72

Kapitel 4. RESULTS

4.1 Alterations of the p53 tumor suppressor gene in ovarian cancer

4.1.1 p53 mutations

In a cohort of 178 ovarian carcinomas we found a total of 145 p53 sequence alterations by PCR-SSCP and direct sequencing (66%) (Table 7). Among the sequence alterations, 100 p53 mutations were identified in 99 cases, one of which had a mutation in both exon 7 and exon 8. In addition, four silent mutations without alteration of the amino acid sequence were identified and were considered polymorphisms. Forty-five of the sequence alterations were polymorphisms or intron alterations of undetermined significance.

Table 7: Summary of p53 sequence alterations in 178 ovarian carcinomas

Type of sequence alteration	n	% cases
Mutations with amino acid alterations in exon 2-11	92	51.1
(in 91 cases, double mutation in case #2341)
Splice site mutations	8	4.5
Polymorphisms in exon 2-11
exon 4, codon 36	1	0.6
exon 4, codon 72	9	5.1
exon 6, codon 213	1	0.6
exon 6, codon 224	1	0.6
exon 7, codon 231	1	0.6
Intron alterations (excluding splice site mutations)
intron 3 polymorphism (16 bp repeat)	24	13.5
intron 6 alteration (nt 13964)	2	1.1
intron 10 alteration (nt 17708)	2	1.1
intron 10 alteration (nt 18550)	4	2.3
Total number of sequence alterations (in 117 cases)	145	65.7

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Fig. 10 A-C: p53 mutation and overexpression
A: Single Strand Conformation Polymorphism (SSCP) analysis of the p53 gene, exon 5 in ovarian carcinomas.
Arrows in lanes 7 and 8 indicate band shifts suspicious for mutations.
B: DNA sequence analysis of exon 5 of the p53 gene in endometrioid ovarian carcinoma #2335.
Missense mutation in codon 141, TGC to TGG (amino acid exchange cystein to tryptophan).
C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335).

74

Fig. 11 A-C: p53 mutation and overexpressio
A: Single Strand Conformation Polymorphism (SSCP) analysis of p53, exon 9 in ovarian carcinomas. Arrows in lanes 3 and 10 (case #3402) indicate band shifts suspicious for mutations.
B: DNA sequence analysis of exon 9 of the p53 gene in endometrioid ovarian carcinoma #3402. A deletion mutation of one basepair in codon 320 causes a frameshift.
C: Immunostaining shows overexpression of the p53 protein in the tumor nuclei (case #2335.)

75

One hundred p53 mutations were identified by PCR-SSCP and direct sequencing in 99 ovarian cancer patients (56%) (Fig. 10-12, Table 8). 64 of these alterations have been previously described in a study of 105 ovarian carcinoma patients (Wen et al. 1999). One ovarian cancer patient had a mutation in exon 7 and exon 8, both with alteration of the amino acid sequence. The majority of mutations (86%) was found in exon 5 (22 mutations), exon 6 (12 mutations), exon 7 (26 mutations) and exon 8 (28 mutations) (Fig. 11, Table 8). However, 14% of the mutations were found outside codons 126-306 in exon 4 (3 mutations), exon 9 (3 mutations), intron 4 (3 splice junction mutations), intron 5 (1 splice junction mutation), intron 6 (1 splice junction mutation), intron 7 (2 splice site muations), and intron 8 (1 splice junction mutations).

Fig. 12: p53 mutations in ovarian cancer. Mutations of the p53 gene were identified in 99/178 epithelial ovarian carcinomas. The majority of mutations were missense mutations.

Out of 100 mutations, 72% were missense mutations (Fig. 12), 7% were single base substitutions which resulted in the introduction of a premature stop codon, 3% were insertions of one to six basepairs, 10% were deletions of one to seven basepairs and 8% were splice junction mutations (Table 8). Four „silent„ DNA sequence alterations, which did not result in an amino acid alteration, were considered polymorphisms and were located in exon 4 (codon 36), exon 6 (codon 213 and 224), and exon 7 (codon 231).

