29872083

Comprehensive analysis of CTNNB1 in adrenocortical carcinomas: Identification of novel mutations and correlation to survival.

Rajani Maharjan*, Samuel Backman, Tobias Åkerstöm, Per Hellman, Peyman Björklund.

Affiliations: Department of Surgical Sciences, Uppsala University, Uppsala, Sweden.

Corresponding Author: Rajani Maharjan Experimental Surgery Group, Department of Surgical Sciences. Rudbeck Laboratory, R3 75185 Uppsala, Sweden. Phone: +46184714706 Email: rajani.maharjan@surgsci.uu.se

APC

p. T1556Nfs Ter3

p.E1462Gfs Ter8

c.4666_4667insA

c.4391 4394del

ACC25

Tumour

Tumour

G

A

A

A

A

A

A

-

C

T

A

T

T

G

A

G

A

G

A

G

A

G

A

G

T

G

G

Control

Control

CTNNB1

p.S45P

p.135N c. 104T>A

ACC26

C

T

G

G

A

A

T

C

C

A

T

T

C

Tumour

G

C

T

C

C

T

T

C

T

C

T

G

A

C

T

G

G

A

A

T

C

C

A

T

T

C

Control

Control

p.S45Y c. 136C>A

p.S45C c. 109C>G

ACC43

C

C

À

T

T

G

T

G

G

T

G

C

Tumour

C

T

C

C

T

T

C

T

C

T

G

A

G

T

C

C

A

T

T

C

T

G

G

T

G

C

Control

Control

p.S45F

ACC53

C

T

C

C

T

T

T

T

C

G

A

G

G

A

C

T

C

T

G

G

A

A

T

C

C

C

T

C

C

T

T

C

T

C

T

G

A

G

Control

Control

ZNRF3

p.H216Y c.646C>T

ACC31

C

G

G

T

T

T

A

C

A

G

G

A

Tumour

C

G

G

T

T

T

C

À

C

A

G

G

À

C

T

C

C

T

T

C

T

C

T

G

A

G

ACC28

c. 133T>C

ACC6

G

C

T

C

C

T

T

C

T

C

T

G

A

Tumour

A

A

A

A

A

A

A

C

T

A

T

T

G

ACC51

A

G

A

G

A

G

A

G

A

A

T

G

T

c. 134C>T

G

A

C

T

C

T

G

G

A

A

T

C

C

Tumour

Tumour

T

Tumour

p.G34R c. 105G>A

ACC46

T

T

Control

Supplementary Figure S1. Electropherogram showing CTNNB1, ZNRF3 and APC mutations.

CTNNB1 amino acid residue

34 35 3 25

Danio rerio MATQSDLMELEMAMDPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEDDDVD-N Xenopus tropicalis MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEDEDVDTN Homo sapiens MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTS Mus musculus MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTS ******************************************************:*** .

ZNRF3 amino acid residue 216

Danio rerio DGEELRVIPCTHRFHKRCVDPWLLQNHTCPHCRHNIIEQKKGGHGPVCVENSSNRGRQQQ Xenopus tropicalis DGEELRVIPCAHRFHKKCVDPWLLQHHTCPHCRHNIIDQKKGNPGAVCLDPGNPV — HGR Homo sapiens DGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQ-KGNPSAVCVETSNLS — RGR Mus musculus DGEELRVIPCTHRFHRKCVDPWLLQHHTCPHCRHNIIEQ-KGNPGAVCVETSNLT — RGR **********:****::********************** **. . **:: .. : :

Supplementary Figure S2. Protein sequence alignment and comparison between Danio rerio, Xenopus tropicalis, Homo Sapiens and Mus musculus. The amino acid residues affected by the mutations are highlighted.

Supplementary Figure S3a) Gel electrophoresis on the cDNA. PCR products showing four ACC tumours with shorter product (630 bases) in addition to the wildtype product (920 bases). b) Electropherogram of ACC 18, ACC 21, ACC 37 and ACC 40 showing deletion of exon 2 and 3 in cDNA unlike non mutant control.

a

b

ACC 37

ACC 40

ACC 21

ACC 18

Control

1000bp

C

C

T

G

A

G

4

T

A

T

T

G

500bp

ACC 18

250bp

C

C

T

G

A

G

A

T

1

I

T

G

ACC 21

A2+3 deletion

C

C

T

G

À

G

A

T

A

I

T

G

ACC 37

C

C

T

G

A

G

A

T

A

T

T

G

ACC 40

Exon 1

Exon 4

T

AGCTGAT

A

T

T

G

Non mutant

Exon 3

Exon 4

Supplementary Figure S4. Schematic illustration showing simplified ß-Catenin mRNA and protein structure (based on crystal structure studies1,2) with different binding regions and A(2+3) deletion site.

