Introduction
Materials and methods
Patients and tumor samples
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 = 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, p.Tyr107_Gly112dup | No | Hypermethylated | ||
3 | F | 39 | P | AIII | 60 | Exon 4, c.12253G>T, p.ArgR110Leu/Exon 8, c.14486C>T, 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, 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 = 27) | ||||||||||
18a
| 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, p.Arg158His | No | Hypermethylated |
R | GBM | 30 | Exon 4, c.12253G>C, p.ArgR110Pro/Exon 5, c.13152G>A, 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, p.Arg273His | No | Unmethylated |
R | OAII c
| 60 | Exon 6, c.13419A>G, p.Tyr220Cys/Exon 8, c.14487G>A, 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 | |||||
34a
| 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, p.Arg248Gln | No | Hypermethylated |
R | GBM | >80 | Exon 5, c.13197T>A, p.Val173Glu/Exon 7, c.14070G>A, p.Arg248Gln | No | Hypermethylated | |||||
36a
| 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, p.Glu326 fs | Yes | Unmethylated | |||||
44a
| 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 = 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 |
DNA extraction
Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for MGMT
MS-MLPA data analysis
Mutation analysis of TP53
Exon | Size (bp)a
| Forward primer | Reverse primer |
---|---|---|---|
A Nucleotide sequences of primers used for 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 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′ |
Loss of heterozygosity (LOH) analysis of the TP53 locus (17p13)
Immunohistochemistry (IHC) for p53
Statistical analysis
Results
Patients and tumor histology
Hypermethylation analysis of the MGMT promoter
TP53 mutation analysis and p53 expression
LOH analysis of the TP53 locus (17p13)
Correlation between MGMT promoter hypermethylation and TP53 mutations
MGMT hypermethylated (n = 29)a | MGMT not hypermethylated (n = 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 |