Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing. Alexander J Cole, Trisha ...
Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing
Alexander J Cole, Trisha Dwight, Anthony J Gill, Kristie-Ann Dickson, Ying Zhu, Adele Clarkson, Gregory B Gard, Jayne Maidens, Susan Valmadre, Roderick Clifton-Bligh and Deborah J Marsh
Supplementary Table S1. Massively Parallel Sequencing data of all samples identified to contain a TP53 mutation; cDNA aligned to the TP53 sequence NM_000546. Sample which were unable to be confirmed by Sanger sequencing are labelled with ^. a) Displays data from the sixtyeight HGSOCs containing a TP53 mutation. b) Displays data from the three ovarian cancer cell lines OV202, OV207 and OV167. a) Sample ID
Genomic position (chr:startend)
Reference allele (%)
Variant allele (%)
Exon
10-04
17:757953 0-7579530 17:757755 0-7577550
-
T (41%)
4
14-04
cDNA change
c.156_157insA
Protein effect
p.Trp53Metfs*3
Mutation type
Tumor reference allele read count
Frameshift
Tumor variant allele read count 824
1217 C (20%)
A (80%)
7
c.731G>T
p.Gly244Val
Missense
Tumor variant allele ratio
0.41
2439 0.80 608
18-04
17:757753 6-7577536
T (9%)
C (91%)
7
c.745A>G
p.Arg249Gly
Missense
2159 0.91 221
40-05 47-05 64-05 74-05
17:757855 6-7578556 17:757399 6-7573996 17:757753 9-7577539 17:757759 9-7577599
T (34%)
C (66%)
Intron 4
c.376-2A>G
-
Splice
A (32%)
T (68%)
10
c.1031T>A
G (75%)
A (25%)
7
c.742C>T
p.Leu344Gln p.Arg248Trp
Missense Missense
-
A (90%)
7
c.681_682insT
p.Asp228fs*1
Frameshift
758 388 1075 506 392 1182
0.66 0.68 0.25
824 0.90 909
76-05 105-06 106-06 109-06
17:757710 0-7577100 17:757840 6-7578406 17:757840 2-7578402 17:757400 3-7574003
T (37%)
C (63%)
8
c.838A>G
p.Arg280Gly
Missense
C (58%)
T (42%)
5
c.524G>A
p.Arg175His
Missense
G (49%)
C (51%)
5
c.528C>G
G (27%)
A (73%)
10
c.1024C>T
1356 807 1166 1640
Missense p.Cys176Trp p.Arg342*
1444 1392
Nonsense
0.63 0.42 0.51
1219 0.73 457
94-06
17:757855 4-7578554
A (24%)
C (76%)
5
c.376T>G
p.Try126DAsp
Missense
709 0.76 223
99-06
17:757852 7-7578527
A (18%)
C (82%)
5
c.403T>G
p.Cys135Gly
Missense
2251 0.82 410
123-07 133-07 156-07
17:757753 8-7577538 17:757820 8-7578208 17:757827 2-7578272
C (37%)
T (63%)
7
c.743G>A
T (46%)
C (54%)
6
c.641A>G
G (46%)
A (54%)
6
c.577C>T
Missense p. Arg248Gln
1441 839
Missense p.His214Arg
2290 1912
Missense p.His193Tyr
1820 1580
0.63 0.54 0.54
LOH ≥70 %
No. of cases with variant
Functional classificati on (SIFT)
Database Presence (IARC)
No
1
-
No
Loss of WT Loss of WT No
1
Deleterious
Yes
1
Deleterious
Yes
1
-
Yes
No
1
Deleterious
Yes
No
2
Deleterious
Yes
Loss of WT No
1
-
Yes
1
Deleterious
Yes
No
3
Deleterious
Yes
No
1
Deleterious
Yes
Loss of WT Loss of WT Loss of WT No
2
Percent Tumour composition
70
80
80 80 60 60^
80 10 40 70
Yes
1
NA Deleterious
Yes
50
1
Deleterious
Yes
4
Deleterious
Yes
No
2
Deleterious
Yes
No
1
Deleterious
Yes
80
60 70 70 60
157-07 198-08 206-08 230-08
17:757826 5-7578265 17:757705 8-7577058 17:757711 7-7577117 17:757843 1-7578431
A (67%)
G (33%)
6
c.584T>C
p.IIe195Thr
Missense
868 1746
C (57%)