C

N

Protein

4(2+3)del-

11

141

Armadilo repeats

664

781

N

1

2

3

4 5

mRNA

6

7

8

9

10

11

12

C

ß- TrCP

TCF

GSK3ß, CK1 phosphorylation sites

Axin

E-cadherin

APC

LEF-1

Supplementary Figure S5. Original Western blot for Figure 3a.

B-catenin

Actin

Cyclin D1

Cyclin D1

Supplementary Figure S6. mRNA expression of Cyclin D1 in tumour samples harboring APC mutation, CTNNB1 mutation and 4(2+3) deletion in comparison to rest of the cohort devoid of CTNNB1 mutation or deletion (*, p=0.0144).

p=0.1839

95053

御

p=0.1818

* ☒

Relative expression

王

2

1

0

ACC wildtype

APC mutation

CTNNB1mutation

CTNNB14(2+3) del

Supplementary Figure S7. mRNA expression of ZNRF3, AXIN2 and LEF1 in tumour samples harboring APC mutation, ZNRF3 alterations, CTNNB1 missense mutation and A(2+3) deletion in comparison to rest of the cohort (p values for AXIN2 and ZNRF3 for ACC wildtype vs APC mutation are 0.1239 and 0.5813 respectively).

ZNRF3

AXIN2

LEF1

4

*

4

4

**

1

4

**

1

**

Relative expression

*

1

---

4

Relative expression

**

1

4

*

1

Relative expression

**

ns

1

ns

**

3

1

4

4

3

3

2

2

E

2

1

1

1

0

ACC wildtype

APC mutation

ZNRF3 alterations

CTNNB1 mutation

CTNNB1 A(2+3)del

O

ACC wildtype

APC mutation

ZNRF3 alterations

CTNNB1 mutation

CTNNB1 A(2+3)del

0

ACC wildtype

APC mutation

ZNRF3 alterations

CTNNB1 mutation

CTNNB1 A(2+3)del

Supplementary Figure S8. SNP array analysis showing a) summary of events at CTNNB1 locus 3p22.1 in the tumours without CTNNB1 mutations b) summary of events at CTNNB1 locus 3p22.1 for tumours with CTNNB1 missense mutation and A(2+3) deletion (top panel) followed by details of each mutant sample. In the figure; red represents loss, blue represents gain and tan yellow represents loss of heterozygosity.

a

100%

50%

50%

100%

H

CTNNB1

HH HHHH 5678

5

H1

HH

16

17

A

b

100%

50%

50%

100%

H

CTNNB1

HH HHHH

5 678

5

답

3

HH

17

ACC 6

ACC 26

A(2+3) del CTNNB1 Mutation

ACC 28

ACC 43

ACC 46

ACC 53

ACC 18

ACC 21

ACC 37

ACC 40

Total ß-catenin

ACC 47

Actin

ACC 32

ACC 42

Active ß-catenin

ACC 24

Actin

ACC 27

ACC 33

ACC 19

ACC 35

Total ß-catenin

ACC 29

ACC 25

ACC 11

ACC 46

Actin

ACC 43

ACC 15

ACC 50

Active ß-catenin

ACC 18

ACC 53

ACC 37

ACC 16

Actin

ACC 40

ACC 20

ACC 7

ACC 17

ACC 23

Total ß-catenin

ACC 22

ACC 2

ACC 41

ACC 38

ACC 4

ACC 52

Actin

ACC 28

ACC 10

Active ß-catenin

ACC 31

ACC 21

ACC 34

ACC 1

Actin

ACC 44

ACC 8

ACC 9

ACC 30

ACC 51

Total ß-catenin

ACC 6

ACC 14

Actin

ACC 55

ACC 49

Active ß-catenin

ACC 26

ACC 5

ACC 54

ACC 48

Actin

ACC 13

Supplementary Figure S9. Western blot on ACC tumours showing expression levels of total and active ß-catenin and actin. The samples with APC mutation, CTNNB1 missense mutation, CTNNB1 4(2+3) deletion and ZNRF3 deletions are highlighted in green, pink , red and blue respectively. Uncropped original blots are shown in Supplementary Fig S16.

