astrocytomas = is=20 frequently associated with G:C → A:T mutations of = the TP53 tumor=20 suppressor gene. Carcinogenesis 22:1715=961719

24.	Bello MJ, Alonso ME, Aminoso C et al (2004)=20 Hypermethylation of the DNA repair gene MGMT: association with = TP53 G:C to=20 A:T transitions in a series of 469 nervous system tumors. Mutat = Res=20 554:23=9632

25.	Watanabe T, Katayama Y, Komine C et al (2005) = O6-methylguanine-DNA methyltransferase methylation and TP53 = mutation in=20 malignant astrocytomas and their relationships with clinical = course. Int J=20 Cancer 113:581=96587

26.	Zawlik I, Vaccarella S, Kita D et al (2008) = Promoter=20 methylation and polymorphisms of the MGMT gene in glioblastomas: a = population-based study. Neuroepidemiology 32:21=9629

27.	Louis DNOH, Wiestler OD, Cavenee WK (eds) = (2007) WHO=20 classification of tumours of the central nervous system, 4th edn. = IARC,=20 Lyon

28.	van der Sijp JR, van Meerbeeck JP, Maat AP et = al=20 (2002) Determination of the molecular relationship between = multiple tumors=20 within one patient is of clinical importance. J Clin Oncol=20 20:1105=961114

29.	Jeuken JW, Cornelissen SJ, Vriezen M et al = (2007)=20 MS-MLPA: an attractive alternative laboratory assay for robust, = reliable,=20 and semiquantitative detection of MGMT promoter hypermethylation = in=20 gliomas. Lab Invest 87:1055=961065

30.	Worsham MJ, Chen KM, Meduri V et al (2006) = Epigenetic=20 events of disease progression in head and neck squamous cell = carcinoma.=20 Arch Otolaryngol Head Neck Surg 132:668=96677

31.	Chen K, Sawhney R, Khan M et al (2007) = Methylation of=20 multiple genes as diagnostic and therapeutic markers in primary = head and=20 neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg=20 133:1131=961138

32.	Hatanpaa KJ, Burger PC, Eshleman JR et al = (2003)=20 Molecular diagnosis of oligodendroglioma in paraffin sections. Lab = Invest=20 83:419=96428

33.	Idbaih A, Boisselier B, Marie Y et al (2007) = TP53=20 codon 72 polymorphism, p53 expression, and 1p/19q status in=20 oligodendroglial tumors. Cancer Genet Cytogenet = 177:103=96107

34.	Nakasu S, Fukami T, Jito J et al (2007) = Prognostic=20 significance of loss of O6-methylguanine-DNA methyltransferase = expression=20 in supratentorial diffuse low-grade astrocytoma. Surg Neurol=20 68:603=96608

35.	Brandes AA, Franceschi E, Tosoni a et al = (2010)=20 O⁶-methylguanine DNA-methyltransferase methylation = status can=20 change between first surgery for newly diagnosed glioblastoma and = second=20 surgery for recurrence: clinical implications. Neuro-Oncology=20 12:283=96288

36.	Ren ZP, Olofsson T, Qu M et al (2007) = Molecular=20 genetic analysis of p53 intratumoral heterogeneity in human = astrocytic=20 brain tumors. J Neuropathol Exp Neurol 66:944=96954

37.	Park CC, Hartmann C, Folkerth R et al (2000) = Systemic=20 metastasis in glioblastoma may represent the emergence of = neoplastic=20 subclones. J Neuropathol Exp Neurol 59:1044=961050

38.	Sarkar C, Ralte AM, Sharma MC, Mehta VS = (2002)=20 Recurrent astrocytic tumours=96=96a study of p53 immunoreactivity = and=20 malignant progression. Br J Neurosurg 16:335=96342

39.	Esteller M, Toyota M, Sanchez-Cespedes M et = al (2000)=20 Inactivation of the DNA repair gene O6-methylguanine-DNA = methyltransferase=20 by promoter hypermethylation is associated with G to A mutations = in K-ras=20 in colorectal tumorigenesis. Cancer Res 60:2368=962371

40.	Whitehall VL, Walsh MD, Young J et al (2001)=20 Methylation of O-6-methylguanine DNA methyltransferase = characterizes a=20 subset of colorectal cancer with low-level DNA microsatellite = instability.=20 Cancer Res 61:827=96830

41.	Park TJ, Han SU, Cho YK et al (2001) = Methylation of=20 O(6)-methylguanine-DNA methyltransferase gene is associated = significantly=20 with K-ras mutation, lymph node invasion, tumor staging, and = disease free=20 survival in patients with gastric carcinoma. Cancer = 92:2760=962768

42.	Jesien-Lewandowicz E, Jesionek-Kupnicka D, = Zawlik L=20 et al (2009) High incidence of MGMT promoter methylation in = primary=20 glioblastomas without correlation with TP53 gene mutations. Cancer = Genet=20 Cytogenet 188:77=9682