76-77

Table 8: p53 mutations and protein overexpression in ovarian carcinomas

Case	Histo-	SSCP	Exon	Codon	Amino	DNA	Amino Acid	Mutation	Im**	TS/TV***
#	patho-	Result		#	Acids	Sequence	Sequence	Type	p53	CpG
	logy				Cons*
3379	Ser	+	4	36	NC	CCG to CCA	Pro / Pro	Polymorph	+	(TS)
2722	Ser	+	4	42	NC	GAT to TAT	Asp to Tyr	Missense	+	TV
3401	Ser	+	4	49, 50	NC	GAT ATT to GA..TT	frameshift	2 bp Del	-	-
715	Ser	-	4	73	NC	GTG to TTG	Val to Leu	Missense	+	TV
3391	Endo	+	4	nt 12301	Co	CGgt to CGga	intron 4	5' splice jun	-	TV
2658	Undiff	+	5	nt 13053	Co	agTA to ggTA	intron 4	3' splice jun	+	TS
3355	Endo	+	5	nt 13053	Co	agTA to ggTA	intron 4	3' splice jun	+	TS
1557	Ser	+	5	130	Co	CTC to GTC	Leu to Val	Missense	+	TV
3400	Ser	+	5	132	Co	AAG to AGG	Lys to Arg	Missense	+	TS
1282	Ser	+	5	132	Co	AAG to ACG	Lys to Thr	Missense	+	TV
ov 70	Ser	+	5	135	Co	TGC to TGG	Cys to Trp	Missense	+	TV
3367	Ser	+	5	136	Co	CAA to TAA	Gln to Stop	Nonsense	-	TS
687	Ser	+	5	136	Co	CAA to TAA	Gln to Stop	Nonsense	-	TS
2335	Endo	+	5	141	Co	TGC to TGG	Cys to Trp	Missense	+	TV
2745	Ser	+	5	151	NC	CCC to CAC	Pro to His	Missense	+	TV
3389	Mixed	+	5	158	NC	CGC to CCC	Arg to Pro	Missense	+	TV
824	Mixed	+	5	159	NC	GCC to GTC	Ala to Val	Missense	+	TS;CpG
704	Ser	+	5	160	NC	ATG to AAG	Met to Lys	Missense	+	TV
769	Ser	+	5	163	NC	TAC to TGC	Tyr to Cys	Missense	+	TS
2354	Brenner	+	5	163	NC	TAC to TGC	Tyr to Cys	Missense	+	TS
713	Ser	+	5	166	NC	TCA to TGA	Ser to Stop	Nonsense	-	TV
2650	Undiff	+	5	175	Co	CGC to CAC	Arg to His	Missense	+	TS;CpG
3371	Mixed	+	5	175	Co	CGC to CAC	Arg to His	Missense	+	TS;CpG
762	Ser	+	5	175	Co	CGC to CAC	Arg to His	Missense	+	TS;CpG
1270	Ser	+	5	175	Co	CGC to CAC	Arg to His	Missense	+	TS;CpG
3354	Ser	+	5	179	Co	CAT to GAT	His to Asp	Missense	+	TV
3529	Ser	+	5	179	Co	CAT to TAT	His to Tyr	Missense	+	TS
698	Adenoca	+	5	179	Co	CAT to CGT	His to Arg	Missense	+	TS
2716	Mixed	+	5	179	Co	CAT to CGT	His to Arg	Missense	+	TS
2721	Ser	+	5	nt 13240	-	TGgt to TGgc	intron 5	5'-splice jun	-	TS
3353	undiff	+	6	192	NC	CAG to TAG	Gln to Stop	Nonsense	-	TS
ov 59	Ser	+	6	193	NC	CAT to CGT	His to Arg	Missense	+	TS
2719	Ser	+	6	193-195	NC	CATCTTATC to GCCCCT	His,Leu,Ile to Ala, Pro	Del / Ins	+	-
2309	Undiff	+	6	195	NC	ATC to AAC	Ile to Asn	Missense	+	TV
805	Ser	+	6	201	NC	TTG to TTT	Leu to Phe	Missense	+	TV
2352	Ser	+	6	203	NC	GTG to TTG	Val to Leu	Missense	+	TV
2733	Ser	+	6	213	NC	CGA to CTA	Arg to Leu	Missense	-	TV
2340	Ser	+	6	213	NC	CGA to CGG	Arg / Arg	Polymorph	-	(TS)
2737	Ser	-	6	215	NC	AGT to GT	frameshift	1bp Del/Nonsense	-	-
2349	Ser	+	6	216	NC	GTG to TTG	Val to Leu	Missense	+	TV
241	Ser	-	6	220	NC	TAT to TGT	Tyr to Cys	Missense	+	TS
808	Clear	-	6	224	NC	GAG to GAA	Glu / Glu	Polymorph	-	(TS)
3373	Ser	+	7	nt 13999	-	agGT to aaGT	intron 6	3' splice jun	+	TS
ov 49	Ser	+	7	227	NC	TCT to TCA	Asp to Ser	Missense	+	TV
2705	Mixed	+	7	231	NC	ACC to ACA	Thr / Thr	Polymorph	+	(TV)
3379	Ser	+	7	234	Co	TAC to TCC	Tyr to Ser	Missense	+	TV
2344	Clear	+	7	234	Co	TAC to TGC	Tyr to Cys	Missense	-	TS
2717	Ser	+	7	234	Co	TAC to TGC	Tyr to Cys	Missense	+	TS
2656	Endo	+	7	237	Co	ATG to ATT	Met to Ile	Missense	+	TV
3344	Endo	+	7	239 / 240	Co	AAC to AAC TTA	Leu ins	3 bp Ins	+	-
2659	Endo	+	7	242	Co	TGC to TAC	Cys to Tyr	Missense	+	TS
790	Mixed	+	7	242	Co	TGC to TTC	Cys to Phe	Missense	+	TV
2338	Endo	+	7	242	Co	TGC to TTC	Cys to Phe	Missense	+	TV
2724	Endo	+	7	242	Co	TGC to AGC	Cys to Ser	Missense	+	TV
2730	Ser	+	7	243	Co	ATG to GTG	Met to Val	Missense	+	TS
665	Ser	+	7	244	Co	GGC to AGC	Gly to Ser	Missense	+	TS
1752	Muc	+	7	245	Co	GGC to AGC	Gly to Ser	Missense	+	TS;CpG
1559	Ser	+	7	245	Co	GGC to GAC	Gly to Asp	Missense	+	TS;CpG
3540	Ser	+	7	248	Co	CGG to CAG	Arg to Glu	Missense	+	TS;CpG
802	Endo	+	7	248	Co	CGG to TGG	Arg to Trp	Missense	+	TS;CpG
249	Ser	+	7	248	Co	CGG to CAG	Arg to Gln	Missense	+	TS;CpG
3403	Undiff	+	7	249, 250	Co	AGG CCC to AGT TCC	Arg,Pro to Ser,Ser	Dble-Missense	+	TV,TS
2364	Ser	+	7	251, 252	Co	ATC CTC to ATC	Leu del	3 bp Del	+	-
1589	Ser	+	7	253	Co	ACC to CCC	Thr to Pro	Missense	+	TV
2651	Undiff	+	7	254 - 256	Co	ATC ATC ACA to A	frameshift	7 bp Del	-	-
2001	Ser	+	7	255	Co	ATC to AGC	Ile to Ser	Missense	-	TV
2337	Endo	+	7	258	Co	GAA to TAA	Glu to Stop	Nonsense	+	TV
2341	Ser	+	7	258	Co	GAA to TAA	Glu to Stop	Nonsense	-	TV
3362	Undiff	+	7	259	NC	GAC to TAC	Asp to Tyr	Missense	+	TV
3347	Ser	+	7	nt 14110	-	AGgt to AGtt	intron 7	5' splice jun	-	TV
2725	Ser	+	8	nt 14449	-	tagTG to aagTG	intron 7	3' splice jun	+	TV
767	Ser	+	8	264	NC	CTA to TA	frameshift	1 bp Del	+	-
696	Ser	+	8	264, 265	NC	CTA CTG to CTG	Leu,Leu to Leu	3 bp Del	+	-
3364	Muc	+	8	266	NC	GGA to CGA	Gly to Arg	Missense	+	TV
ov45	Ser	+	8	266	NC	GGA to AGA	Gly to Arg	Missense	+	TS
2341	Ser	+	8	267	NC	CGG to CCG	Arg to Pro	Missense	-	TV
2332	Endo	+	8	267	NC	CGG to CCG	Arg to Pro	Missense	+	TV
2708	Mixed	-	8	271	Co	GAG to TAG	Glu to Stop	Nonsense	-	TV
2155	Ser	-	8	272	Co	GTG to ATG	Val to Met	Missense	+	TS
2732	Ser	-	8	272	Co	GTG to TTG	Val to Leu	Missense	+	TV
3346	Ser	+	8	273	Co	CGT to CAT	Arg to His	Missense	+	TS;CpG
3358	Undiff	+	8	273	Co	CGT to CAT	Arg to His	Missense	+	TS;CpG
3382	Endo	+	8	273	Co	CGT to CTT	Arg to Leu	Missense	+	TV
2715	Ser	-	8	273	Co	CGT to TGT	Arg to Cys	Missense	+	TS;CpG
693	Ser	+	8	273	Co	CGT to TGT	Arg to Cys	Missense	+	TS;CpG
700	Ser	-	8	273	Co	CGT to GGT	Arg to Gly	Missense	+	TV
2718	Ser	+	8	275	Co	TGT to TAT	Cys to Tyr	Missense	+	TS
ov 93	Ser	+	8	275	Co	TGT to GGT	Cys to Gly	Missense	+	TV
ov 164	Ser	+	8	275	Co	TGT to GGT	Cys to Gly	Missense	+	TV
2326	Mixed	+	8	276	Co	GCC to GTC	Ala to Val	Missense	+	TS
3385	Endo	+	8	280	Co	AGA to AGT	Arg to Ser	Missense	+	TV
2328	Ser	+	8	280	Co	AGA to ATA	Arg to Ile	Missense	+	TV
1751	Ser	+	8	281	Co	GAC to GAA	Asp to Glu	Missense	+	TV
2182	Endo	+	8	281	Co	GAC to GAG	Asp to Glu	Missense	-	TV
ov 72	Ser	+	8	281	Co	GAC to CAC	Asp to His	Missense	+	TV
3351	Ser	+	8	282	Co	CGG to TGG	Arg to Trp	Missense	+	TS;CpG
1756	Clear	+	8	282	Co	CGG to TGG	Arg to Trp	Missense	+	TS;CpG
1254	Ser	+	8	286	Co	GAA to GTA	Glu to Val	Missense	+	TV
3386	Ser	+	8	300	NC	CCC to ..CC	frameshift	1 bp Del	+	-
3461	Endo	+	8	301	NC	CCA to CCC A	frameshift	1 bp Ins	-	-
2339	Ser	+	9	nt 14680	-	agCA to aaCA	intron 8	3' splice jun	-	TS
1749	Undiff	+	9		NC	AAC to AA..	frameshift	1 bp Del	-	-
3402	Endo	+	9		NC	AAG to A..G	frameshift	1 bp Del	+	-
3394	Undiff	+	9	324	311	GAT to G..T	frameshift	1 bp Del	+	-
					320