		Cytoplasmic
		Negative	Positive
			Low	High
Nuclear		☒	☒ ☒ ☒	☒ ☒
	Negative	☒ ☒	☒ ☒ ☒ ☒ ☒ ☒	☒ ☒
				☒ ☒ ☒
	Low Positive		☒ ☒ ☒	☒ ☒
	Focal High			☒ ☒ ☒
	Low Positive High	☒	☒ ☒	☒ ☒ ☒
			☒	☒
			☒	☒ ☒ ☒ ☒ ☒ ☒ ☒ ☒

Supplementary Figure S10. Tumours grouped according to the immunohistochemical staining of B-catenin. CTNNBI mutants are marked in red and the rest of the samples are marked in black. The samples were scored and categorized as “negative” or “positive” for cytoplasmic staining and as “negative”, “focal positive” (heterogeneous) and “positive” for nuclear staining. For positive cases the nuclear and cytoplasmic staining intensity were scored manually ascending from 1+ to 5+. Samples with 1+ to 2+ scoring were grouped as “low” and those with 3+to 5+ scoring were grouped as “high”.

ACC 6

ACC 26

ACC 28

€

€

ACC 43

ACC 46

ACC 53

ACC 21

ACC 37

ACC 40

b)

ACC 18

ACC 5	ACC 15	ACC 25
ACC 7	ACC 16	ACC 27
ACC 8	ACC 17	ACC 30
ACC 9	ACC 22	ACC 31
ACC 13	ACC 24	ACC 32

ACC 33	ACC 38		ACC 52
ACC 34	ACC 41	ACC 55
			A
ACC 35	ACC 50

ACC 4		ACC 19	ACC 44
ACC 10		ACC 20	ACC 47
ACC 11		ACC 29	ACC 48
ACC 12		ACC 23	ACC 49
ACC 14		ACC 42	ACC 54

ACC 51

Supplementary Figure S11. Photomicrographs showing different groups of ACC in terms of ß- catenin nuclear expression. a) tumours with CTNNB1 mutation and nuclear expression b) tumour with CTNNB1 mutation but without nuclear expression; c) tumours without CTNNB1 mutation and nuclear expression d) tumours without CTNNB1 mutation and with nuclear expression.

Supplementary Fig S12: Comparative analysis of active beta catenin expression and nuclear expression in ACC.

40

30

Relative active ß-catenin expression

20

10

3

2

1

0

ß-catenin Nuclear expression

Hormone

ß-catenin mutation status

ß-catenin nuclear expression

Hormone

☒ CTNNB1 missense mutation

☐ Nuclear expression present

☐ Cortisol

☒ CTNNB1 wildtype

☐ Nuclear expression absent

☐ Androgen

☒ Not available

☐ Aldosterone

b

Supplementary Figure S13. a) Age at operation and b) tumour size comparison between group of tumours with APC/CTNNB1/ZNRF3 alterations versus tumours wildtype for APC/CTNNB1/ZNRF3.

a

Age

Tumour Size

100

p=0.3517

40

p=0.5731

80

age in years

length in cm

30

60

20

40

20

10

0

APC/CTNNB1/ZNRF3 alterations

APC/CTNNB1/ZNRF3 wildtype

0

APC/CTNNB1/ZNRF3 alterations

APC/CTNNB1/ZNRF3 wildtype

Supplementary Figure S14. Disease-free survival graph for a) patients with tumour harbouring APC/CTNNB1/ZNRF3 alterations versus the wildtype b) patients with tumour harbouring CTNNB1 mutations alone versus the wildtype.

a

Disease-free Survival

b

Disease-free Survival

100

- APC/CTNNB1/ZNRF3 wildtype

100

- CTNNB1 mutation absent

Disease-free survival

-L. APC/CTNNB1/ZNRF3 alterations Log rank p=0.1152

Disease-free survival

-L. CTNNB1 mutation present Log rank p=0.1148

50

50

0

0

0

100

200

300

400

0

100

200

300

400

Time (months)

Time (months)

Survival

100

nuclear expression absent

Overall survival

nuclear expression present

Log rank p= 0.1555

50

0

0

100

200

300

400

Time (months)