A (43%)
8
c.880G>T
p.Glu294*
Nonsense
A (65%)
G (35%)
8
c.821T>C
p.Val274Ala
Missense
-
T (24%)
5
c.499dupA
p.Gln167Thrfs* 13
Frameshift
826 1098 676 1242
0.33 0.43 0.35
884
No
1
Deleterious
Yes
No
2
NA
Yes
No
1
Deleterious
Yes
No
1
-
No
No
2
Deleterious
Yes
Loss of WT No
1
No
1
No
1
No
2
No
2
NA NA
Yes
No
1
Deleterious
Yes
No
1
Deleterious
Yes
No
1
-
No
No
2
15
50
0.24 3747
237-08 309-09
17:757820 8-7578208 17:757398 2-7573982
T (58%)
C (42%)
6
c.641A>G
p.His214Arg
Missense
C (11%)
A (89%)
10
c.1045G>T
p.Glu349*
Nonsense
50 1755
2382
0.42
1324 0.89 166
337-09 353-09 381-09 416-09 466-10 471-10 472-10 490-10 497-10 521-10
17:757753 8-7577538 17:757704 6-7577046 17:757931 1-7579311 17:757821 2-7578212 17:757400 3-7574003 17:757840 4-7578404 17:757844 0-7578440 17:757686 2-7576862 17:757821 2-7578212 17:757817 6-7578176
C (76%)
T (24%)
7
c.743G>A
p.Arg248Gln
Missense
C (72%)
A (28%)
8
c.892G>T
p.Glu298*
Nonsense
C (79%)
T (21%)
Intron 4
c.375+1G>A
-
Splice
G (87%)
A (13%)
6
c.637C>T
p.Arg213*
Nonsense
578 1879 623 1611 320 1224 570 3767
G 59%)
A (41%)
10
c.1024C>T
p.Arg342*
Nonsense
A (43%)
T (57%)
5
c.526T>A
p.Cys176Ser
Missense
T (39%)
C (61%)
5
c.490A>G
p.Lys164Glu
Missense
-
A (38%)
9
c.983_984insT
p.Phe328Hisfs* 7 p.Arg213*
Frameshift
856 1231 2158 1629 2239 1454
G (52%)
A (48%)
6
c.637C>T
423 1109
Nonsense
2059 2252
C (30%)
T (70%)
Intron 6
c.672+1G>A
-
Splice
0.24 0.28 0.21 0.13 0.41 0.57 0.61 0.38 0.48
1716 0.70 749
531-10
17:757753 8-7577538
C (21%)
T (79%)
7
c.743G>A
p.Arg248Gln
Missense
1748 0.79 461
537-10 543-10
17:757750 67577506 17:757710 6-7577106
C (71%)
A (29%)
7
c.775G>T
p.Asp259Try
Missense
G (19%)
A (81%)
8
c.832C>T
p.Pro278Ser
Missense
846 2074
0.29
2248 0.81 514
565-10 568-10 586-11
17:757692 7-7576927 17:757820 5-7578205 17:757839 8-7578398
C (64%)
T (36%)
Intron 8
c.920-1G>A
-
Splice
C (82%)
A (18%)
6
c.644G>T
p.Ser215Iie
Missense
-
C (78%)
5
c.531_532insG
p.His178Alafs*2
Frameshift
660 1159 661 2980
0.36 0.18
1571 0.78 2018
612-11
17:757840 6-7578406
C (41%)
T (59%)
5
c.524G>A
p.Arg175His
10
Missense
1433 1012
0.59
30 Yes NA 4
40 Yes
Deleterious Yes NA -
Yes Yes
30^ 7 15 10^ 30 80 80
Yes NA
Loss of WT Loss of WT No
1
15 70
Yes
4
Deleterious
Yes
80
1
Deleterious
Yes
Loss of WT No
1
Deleterious
Yes
No
1
Loss of WT No
1
Deleterious -
No
3
Deleterious
Yes
80 50^
60 1
Yes -
30 Yes
20^ 85
85
614-11
17:757844 9-7578449
C (20%)
17:757400 0-7574000
C (24%)
T (80%)
5
c.481G>A
p.Ala161Thr
Missense
1417 0.80 354
630-11
A (76%)
10
c.1027G>T
p.Glu343*
Nonsense
1125 0.76 358
631-11 634-11 638-11
17:757840 6-7578406 17:757819 0-7578190 17:757848 5-7578485
C (53%)
T (47%)
5
c.524G>A
p.Arg175His
Missense
T (54%)
C (46%)
6
c.659A>G
p.Try220Cys
Missense
A (17%)
- (83%)
5
c.445delT
p.Ser149Profs* 20
Frameshift
1527 1707 1314 1553
0.47 0.46
2286 0.83 456
651-11
17:757759 4-7577594
A (13%)
17:757750 9-7577509
C (21%)
T (87%)
7
c.687T>A
p.Cys229*
Nonsense
907 0.87 133
666-11
T (79%)
7
c.772G>A
p.Glu258Lys
Missense
2084 0.79 562
676-11 679-11
694-11 702-11 711-11 764-12 767-12 778-12 787-12 849-13 862-13 879-13 880-13