* according to Cho et al. 1994
** Im p53: Immunostaining for p53
*** TS / TV: Transition / Transversion
Histopathology: Ser: Serous, Endo: Endometrioid, Undiff: Undifferentiated
Muc: Mucinous, Clear: Clear Cell, Mixed: Mixed Epithelial

78

Eleven cases had deletions of 1-9 bp, four of which (cases # 2719, 2737, 767, 696) have been described previously (Wen et al. 1999). Of the newly identified deletions, four were 1 bp deletions, one in exon 8 (case # 3386) and three in exon 9 (case # 1749, 3402, 3394), all resulting in the introduction of a premature stop codon at codon 344. Three of these cases had p53 protein overexpression. One case had a 3 bp deletion in exon 7, resulting in the loss of a leucine residue (case #2364) and one case had a 2 bp deletion in exon 4, causing the introduction of a stop codon at codon 50 (case #3401). This case was negative in immunohistochemistry. A seven bp deletion in exon 7 (case #2651), affecting codons 254-256, resulted in a frameshift affecting the entire sequence between codon 256 and a premature stop at codon 344.

Furthermore, we identified 5 splice junction mutations (cases # 3391, 2658, 3355, 3373, 3347) in addition to three cases described in our previous study (Wen et al. 1999). Two of these affected the donor site at exon 4/intron 4 and exon 7/intron 7 respectively (Table 8). Both cases were negative for immunohistochemistry. Three splice site mutations involved the consensus acceptor site at intron 4/exon5 (2 cases) and intron 6/exon 7. These three cases had p53 protein overexpression (Table 8).