Survival

Supplementary Figure S15. Overall survival graph for patients with tumour showing nuclear expression of ß-catenin versus those lacking the expression in whole cohort (top) and in adult ACC (bottom).

nuclear expression absent

100

nuclear expression present

Overall survival

Log rank p=0.0897

50

0

0

100

200

300

400

Time (months)

Pannel 2

Pannel 1

Total ß-catenin

Total ß-catenin

Actin

Actin

Active ß-catenin

Active ß-catenin

Actin

Actin

Pannel 3 Pannel 4

Total B-catenin

Total ß-catenin

Actin

Actin

Active ß-catenin

Active ß-catenin

Actin

Actin

Pannel 5

Total ß-catenin

Actin

Active ß-catenin

1

Actin

Pannel 6 Total ß-catenin

Actin

Active ß-catenin

Actin

Pannel 7

Total B-catenin

Total ß-catenin

Pannel 8

Actin

Actin

Active ß-catenin

Active ß-catenin

Actin

Actin

Supplementary Figure S16. Original blots for Supplementary Figure S9. The samples enclosed by the red box represent the samples included in Supplementary Figure S9. Panel 1, 2, 3, 4, 5, 6, 7, and 8 in the figure corresponds to the panel comprising samples ACC 47 - ACC 25, ACC 46 - ACC 52, ACC 29 - ACC 40, ACC 17- ACC 8, ACC 22 - ACC 44, ACC 9 - ACC 48, ACC 30 - ACC 54, and ACC 13 in Supplementary Figure S9 respectively.

Supplementary Table S1: Missense mutation details

Sample ID	CTNNB1 mutation		Mutation Type	SIFT	Polyphen-2	PROVEAN
	cDNA	Protein
ACC6	c.133T>C	p.S45P	Missense	Damaging (0)	Prob. Damaging (0.988)	Deleterious (-2.928)
ACC 26	c.104T>A	p.I35N	Missense	Damaging (0)	Prob. Damaging (1.00)	Deleterious (-5.175))
ACC 28	c.136C>A	p.S45Y	Missense	Damaging (0)	Prob. Damaging (0.999)	Deleterious (-3.620)
ACC 43	c.109C>G	p.S37C	Missense	Damaging (0)	Prob. Damaging (1.00)	Deleterious (-3.951)
ACC 46	c.105G>A	p.G34R	Missense	Damaging (0)	Prob. Damaging (1.00)	Deleterious (-6.200)
ACC 53	c. 134C>T	p.S45F	Missense	Damaging (0)	Prob. Damaging (0.996)	Deleterious (-3.849)

Sample ID	ZNRF3 mutation		Mutation Type	SIFT	Polyphen-2	PROVEAN
	cDNA	Protein
ACC31	c.646C>T	p.H216Y	Missense	Damaging (0)	Prob.Damaging (0.999)	Deleterious (-5.577)

Sample ID	APC mutation		Mutation Type	SIFT	Polyphen-2	PROVEAN
	cDNA	Protein
ACC25	c.4666_4667insA	p.T1556NfsTer3	Insertion	-	-	-
ACC51	c.4391_4394del	p.E1462GfsTer8	Deletion	-

Supplementary Table S2: Primer used for detection of deletion in DNA
ACC 18
Fw primer	GCTTCACTGTATGTATGATC
Rw primer	CTGACTTTCAGTAAGGCAATG
ACC 21
Fw primer	CTAGAACTTCTGGTGATATGG
Rw primer	CTGTGTAGATGGGATCTGC
ACC 37
Fw primer	CTGCTAGTTTCACCATATCC
Rw primer	CTCACTATCCACAGTTCAGC
ACC 40
Fw primer	CTGCTAGTTTCACCATATCC
Rw primer	CTGACTTTCAGTAAGGCAATG

Supplementary Table S3: Details of deletion mutation

Sample	Deletion (genome)	Mutation(coding DNA)	Mutation(RNA)
ACC18	g.41217632_g.41224675del	c .- 48-6389_163del	r .- 48_241del
ACC21	g. 41220754_g.41224725delinsA	c .- 48-3267_213del	r .- 48_241del
ACC37	g. 41218557_g. 41224902delinsT	c .- 48-5464_241-51del	r .- 48_241 del
ACC40	g.41218317_g.41224723del	c .- 48-5704_211del	r .- 48_241del