938-13 943-13
17:757931 0-7579310 17:757757 4-7577574
A (84%)
C (16%)
Intron 4
c.375+2T>G
-
Splice
T (14%)
C (86%)
7
c.707A>G
p.Try236Cys
Missense
17:757825 3-7578254 17:757841 3-7578413 17:757756 6-7577566 17:757934 1-7579345 17:757844 6-7578447 17:757400 3-7574003 17:757753 8-7577538 17:757855 3-7578553 17:757846 1-7578461 17:757753 9-7577539 17:757755 7-7577590
C (34%) C (34%) C (52%)
A (66%) A (66%) A (48%)
6
T (76%)
17:757711 4-7577114 17:757840 7-7578407
268 1417
0.16
1720 0.86
p.Gly199Leu
Missense
5
c.[595G>T];[596G> T] c.517G>T
p.Val173Leu
Missense
C (23%)
7
c.715A>G
p.Asn239Asp
Missense
AATGC (68%) TG (48%)
- (32%)
4
c.342_346del
Frameshift
- (52%)
5
c.483_484del
G (64%)
- (36%)
10
c.1024delC
C (31%)
T (69%)
7
c.G743G>A
p.Ser116Phefs* 32 p.Ala161Leufs* 17 p.Arg342Glufs* 2 p.Arg248Gln
T (59%)
C (41%)
5
c.377A>G
p.Try126Cys
Missense
270 1425
2739
533 1750 862 1821
Frameshift
964 900
Frameshift
515 1430
Missense
1246 567 472 680
C (45%)
A (55%)
5
c.469G>T
p.Val157Phe
Missense
G (41%)
A (59%)
7
c.742C>T
p.Arg248Trp
Missense
-
GGAACTGTTA CACATGTAGT TGTAGTGGAT GGT A (26%)
7
c.723_724dupACC ATCCACTACAAC TACATGTGTAAC AGTTCC c.824G>T
p.Ser241_Cys2 42ThrIleHisTry AsnTyrMetCys AsnSerSerCys p.Cys275Phe
In-frame Insertion
1619 1333 1184 834
8
C (45%)
5
c.523C>G
Deleterious
Yes
3
NA Deleterious
Yes
No
1
Deleterious
Yes
Loss of WT Loss of WT Loss of WT No
1
-
Yes
1
NA
Yes
1
Deleterious
Yes
1
-
Yes
Loss of WT No
1
Deleterious
Yes
1
Deleterious
Yes
No
1
Deleterious
Yes
No
1
Deleterious
Yes
No
1
-
No
No
1
-
No
No
1
-
Yes
No
4
Deleterious
Yes
No
1
Deleterious
Yes
No
1
Deleterious
Yes
No
2
Deleterious
Yes
No
1
-
No
60 1
Yes 35 85 85 90
40
70 5^
80
p.Arg175Cys
0.48 0.23 0.32 0.52 0.36 0.69 0.41 0.55 0.59
50
Missense
N/A 652
Missense
936 1152
0.26 0.45
15^ 20 60 25 70
80 50 50
N/A N/A 1815
G (55%)
1
50 1369 1479
C (74%)
0.66
Loss of WT Loss of WT No
10 No
1
Deleterious
Yes
No
1
Deleterious
Yes
60 40
949-13
17:757953 6-7579536
C (8%)
17:757853 0-7578530 17:757853 0-7578530
A (70%)
17:757712 1-7577121
G (11%)
A (92%)
4
c.151G>T
p.Glu51*
Nonsense
1302 0.92 111
958-13 1001-14
G (30%)
5
c.400T>C
p.Phe134Leu
Missense
467 1081
A (14%)
C (86%)
5
c.400T>G
p.Phe134Val
Missense
0.30
2129 0.86 343
1004-14
A (89%)
8
c.817C>T
p.Arg273Cys
Missense
1394 0.89 164
966-14 969-14
17:757712 0-7577120 17:757845 4-7578407
C (60%)
T (40%)
8
c.818G>A
p.Arg273His
Missense
G (20%)
A (80%)
5
c.476C>T
p.Ala159Val
Missense
651 980
0.40
2118 0.80 534
985-14
17:757705 8-7577058
C (35%)
A (65%)
8
c.880G>T
p.Glu294*
Nonsense
978 519
0.65
Loss of WT No
1
Yes
1
NA Deleterious
Yes
Loss of WT Loss of WT No
1
Deleterious
Yes
1
Deleterious
Yes
1
Deleterious
Yes
Loss of WT No
1
Deleterious
Yes
80 30
80
90 30
80 2
Yes NA
20
b) Sample ID
Genomic position (chr:startend)
Reference allele (%)
Variant allele (%)
Exon
OV202
17:75775597577559
G (1%)
A (99%)
7
17:75771207577120
C (1%)
17:75770467577046
C (1%)
cDNA change
c.722C>T
Protein effect
p.Ser241Phe
Mutation type
Tumor reference allele read count
Missense
Tumor variant allele read count