Four regions of the translated sequence of p53 have been highly conserved throughout evolution and span codon 117-124 (II), 171-181 (III), 234-258 (IV) and 270-286 (V). These regions span a total of 79 codons. In 62% (62/100) of the cases, mutations were located in these highly conserved regions (Fig. 13). Region II, which spans part of exon 4 and exon 5, contained 10 mutations including 3 splice site mutations in intron 4 (10%). Region III contained 8 mutations (8%), region IV 23 mutations (23%) and region V a total of 21 mutations (21%). Thirty-eight percent of the mutations were found in non-conserved regions of the gene. The codons most frequently mutated were codons 175, 179, 234, 242, 248, 273, 275 and 281 which were each mutated in three or more different carcinomas (Fig. 13).

79

Fig. 13: p53 mutations in evolutionary highly conserved domains of the gene. The upper part of the figure shows the number of mutations in codons 36 through 324 of the p53 gene. Mutations in evolutionary highly conserved regions are marked in yellow, mutations in non-conserved regions in dark blue and splice site mutations in white. The lower part of the figure shows the corresponding DNA interacting regions of the p53 protein.

80

Fourty-two mutations (42%) were classified as transition mutations, 46 (45%) as transversions, and 13% were deletions or insertions. We found 29 G:C to A:T transitions and 13 A:T to G:C transitions. Among the G:C to A:T transitions 16 (55%) were located in CpG sites, which are known to be potential sites of DNA methylation. Out of 46 transversions, we found 20 G:C to T:A mutations, 12 G:C to C:G, 8 T:A to A:T and 6 A:T to G:C mutations.

4.1.2 p53 polymorphisms and intron alterations

A total number of 45 p53 polymorphisms and intron alterations of unknown significance were found in 33 cases (19%). In 15/33 cases (46%) with polymorphisms or intron alterations additional p53 mutations with an amino acid exchange had been identified. This accounted for 15% of the cases with mutations. Only 61/178 ovarian cancer cases (34%) were found to have a completely normal p53 sequence with neither mutations nor other sequence alterations in the DNA segments which were analyzed.

The most frequent exon polymorphism was located in exon 4 at codon 72 (Fig. 14), and was found in 9 cases (5.1%). This CGCCCC polymorphism at codon 72 results in an arginine to proline amino acid exchange (Harris et al. 1986, Matlashewski et al. 1987). In one case (#3379), an additional CCG to CCA (Pro/Pro) polymorphism was identified at codon 36 of exon 4 (Table 9 and 10).

A further exon polymorphisms, CGACGG (Arg/Arg), was identified in exon 6 at codon 213. Another silent mutation which was considered a polymorphism, occured at codon 224 of exon 6 as a GAGGAA (Glu/Glu) polymorphism without amino acid alteration. In exon 7 an ACCACA (Thr/Thr) polymorphism was identified at codon 231 (Table 9 and 10).

An intron 3 polymorphism (Fig. 15), which is known as a 16 bp repeat (Lazar et al. 1993) at nucleotide 11951, was found in 24 (14%) of the cases. Six (25%) of the cases were identified by SSCP as homozygote and 18 (75%) were heterozygote for this intron 3 alteration. Five cases were sequenced to confirm the polymorphism suspected by the SSCP alteration.

Eight p53 sequence alterations were identified in intron sequences, flanking the exons 7, 10 and 11 (Table 9 and 10). In all of these cases, a shift in the SSCP bands was noticed without evidence of a mutation in the exon sequence. In intron 6 at position 13964, a gc base exchange was identified in two cases. No normal tissue was available in these cases to verify the polymorphism.

81

Fig. 14: CGC CCC (Arg Pro) polymorphism at codon 72 of the p53 gene
A: SSCP analysis of p53, exon 4 in ovarian carcinomas. DNA in lanes 2,4,5,6,and 8 shows
band shifts suspicious for sequence alterations and was analyzed by DNA sequencing.
B: DNA sequence analysis of p53, exon 4 shows a CGC to CCC poymorphism at codon 72,
resulting in an arginine to proline amino acid exchange.

82

Fig. 15: Intron 3 polymorphism of the p53 gene
A: Single Strand Conformation Polymorphism (SSCP) of p53, exon 3 with the adjacent intron
sequence shows a band shift in lanes 2,4,8 and 9 indicating the intron 3 polymorphism.
B: Wild type (lanes 3 and 4) and the polymorphism sequence (lanes 1 and 2) of p53, intron 3
with a 16 bp insertion (lanes 1 and 2) and adjacent sequence are shown as
5‘ to 3‘: GCTggggacctggagggctGGGGGG

83

Multiple polymorphisms were identified in 11/178 (6%) of all ovarian cancer cases and 11/33 (33%) of the cases with polymorphisms. In case #3379, three polymorphisms were identified, two in exon 4 (codon 36 and codon 72) and one in intron 3. The other cases had two polymorphisms. Eight out of nine cases with exon 4 polymorphisms (89%) and 3/6 cases with intron 10 alterations (50%) had an additional intron 3 polymorphism.