Transcript ID: ENST00000349496.9

Supplementary Table S4. Analysis of sequences at deletion junctions.
Sample	Deletion	Sequence at the site of deletion
ACC18	g.41217632_g.41224675del	CAAAGGGTAGttacacttta cctgaggaagAGGATGTGG
ACC40	g.41218317_g.41224723del	TAATACAATActtgaattgc cagggattttCTCAGTCCTT
ACC37	g. 41218557_g.41224902delinsT	GTTAAACTAAgagttgtttt. tagcaaatacTTAGGTAAA
ACC21	g.41220754_g.41224725delinsA	ATTATGAATTgctagtataa gggattttctCAGTCCTTCA

Two of the samples harboring the 4(2+3) deletion (ACC 18, ACC 40) displayed sequence microhomology whereas the other two samples (ACC 37, ACC 21) harbored reverse complement sequence at the breakpoint. The sequences in lowercase font depict the deleted regions and the uppercase font on the left and right depict deletions start and end sites. The microhomologous sequences are underlined whereas the reverse complement sequences are bold and underlined. The samples harboring reverse complement sequences also had similar patterns of deletion where an additional nucleotide was inserted at the breakpoint, unlike the ones displaying microhomology that harbored a defined breakpoint.

Supplementary Table S5: Sequence at deletion site.
ACC18
	deletion start
CAAAGGGTAG	TTACACTTTA
CCTGAGGAAG	AGGATGTGGA
deletion end
ACC21
	deletion start
ATTATGAATT	GCTAGTATAAAA
GGGATTTTCT	CAGTCCTTCACT
deletion end
ACC37
	deletion start
GTTAAACTAA	GAGTTGTTTT
TAGCAAATAC	TTAGGTAAAT
deletion end
ACC40
	deletion start
TAATACAATA	CTTGAATTGC
CAGGGATTTT	CTCAGTCCTT
deletion end

Several small identical (flanking microhomology), complement or reverse complement sequences near the breakpoints were observed. Similar sequence are highlighted in red, reverse complement sequence in green and complementary sequence in blue

Supplementary Table S6: Nucleotide percentile in Deletion start site

Tumours	Nos. (T)	Nos. (A)	Nos. (G)	Nos. (C)	AT(percent)
ACC18	36T	34A	17G	13C	70%
ACC21	43T	30A	20G	07C	73%
ACC 37	31T	35A	21G	13C	72%
ACC 40	47T	25A	11G	17C	72%

Supplementary Table S7. Cohort details

ACC case	Mutations	Gene	Nuclear Accumulation	Age	Gender	Event (OS) (yr/mnth)	DFS (yr/mnth)	Tumour Size(cm)	TNM	ENSAT Stage	Hormone Production
1	No		NA	32	M	28 y/8 m	28 y/8 m	7x5x5	T2 N0 M0	II	Cortisol
2	No		NA	60	F	₮ 11 y/10 m	*11y10m	9x8x6	T2 N0 M0	II	None
3	No		NA	45	F	1 1 y/2 m	fly/2 m	10x6x3	T2 N0 M0	III	Cortisol
4	No		+	66	F	₮ 18 y/6 m	+4y/5m	7x6	T2 N0 M0	II	None
5	No		-	53	F	1 7 y/3 m	12y/5m	4.5x7.5x10	T3 N0 M0	III	Androgen
6	c.133T>C	CTNNB1	+	32	F	£8 m	+8m	8x6	T2 N0 M0	II	None
7	No		-	60	M	1 5 y/2 m	14y	20x20	T2 N0 M0	II	Cortisol+Aldosteron
8	No		-	21	M	13 y/10 m	+6m	18.5x14x10	T2 N0 M0	II	Androgen
9	No		-	20	F	24 y/11 m	24y/11 m	5x5	T1 N0 M0	I	Androgen
10	No		+	69	F	Ily	₹11m	30x25×20	T3 N1 M0	III	None
11	No		+	45	F	1 4 y/7 m	13y/5m	9x5x4	T2 N0 M0	II	Cortisol+Androgen
12	No		+	60	F	1 3 y/4 m	fly	9x9x10	T3N1M0	III	Androgen
13	No		-	63	F	1 2 y/3m	12y/3m	6.5x5x9	T3N1M0	III	Cortisol
14	No		+	36	F	1 1 y/6 m	*1y/6 m	8.5x7x11	T3 N1 M0	III	None
15	No		-	46	M	1 4 y/2 m	12y/4m	12×14	T3 N1 M0	III	Cortisol
16	No		-	58	F	22 y/8 m	22 y/8 m	5x5.5x6	T2 N0 M0	II	None
17	No		-	38	F	1 2 y/1 m	+2 y/1 m	10 x 5	T3 N0 M0	III	None
18	c .- 48- 6389_163del	CTNNB1	-	29	F	1 3 y/7 m	+9m	11x10x7	T2 N0 M0	II	Cortisol+Androgen
19	No		+	NA	F	NA	NA	NA	NA	NA	NA
20	No		+	62	M	1 2 y/1 m	*ly/2m	6x4x5	T3 N0 M0	III	Cortisol
21	c .- 48- 3267_213del	CTNNB1	+	41	M	Ily	15m	15x11x8	T3 N0 M0	III	Cortisol
22	No		-	52	M	1 4 y/1 m	*ly/10m	5x3.5	T3 N0 M0	III	Cortisol+Aldosteron e
23	No		+	70	M	£ 5 m	-	10x7x5	T4 N0 M1	IV	Androgen
24	No		-	26	F	£ 10 y/1 m	-	10×14	T4 N0 M1	IV	Cortisol+Androgen