Tumor variant allele ratio
2257 0.99 14
OV207
T (99%)
8
c.818G>A
p.Arg273His
Missense
2139 0.99 19
OV167
A (99%)
8
c.892G>T
p.Glu298*
Nonsense
3026 0.99 14
LOH ≥70%
No. of cases with variant
Database Presence (IARC)
Percent Tumour composition
Loss of WT Loss of WT Loss of WT
Not applicable
Deleterious
Yes
NA
Not applicable
Deleterious
Yes
NA
Not applicable
NA
Yes
NA
Functional classification (SIFT)
Supplementary Table S2. p53 Immunohistochemical staining of (a) 72 high-grade serous ovarian cancer samples and (b) 4 low-grade serous ovarian cancer samples. a) Sample ID
% cells stained positive for p53
IHC STATUS
TP53 Status
10-04
2
Low
Frameshift
14-04
100
High
Missense
18-04
100
High
Missense
40-05
100
High
Splice
47-05
100
High
Missense
64-05
100
High
Missense
74-05
2
Low
Frameshift
76-05
100
High
Missense
105-06
100
High
Missense
106-06
100
High
Missense
109-06
100
High
Nonsense
94-06
100
High
Missense
99-06
100
High
Missense
123-07
100
High
Missense
133-07
100
High
Missense
156-07
100
High
Missense
157-07
100
High
Missense
198-08
4
Low
Nonsense
206-08
100
High
Missense
230-08
0
Low
Frameshift
237-08
100
High
Missense
309-09
100
High
Nonsense
337-09
100
High
Missense
353-09
55
Intermediate
Nonsense
381-09
0
Low
Splice
416-09
1
Low
Nonsense
427-09
65
Intermediate
Wild-type
466-10
90
High
Nonsense
471-10
100
High
Missense
472-10
100
High
Missense
490-10
100
High
Frameshift
493-10
100
High
Wild-type
497-10
2
Low
Nonsense
521-10
2
Low
Splice
531-10
100
High
Missense
537-10
100
High
Missense
543-10
100
High
Missense
565-10
85
High
Splice
568-10
100
High
Missense
586-11
0
Low
Frameshift
612-11
100
High
Missense
614-11
100
High
Missense
630-11
85
High
Nonsense
631-11
100
High
Missense
634-11
100
High
Missense
638-11
0
Low
Frameshift
651-11
0
Low
Nonsense
666-11
100
High
Missense
676-11
0
Low
Splice
679-11
100
High
Missense
694-11
95
High
Missense
695-11
0
Low
Wild-type
702-11
100
High
Missense
711-11
100
High
Missense
764-12
0
Low
Frameshift
767-12
0
Low
Frameshift
778-12
100
High
Frameshift
787-12
100
High
Missense
849-13
100
High
Missense
862-13
100
High
Missense
879-13
100
High
Missense
880-13
100
High
In-frame Insertion
881-13
40
Intermediate
Wild-type
938-13
80
High
Missense
943-13
100
High
Missense
949-13
0
Low
Nonsense
958-13
100
High
Missense
1001-14
100
High
Missense
1004-14
100
High
Missense
966-14
100
High
Missense
969-14
100
High
Missense
985-14
1
Low
Nonsense
b) Sample ID
% cells stained positive for p53
IHC STATUS
TP53 Status
544-10
8
Intermediate
Wild-type
624-11
40
Intermediate
Wild-type
909-13
65
Intermediate
Wild-type
730-12
55
Intermediate
Wild-type
Supplementary Table S3. Clinical data for a) 72 high-grade serous ovarian cancer; and, b) 4 low-grade serous ovarian cancer. a)
3C 1C
Status at last Follow Up (0Alive, 1-Dead) 1 1
Duration of follow-up (months) 34 18
High
3B
1
77
High High High High High High High High High High High High High High High High High
3A 2B 2A 2C 3C 3B 3C 3C 3C 3C 3C 3C 3C 3B 3C 3C 3C
0 1 1 0 1 1 0 1 1 0 1 0 0 1 1 0 0
0 56 10 62 44 23 89 36 23 85 1 80 75 47 14 73 70