4.1.3 Confirmation of p53 polymorphisms in normal tissue DNA

In cases with exon 4, exon 6 or exon 7 polymorphisms, and in cases with intron 6 or intron 10 alterations, normal tissue of the ovarian cancer patients, which had been formalin fixed and paraffin embedded at the time of surgery, was sequenced at the same DNA site (Table 10). Normal tissue was available in 12/20 of these cases. In three cases with a codon 72 ArgPro polymorphism, the same sequence alteration was found in normal tissue. In two heterozygote cases, in which the CGC sequence was stronger than the CCC sequence and in one case with equally strong G and C band only, the CGC (arginin) sequence was identified in normal tissue. In two cases with a homozygote CGCCCC polymorphism in the tumor, only CGC was identified in normal tissue. In the cases with exon 6, intron 6 and exon 7 polymorphisms no normal tissue was available

The intron 10 alteration at nucleotide 17708 was confirmed in one case, in which normal tissue was available. We have identified this alteration in two ovarian carcinoma cases, which had shown SSCP band shifts. We assume that this is an at polymorphism at nucleotide 17708, which has not been described so far. Furthermore, the intron 10 ct sequence alteration at nucleotide 18550 was found in four ovarian carcinoma cases and could be confirmed in all three cases, in which normal tissue had been analyzed (Table 10). We therefore consider these intron alterations as two novel intron 10 polymorphisms.

4.1.4 p53 protein overexpression

p53 protein overexpression, using a cut-point of 10% positively stained nuclei, was found in 62% (110/178) of the cases. The cut-off for p53 overexpression was chosen as 10%, because we found frozen sections from proliferating normal

84

Fig. 16: Intron 10 polymorphism of the p53 gene
at nucleotide 17708
A novel polymorphism of the p53 gene was identified
at nucleotide 17708 (a>t) of intron 10 by SSCP analysi
and DNA sequencing in the ovarian cancer case #3402.

85

Fig. 17: Intron 10 polymorphism of the p53 gene at nucleotide 18550
A: SSCP analysis of p53 exon 11 and flanking intron sequence in ovarian carcinomas.
The band shift in lane 5 (case #3394) is suspicious for DNA sequence alterations.
B: DNA sequence analysis of p53 exon 11/ intron 10 reveals a novel c>t polymorphism
at nucleotide 18550 of intron 10 in the ovarian cancer cases #2660 and #3344.
The polymorphism was confirmed in normal tissue DNA.

86

Table 9: p53 polymorphisms and overexpression in ovarian carcinomas

Case #	Histo-	SSCP	p53	Exon/	Codon/	DNA	Amino Acid	Polymor-	Immuno-
	patho-	Result	Mut	Intron	Nucleotide	Sequence	Sequence	phism Type	staining
	logy				#				p53
1749	Undiff	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
1751*	Ser	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
1754	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
2341	Ser	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
2353	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
2364	Ser	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
2653*	Undiff	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
2660*	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3348	Endo	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3350*	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3351	Ser	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3355	Endo	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3357	Endo	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3359	Endo	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3362	Undiff	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3365	Endo	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3370*	Muc	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3376*	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3377*	Ser	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3379*	Ser	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3381*	Endo	+	-	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	-
3385*	Endo	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3402*	Endo	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3403	Undiff	+	+	Intron 3	nt 11951	gggtcgggaggtccag	-	16 bp repeat	+
3379*	Ser	+	+	Exon 4	36	CCG to CCA	Pro / Pro	known PM	+
1751*	Ser	+	+	Exon 4	72	CGC to CCC	Arg to Pro	known PM	+
3350*	Ser	+	-	Exon 4	72	CGC to CCC	Arg to Pro	known PM	-
3354	Ser	+	+	Exon 4	72	CGC to CCC	Arg to Pro	known PM	+
3370*	Muc	+	-	Exon 4	72	CGC to CCC	Arg to Pro	known PM	-
3376*	Ser	+	-	Exon 4	72	CGC to CCC	Arg to Pro	known PM	-
3377*	Ser	+	-	Exon 4	72	CGC to CCC	Arg to Pro	known PM	-
3379*	Ser	+	+	Exon 4	72	CGC to CCC	Arg to Pro	known PM	+
3381*	Endo	+	-	Exon 4	72	CGC to CCC	Arg to Pro	known PM	-
3385*	Endo	+	+	Exon 4	72	CGC to CCC	Arg to Pro	known PM	+
2340	Ser	+	-	Exon 6	213	CGA to CGG	Arg / Arg	known PM	-
808	Clear	-	-	Exon 6	224	GAG to GAA	Glu / Glu	possible PM	-
3529	Ser	+	+	Intron 6	nt 13964	gcgca to gccca	-	possible PM	+
3390	Ser	+	-	Intron 6	nt 13964	gcgca to gccca	-	possible PM	-
2705	Mixed	+	-	Exon 7	231	ACC to ACA	Thr / Thr	possible PM	+
2653*	Undiff	+	-	Intron 10	nt 17708	ctact to cttct	-	possible PM	+
3402*	Endo	+	+	Intron 10	nt 17708	ctact to cttct	-	possible PM	+
2660*	Ser	+	-	Intron 10	nt 18550	ccctc to ccttc	-	possible PM	+
3344	Endo	+	+	Intron 10	nt 18550	ccctc to ccttc	-	possible PM	+
3394	Undiff	+	+	Intron 10	nt 18550	ccctc to ccttc	-	possible PM	+
3464	Ser	+	-	Intron 10	nt 18550	ccctc to ccttc	-	possible PM	+