25	c.4666 4667i nsA	APC	-	47	F	1 6y/5 m	15y/10m	5x5	T1 N0 M0	I	None
26	c. 104T>A	CTNNB1	+	74	F	₮ 11y/8 m	*11y/8 m	16x15x12	T4 N0 M0	III	Cortisol+Androgen
27	No		-	67	M	1 9 y/6 m	*1y/7m	25x17x11	T3 N0 M0	III	None
28	c.136C>A	CTNNB1	+	60	F	₮ 11 y/8 m	*11 y/8	7.5x7.5	T2 N0 M0	II	Cortisol+Androgen
29	No		+	34	M	14 y/3 m	14 y/3 m	15x15	T2 N0 M0	II	None
30	No		-	62	F	₮ 13 y/2 m	+4y/9m	18x18x10	T2 N0 M0	II	None
31	c.646C>T	ZNRF3	-	62	M	1 9 y/7 m	+4y/5m	15x13x9	T2 N0 M0	II	Cortisol
32	No		-	60	M	£ 10 m	+1m	5.5x5.5x6	T4 N0 M0	III	Cortisol
33	No		-	15	F	9 y/3 m	9y/3m	9x8x6	T2 N0 M0	II	Androgen
34	No		-	58	M	7 y/8 m	7y/8m	10x8x6	T2 N0 M0	II	None
35	No		-	57	F	£3 m	12m	19x9x10	T2 N0 M0	II	Cortisol+Androgen
37	c .- 48-5464 241-51del	CTNNB1	+	78	M	₮4 m	-	9x9x10	T2 N0 M1	IV	Cortisol+Aldosteron e
38	No		-	75	M	6y/7m	6y/7m	30x20x8	T2 N0 M0	II	Cortisol
40	c .- 48-	CTNNB1	+	38	F	£13 m	+3m	12x8x5	T4 N0 M0	III	None
41	No		-	64	F	5 y/2 m	5y/2m	11x9x8	T4 N0 M0	III	Cortisol+Androgen
42	No		+	28	M	£4 m	-	10x10x6.5	T4 N0 M1	IV	Cortisol+Aldosteron
43	c.109C>G	CTNNB1	+	3	M	4 y/8 m	4y/8m	7x4.5x4.5	NA	III	Cortisol+Androgen
44	No		+	67	F	5 y/1 m	+3m	13x10.5x7	NA	III	None
46	c.105G>A	CTNNB1	+	67	F	Ily	+1m	8.5x5x5	T3 N1 M0	III	Cortisol
47	No		+	50	F	£ 9 m	+3m	8x8x6	T4 N0 M0	IV	Cortisol
48	No		+	6	F	2 y/9 m	2y/9m	9x8x12	T3 N0 M0	II	Cortisol+Androgen
49	No		+	63	M	2 y/6 m	2 y/6 m	26x15x14	T3 N0 M0	III	Androgen
50	No		-	1	F	2 y/4 m	2y/4m	6x8x6.5	T4 N0 M0	III	Androgen
51	c.4391 4394 del	APC	+	8	M	2 y/1 m	ly/11m	11×8.5x8	T2 N0 M0	II	Cortisol
52	No		-	14	F	7m	7m	5x5x4	T1 N0 M0	I	Cortisol+Androgen
53	c.134C>T	CTNNB1	+	53	F	£1 m	*1m	10×6×4	T4 N0 M0	III	Cortisol
54	No		+	43	F	1 y/8 m	+2m	9x8x7	T3 N1 M0	III	Cortisol
55	No		-	43	M	1 y/2 m	1y	19x14x12	T3 N0 M0	III	Cortisol