Sample ID fi 10-04 14-04
Age
Type
Site
Grade
Stage
58 86
Serous adenocarcinoma Serous adenocarcinoma
High High
18-04
57
Serous adenocarcinoma
40-05 47-05 64-05 74-05 76-05 105-06 106-06 109-06 94-06 99-06 123-07 133-07 156-07 157-07 198-08 206-08 230-08
62 60 68 45 60 77 82 68 63 79 60 59 73 50 77 73 66
Serous adenocarcinoma Serous adenocarcinoma Carcinosarcoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma
Ovary Ovary Fallopian Tube Uterus Ovary Ovary Ovary Ovary Ovary Ovary Ovary Peritoneum Ovary Peritoneum Ovary Peritoneum Ovary Ovary Ovary Ovary
237-08 309-09 337-09 353-09 381-09
63 56 38 62 55
Serous adenocarcinoma Papillary serous carcinoma Serous adenocarcinoma Serous carcinoma Serous adenocarcinoma
416-09
62
Serous adenocarcinoma
427-09 466-10 471-10 472-10 490-10 493-10 497-10
30 79 82 57 64 68 43
Serous adenocarcinoma Serous adenocarcinoma Serous carcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma
521-10
36
Serous adenocarcinoma
531-10 537-10
77 79
Endometrioid adenocarcinoma Serous adenocarcinoma
543-10
46
Serous adenocarcinomainoma
565-10 568-10 586-11 612-11 614-11
61 80 51 54 76
Serous adenocarcinoma Serous carcinoma Serous adenocarcinoma Serous adenocarcinoma Serous adenocarcinoma
630-11
70
Serous adenocarcinoma
Ovary Ovary Peritoneum Ovary Ovary Fallopian Tube Ovary Ovary Ovary Ovary Ovary Ovary Ovary Fallopian Tube Ovary Ovary Fallopian tube Ovary Ovary Ovary Ovary Ovary Fallopian Tube
High High High High High
3C 3b 3B 3B 2A
0 0 1 1 1
62 17 22 9 33
High
3B
1
7
High High High High High High High
3B 3C 3c 3B 3C 3B 3C
0 1
8 32
1 1 1 0
18 37 33 29
High
3C
0
45
High High
2C 3C
1 1
33 34
High
2C
0
42
High High High High High
3B 3b 3C 3C 1A
0 1 0 0 0
29 1 38 31 37
High
3C
0
35
631-11 634-11
61 52
Serous adenocarcinoma Serous papillary carcinoma
638-11
63
Serous carcinoma
651-11
62
Serous carcinoma
666-11
46
Serous adenocarcinoma
676-11
52
Serous adenocarcinoma
679-11
60
Serous adenocarcinoma
694-11 695-11
56 68
Serous adenocarcinoma Serous adenocarcinoma
702-11
81
Serous adenocarcinoma
711-11
62
Serous adenocarcinoma
764-12 767-12
67 82
Serous adenocarcinoma Serous adenocarcinoma
778-12
48
Serous adenocarcinoma
787-12 849-13 862-13
34 56 72
Serous adenocarcinoma Serous carcinoma. Serous carcinoma
879-13
83
Serous carcinoma
880-13 881-13
62 73
Serous carcinoma Serous carcinoma
Ovary Ovary Fallopian tube Fallopian tube Fallopian Tube Fallopian Tube Fallopian Tube Ovary Ovary Fallopian Tube Fallopian Tube Ovary Ovary Fallopian Tube Ovary Ovary Ovary Fallopian tube Ovary Ovary
0 1
36 36
1A
0
35
High
2A
0
34
High
3C
1
16
High
3C
0
31
High High
3C 2B
0 0
26 23
High
2C
1
3
High
3C
1
15
High High
3C 2B
0 0
3 24
High
3C
0
22
High High High
3B 3C 3C
0 0 0
24 20 15
High
1A
0
16
High High
3C 3C
1 0
12 16
High High
3C 3C
High
1C
High
938-13 943-13 949-13 958-13 100114 100414 966-14 969-14 985-14
80 89 60 61
Serous carcinoma Serous carcinoma Serous carcinoma Serous carcinoma
Ovary Ovary Ovary Ovary
High High High High
3C 3C 3C 3C
0
11
0 0
0 9
60
Serous carcinoma
Ovary
High
3C
0
0
55