* multiple p53 polymorphisms

87

Table 10: Normal tissue sequencing results for p53 polymorphisms

Case #	Exon/	Codon/	Tumor	Tumor	Nl tissue	Nl tissue	Immuno-
	Intron	Nucleotide	DNA	Amino Acid	DNA	Amino Acid	staining
		#	Sequence	Sequence	Sequence	Sequence	p53
3379	Exon 4	36	CCG to CCA	Pro / Pro	not available	-	+
1751	Exon 4	72	CGC to CCC	Arg to Pro	CGC	Arg	+
3350	Exon 4	72	CGC to CCC	Arg to Pro	CCC	Pro	-
3354	Exon 4	72	CGC to CCC	Arg to Pro	CGC	Arg	+
3370	Exon 4	72	CGC >> CCC*	Arg >> Pro	CGC	Arg	-
3376	Exon 4	72	CGC to CCC	Arg to Pro	not available	-	-
3377	Exon 4	72	CGC to CCC	Arg to Pro	CCC	Pro	-
3379	Exon 4	72	CGC / CCC	Arg / Pro	CGC	Arg	+
3381	Exon 4	72	CGC >> CCC	Arg >> Pro	CGC	Arg	-
3385	Exon 4	72	CGC to CCC	Arg to Pro	CCC	Pro	+
2340	Exon 6	213	CGA to CGG	Arg / Arg	not available	-	-
808	Exon 6	224	GAG to GAA	Glu / Glu	not available	-	-
3529	Intron 6	nt 13964	gcgca to gccca	-	not available	-	+
3390	Intron 6	nt 13964	gcgca to gccca	-	not available	-	-
2705	Exon 7	231	ACC to ACA	Thr / Thr	not available	-	+
2653	Intron 10	nt 17708	ctact to cttct	-	ctact to cttct	-	+
3402	Intron 10	nt 17708	ctact to cttct	-	not available	-	+
2660	Intron 10	nt 18550	ccctc to ccttc	-	ccctc to ccttc	-	+
3344	Intron 10	nt 18550	ccctc to ccttc	-	not available	-	+
3394	Intron 10	nt 18550	ccctc to ccttc	-	ccctc to ccttc	-	+
3464	Intron 10	nt 18550	ccctc to ccttc	-	ccctc to ccttc	-	+

* base G stronger than C

88

4.1.5 Comparison of p53 overexpression with p53 mutations

p53 overexpression detected by immunohistochemical staining was signifi-cantly correlated with p53 mutations (p<0.001) detected by DNA sequence analysis. The percentage of cases with overexpression was 81% in ovarian carcinomas with p53 mutation and 38% in cases with wildtype p53. The percentage of cases with p53 immunostaining varied according to the type of mutation. The correlation between protein overexpression and mutations was mainly due to the high proportion of missense mutations. 67 out of 71 (94%) of the cases with missense mutations showed p53 overexpression, while immunostaining 10% was only seen in 1/7 (14%) of the cases with nonsense mutations, 8/13 (62%) of the cases with insertions or deletions and 4/8 (50%) of the cases with splice site mutations (Fig. 18 a,b).

Overall only 46% (13/28) of the ovarian carcinomas with nonmissense mutations had p53 overexpression, which is not significantly different from the frequency of overexpression in 30/79 tumors with wildtype p53 sequence (38%) (p=0.43). Nineteen percent (19/99) of the cases with p53 mutations failed to show immunostaining, which is largely caused by nonmissense mutations.

4.1.6 p53 polymorphisms and p53 protein overexpression

Ovarian cancer cases with normal p53 expression respectively p53 overexpression were distributed approximately equally among cases with exon 4 polymorphism (p=0.27) and intron 3 polymorphisms (p=0.41) (Table 11). Interestingly, though, out of eight cases with intron 6 or intron 10 sequence alterations, seven cases had p53 overexpression which is close to reaching statistical significance (p=0.086) (Table 11).

89

Fig. 18a: p53 protein overexpression according to type of p53 mutation.
Overexpression is found in a high percentage of cases with missense mutations,
while the frequency of overexpression in nonmissense mutations is overall only 46%.

Fig. 18b: p53 protein overexpression according to p53 mutation or wildtype p53.
A high proportion of cases (38%) shows p53 protein accumulation (immunostaining in
10% of the nuclei) despite p53 wildtype sequence.

90

Table 11: p53 protein overexpression in ovarian cancer cases with p53 polymorphisms

p53 Polymorphism	p53 Over- expression (%)	Normal p53 Expression n (%)	Total n (%)	p-value
Exon 4 Codon 72
Polymorphism CGC Arg CCC Pro	4 (44)	5(56)	9 (5)
Normal Sequence CGC Arg	106 (63)	63 (37)	169 (95)	0.27
Intron 3
16 bp repeat Polymorphism	14 (54)	12 (46)	26 (15)
Normal Intron 3 Sequence	96 (63)	56 (37)	152 (85)	0.37
Intron 6 / Intron 10
Polymorphism	8 (89)	1 (11)	9 (5)
Normal Sequence	102 (60)	67 (40)	169 (95)	0.086
Total	110 (62)	68 (38)	178 (100)

4.1.7 Correlation of p53 alterations with histopathological and clinical data

p53 mutations were significantly more frequent in ovarian carcinomas with advanced FIGO stages III and IV (p<0.001), poor differentiation (p<0.001), residual tumor burden after debulking surgery (p<0.04), DNA-aneuploidy (p=0.003) and high S-phase-fraction as measured by image cytometry (p<0.001). Patients with p53 mutations also had a higher median age than patients with p53 wildtype (p=0.041). Overexpression of the p53 protein was correlated only with low grade of differentiation (p<0.001) and high S-phase-fraction (p<0.001) (Table 12).

4.1.8 p53 alterations and response to chemotherapy

Patients with p53 overexpressing tumors were significantly more often resistant or refractory to a platinum based chemotherapy (61%) than patients with non-overexpressing tumors (22%) (p=0.001) (Table 13, Fig. 19 A-D).

