Assessing mutant p53 in primary high-grade serous ovarian cancer ...

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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