Serous carcinoma
Ovary
High
3C
0
0
76 76 57
Serous carcinoma Serous carcinoma Serous carcinoma
Ovary Ovary Ovary
High High High
3C 3C 3C
0 0 0
0 5 5
Status at last Follow Up (0Alive, 1-Dead) 0 0 0 0
Duration of follow-up (months) 35 11 11 26
b)
Sample ID ii 544-10 624-11 909-13 730-12
Age
Type
Site
Grade
Stage
61 78 69 47
Serous border tumour Serous border tumour Serous adenocarcinoma Serous adenocarcinoma
Ovary Ovary Ovary Ovary
Low Low Low Low
1C 1A 1A 3C
Supplementary Table S4. Cell line typing for OV207
Marker D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 Amel D5S818 FGA
OV207 13 31.2 8 11 14,18 9.3 12 13 17,24 13,14 15,16 8 12 X 11,12 20
Cell typing was performed by CellBank Australia (Westmead NSW 2145, Australia) using the AmpFl STR Identifiler PCR Amplification Kit (Applied Biosystems), a 16 loci (15 STR loci plus Amelogenin) STR multiplex kit. To our knowledge, a profile for OV207 has not previously been available.
Supplementary Table S5. TP53 primer pairs used from the IARC TP53 Database for Sanger sequencing. Exon Exon 4
Product size (bp) 413
Exons 5 and 6
467
Exon 7
237
Exons 8 and 9
445
Exon 10
260
Exon 11
245
Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5'-3' TGAGGACCTGGTCCTCTGAC AGAGGAATCCCAAAGTTCCA TGTTCACTTGTGCCCTGACT TTAACCCCTCCTCCCAGAGA CTTGCCACAGGTCTCCCCAA AGGGGTCAGAGGCAAGCAGA TTGGGAGTAGATGGAGCCT AGTGTTAGACTGGAAACTTT CAATTGTAACTTGAACCATC GGATGAGAATGGAATCCTAT AGACCCTCTCACTCATGTGA TGACGCACACCTATTGCAAG
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1. Kaplan-Meir analyses comparing overall survival with a) TP53 mutation type (missense, frameshift, splice, nonsense or wild-type (WT)), b) missense versus all other TP53 mutations (excluding the single insertion duplication mutation), c) TP53 mutations located within the p53 DNA binding domain (DBD) versus outside of this domain, and d) p53 expression levels determined by p53 immunohistochemistry; ‘Low’ (L), ‘Intermediate’ (I) or ‘High’ (H). Supplementary Figure S2. Additional representative images showing p53 IHC category, percent positive p53 nuclei and TP53 mutation status. Supplementary Figure S3. Representative images showing p53 staining patterns of wildtype TP53 tumours. ^LGSC; *non-neoplastic, infiltrating lymphocytes staining blue. Supplementary Figure S4. Representative images of cell pellets (p53 null SK-OV-3, wildtype TP53 A2780, nonsense TP53 mutant OV167 and TP53 missense mutant OVCAR-3) showing p53 staining patterns. Supplementary Figure S5. Massively parallel sequencing analysis pipeline. Fresh frozen tissue was collected from women with ovarian cancer by Kolling Institute Gynaecological Tumour Bank staff. DNA was extracted and TP53 analyzed using the Fluidigm Access Array™ system followed by MPS using a MiSeq platform (Illumina). Raw results were processed using a bioinformatics pipeline. Analysis was performed using ANNOVAR. Variants were filtered against the 1000 genomes to exclude common SNPs. SIFT scores were used to remove all tolerated mutations and TP53 mutations were visualised using IGV.
1