91

Table 12. Clinico-pathologic characteristics of patients with epithelial ovarian cancer

			p53 Mutation Analysis			p53 Protein Expression			p53 Alterations
			Characteristic		p53		p53	P	Overex-	normal		P	p53		normal	P
			Mutation	Wildtype	value*	pression	Expression	value*	Alteration	p53	value*
			Patients - no. (%)		99 (56)		79 (44)		110 (62)	68 (38)			132 (74)		46 (26)
			Median age - yrs		57		53	0.041**	57	52		0.06**	57		52	0.056**
			Histological Subtype - no. (%)
	Serous		60 (61)	51 (65)	0.08	67 (61)	44 (65)	0.62	79 (60)	32 (70)	0.4
	Mucinous		2 (2)	6 (8)		4 (4)	4 (6)		4 (3)	4 (9)
	Endometriod		18 (18)	12 (15)		20 (18)	10 (15)		24 (18)	6 (13)
	Clear Cell		1 (1)	5 (6)		2 (2)	4 (6)		4 (3)	2 (4)
	Brenner		1 (1)	0		1 (1)	0		1 (1)	0
	Undifferentiated		8 (8)	2 (3)		7 (6)	3 (4)		9 (7)	1 (2)
	Mixed Epithelial		7 (7)	3 (4)		7 (6)	3 (4)		9 (7)	1 (2)
	Unclassified		2 (2)	0		2 (2)	0		2 (2)	0
			Clinical stage - no. (%)
	FIGO I		11 (11)	25 (32)	0.001	18 (17)	18 (28)	0.11	21 (16)	15 (34)	0.005
	FIGO II		3 (3)	5 (7)		4 (4)	4 (6)		5 (4)	2 (7)
	FIGO III		67 (70)	39 (51)		72 (67)	34 (52)		83 (64)	23 (52)
	FIGO IV		15 (16)	8 (10)		14 (13)	9 (14)		20 (16)	3 (7)
			Grade of Differentiation - no. (%)
	I	(well)	7 (7)	21 (27)	< 0.001	8 (7)	20 (29)	< 0.001	11 (8)	17 (37)	< 0.001
	II	(moderate)	25 (25)	27 (34)		32 (29)	20 (29)		36 (27)	16 (35)
	III	(poor)	67 (68)	31 (39)		70 (64)	28 (41)		85 (64)	13 (28)
			Residual Tumor - no. (%)
	No residual tumor		32 (39)	39 (56)	0.04	36 (40)	35 (56)	0.062	46 (41)	25 (60)	0.044
	< 2 cm		29 (35)	19 (27)		32 (35)	16 (26)		37 (33)	11 (26)
	> 2 cm		22 (27)	12 (17)		23 (25)	11 (18)		28 (25)	6 (14)
			Lymph nodes - no. (%)
	Negative		9 (11)	15 (22)	0.2	13 (15)	11 (17)	0.5	16 (15)	8 (19)	0.24
	Positive		20 (25)	17 (25)		20 (24)	17 (27)		24 (23)	13 (30)
	Not resected		50 (63)	37 (54)		52 (61)	35 (56)		65 (62)	22 (51)
			DNA-ploidy - no. (%) (n=102)
	Diploid		21 (39)	33 (69)	0.003	28 (48)	26 (59)	0.28	31 (44)	23 (74)	0.004
	Aneuploid		33 (61)	15 (31)		30 (52)	18 (41)		40 (56)	8 (26)
			S-Phase-Fraction - no. (%) (n=102)
	low ( < 5%)		4 (7)	22 (46)	< 0.001	6 (10)	20 (45)	< 0.001	9 (13)	17 (55)	< 0.001
	intermediate ( 5% - 14.4%)		26 (48)	21 (44)		29 (50)	18 (41)		35 (49)	12 (39)
	high ( > 14.5%)		24 (44)	5 (10)		23 (40)	6 (14)		27 (38)	2 (6)
			Treatment - no. (%) ***
	Cisplatin - Cyclophosphamide		27 (32)	27 (39)	0.43	34 (37)	20 (33)	0.55	39 (35)	15 (38)	0.98
	Carboplatin - Cyclophosphamide		13 (16)	7 (10)		13 (14)	7 (12)		15 (14)	5 (13)
	Other regimens		25 (31)	16 (23)		26 (29)	15 (25)		31 (28)	10 (25)
	No chemotherapy		17 (21)	19 (28)		18 (20)	18 (30)		26 (23)	10 (25)
	No information - no.		17	10		19	8		21	6

* Chi-Square test for two categories, Mantel-Haenszel test for three or more categories
** Wilcoxon test
*** Chemotherapy regimens are described in the methods section

92

Table 13: Response to platinum-based chemotherapy in correlation to p53 alterations

	Immunostaining		p53 Sequence		Type of Mutation
Response to Chemotherapy	Neg n (%)	Pos n (%)	WT n (%)	MUT n (%)	WT/NonMissense n(%)	Missense n(%)
Sensitive	21 (78)	18 (39)	22 (65)	17 (44)	29 (66)	10 (34)
Resistant / Refractory	6 (22)	28 (61)	12 (35)	22 (56)	15 (34)	19 (66)
p-value		0.001		0.071		0.008
Total (100%)	27	46	34	39	44	29

Interestingly, out of 5 patients in our study who were treated with Taxol/Carboplatin, the only patient (case #3400) who was sensitive to treatment and had no evidence of disease 28 months after diagnosis of a poorly differentiated FIGO III ovarian carcinoma, had high overexpression of p53 protein and a missense mutation in exon 5.

4.1.9 p53 alterations as a predictor of time to progression and overall survival

Ovarian cancer patients with p53 mutations had a significantly shorter time to progression (p=0.029) and overall survival (p=0.014) than patients with wildtype p53 (Fig. 20A, Table 14, 15). In 61/99 (62%) tumors, the p53 mutation was in an evolutionary highly conserved domain. If the clinical outcome of these was

93

Fig. 19 A-B: Estimated probability of not progressing in epithelial ovarian cancer
patients (n=74), who received platinum-based chemotherapy. (A) Ovarian carcinomas with p53 mutations versus wildtype p53. (B) Ovarian carcinomas with p53 proteinoverexpression versus normal p53 expression.

94

Fig. 19 C-D:: Estimated probability of (C) not progressing, and (D) overall
survival respectively in epithelial ovarian cancer patients (n=74), who received
platinum-based chemotherapy. Ovarian cancer with p53 alterations (mutation or
overexpression or both) versus normal p53 status.

95

4.1.10 Univariate and multivariable analysis of prognostic factors

Univariate analysis of time to progression identified p53 mutations, p53 alterations, residual disease, FIGO stage, histologic grade, age, lymph node status, and S-phase-fraction as prognostic factors. Multivariable analysis of time to progression identified only FIGO stage (p=0.001), residual disease (p=0.005) and age (p=0.032) as independent prognostic factors (Table 14).

Univariate analysis of overall survival identified residual disease, FIGO stage, age, grade, lymph node status and S-phase-fraction as prognostic factors besides p53 mutation and p53 alteration (Table 15). In multivariable analysis though, only residual disease (p<0.001), FIGO stage (p=0.001), grade (p=0.009), and patient age (p=0.01) were shown to be independent predictors of survival (Table 15).

96

Fig. 20 A-B: Estimated probability of overall survival in patients with epithelial
ovarian cancer (n=178) according to p53 alterations. (A) p53 mutations versus
wildtype p53. (B) p53 mutations in evolutionary highly conserved domains versus
wildtype p53 or mutations in non-conserved domains.

97

Fig. 20 C-D: Estimated probability of overall survival in epithelial ovarian
patients (n=178) according to p53 alterations. (C) p53 mutations in DNA-
interacting regions versus wildtype p53 or mutations in scaffolding regions of the p53
protein. (D) p53 protein overexpression versus normal expression of the p53 protein.

98

Fig. 20 E-F: Estimated probability of (E) not progressing, and (F) overall survival
respectively in epithelial ovarian cancer patients (n=178). Ovarian cancer with p53
alterations (p53 mutation or p53 overexpression or both) versus normal p53 status.

99

Table 14: Multivariable proportional hazards Cox regression analysis for the identification of independent prognostic factors in time to progression of patients with ovarian carcinoma.

Variables			Univariate Analysis				Multivariable Analysis
Variables in the			Unadjusted				Adjusted
Baseline Model *			Relative Risk	(95% CI)	p-value[1]		Relative Risk	(95%CI)	p-value[2]
FIGO					< 0.001				0,001
I + II			1,00				1,00
III + IV			6,08	(3.16, 11.71)			3,76	(1.70, 8.32)
Residual Disease					< 0.001				0,005
None			1,00				1,00
Any			3,89	(2.50, 6.06)			1,98	(1.19, 3.27)
Histologic Grade					< 0.001				0,44
Well Differentiated			1,00				1,00
Moderately			1,95	(0.99, 3.85)			1,23	(0.54, 2.81)
Poorly			3,00	(1.58, 5.68)			1,52	(0.70, 3.34)
The p53 Variables			Unadjusted				Adjusted
			Relative Risk	(95% CI)	p-value[1]		Relative Risk	(95%CI)	p-value[2]
Immunostaining					0,071				0,67
No			1,00				1,00
Yes			1,42	(0.96, 2.08)			1,10	(0.71, 1.69)
Mutation					0,029				0,82
No			1,00				1,00
Yes			1,51	(1.04, 2.20)			0,95	(0.62, 1.47)
Alteration					0,030				0,59
No			1,00				1,00
Yes			1,59	(1.03, 2.47)			1,15	(0.70, 1.90)
Conserved Regions					0,010				0,47
Non Conserved/ WT p53			1,00				1,00
Cons. Region II, III, IV, V			1,66	(1.14, 2.42)			1,17	(0.77, 1.79)
% p53 IM (continuous)					0,13				0,40
0			1,00				1,00
10			1,03	(0.99, 1.08)			0,98	(0.93, 1.03)
20			1,07	(0.98, 1.16)			0,96	(0.87, 1.05)
50			1,18	(0.95, 1.46)			0,90	(0.71, 1.14)
Other Variables			Unadjusted				Adjusted
of Interest			Relative Risk	(95% CI)	p-value[1]		Relative Risk	(95%CI)	p-value[2]
Age [0]					0,019				0,032
50			1,00				1,00
60			1,19	(1.03, 1.38)			1,21	(1.02, 1.44)
70			1,42	(1.06, 1.90)			1,46	(1.04, 2.06)
Metastasis					0,057				0,63
None			1,00				1,00
Any			1,82	(1.02, 3.23)			1,16	(0.63, 2.15)
Lymph Node Status					< 0.001				0,31
Free of Tumor			1,00				1,00
Infiltrated			4,84	(2.00, 11.73)			1,91	(0.76, 4.76)
Not Resected			3,57	(1.54, 8.29)			1,84	(0.76, 4.44)
Histology					0,18				0,37
Serous			1,00				1,00
Other			0,76	(0.51, 1.14)			1,23	(0.78, 1.95)
S-Phase					0,006				0,54
0.0 - 4.9%			1,00				1,00
5.0 - 14.4%			1,76	(0.83, 3.77)			1,44	(0.54, 3.84)
14.5% +			3,29	(1.51, 7.18)			1,79	(0.61, 5.25)
Ploidy					1,00				0,45
Diploid			1,00				1,00
Tetraploid / Aneuploid			1,00	(0.60, 1.68)			0,79	(0.43, 1.46)