I : Deceased; }:Recurrence; F/M: Female/male; NA: Not Available; TNM : Tumour Node Metastasis

Supplementary Table S8
		Age at operation	tumour size	Female;male	Cortisol producing	ENSAT stage
		(median in yrs)	(median length in cm)			III+IV; I+II
APC/CTNNB1/ZNRF3alterations	n=22	52.5 (n=22)	10 (n= 22)	14;8 (n=22)	15 (n=22)	14;8 (n=22)
APC/CTNNB1/ZNRF3 wildtype	n=30	46 (n=29)	10 (n=29)	17;12 (n=29)	14 (n=29)	15;14 (n=29)
				x2=0.005 p=0.9412	χ2=1.291 p=0.2559	x2=0.320 p=0.5719
Nuclear accumulation	n=25	51.5 (n=24)	10 (n=24)	15;9 (n=24)	14 (n=24)	17;7 (n=24)
No nuclear accumulation	n=24	52.5 (n=24)	10 (n=24)	14;10 (n=24)	13 (n=24)	11;13 (n=24)
		p=0.5159	p=0.8097	x2=0.087 p=0.7679	x2=0.085 p=0.7711	x2=2.143 p=0.1432
Active ß- catenin expression	n=30	46 (n=29)	9 (n=29)	18;11 (n=29)	16 (n=29)	16;13 (n=29)
lack of active ß- catenin expression	n=16	58 (n=16)	10 ((n=16)	9;7 (n=16)	9 (n=16)	8;16 (n=16)
		p=0.3744	p=0.5059	x2=0.004 p=0.9493	x2=0.005 p=0.0.9445	x2=1.723 p=0.1893
Cohort	n=52	52 (n=51)	10 (n=51)	31;20 (n=51)	29 (n=51)	29;22 (n=51)

Supplementary Table S9. Cox regression analysis for overall survival

Univariate analysis

	Cohort (n=51)		Cohort - childhood ACC ( n=46)
	HR (95% CI)	P value	HR (95% CI)	P value
Age at operation	1.012 (0.994 - 1.032)	0.203	1.006 (0.986 - 1.,027	0.578
Cortisol production	1.763 (0.873 - 3.559)	0.114	1.890 (0.939- 3.806)	0.074
ENSAT staging (I/II vs II/IV)	4.076 (1.798 - 9.244)	0.001	4.160 (1.832 - 9.448)	0.001
Gender	1.037 (0.510 - 2.108)	0.919	0.993 (0.491 - 2.025)	0.993
Size	1.009 (0.954 - 1.068)	0.752	1.003 (0.948 -1.062)	0.907
APC/CTNNB1/ZNRF3 alteration	2.493 (1.211-5.131)	0.013	2.481 (1.206-5.107)	0.014

Multuvariate analysis

	Cohort (n=51)		Cohort - childhood ACC ( n=46)
	HR (95% CI)	P value	HR (95% CI)	P value
Grading	3.480 (1.528-7.927)	0.003	3.604 (1.589-8.174)	0.002
APC/CTNNB1/ZNRF3 alteration	1.976 (0.949-4.113)	0.069	2.028 (0.979-4.201)	0.057

Supplementary Table S10
CTNNB1	DNA primers
	Exon 3
	Fw primer	CAATGGGTCATATCACAGATTCTT
	Rw primer	CTGTGTAGATGGGATCTGC
	Exon 5
	Fw primer	GCTCAAGGGGAGTAGTTTCAGA
	Rw primer	TCCACTGGTGAACTGGGAAG
	cDNA Primers
	Fw primer	GAAGGTCTGAGGAGCAGCTTC
	Rw primer	ACAACACAGAATCCACTGGTG
	DNA primers
ZNRF3	Exon 2	GGGTTGAAAAACTGTCCCCA
		CCACCTCATGCAACTTCAGC
	Exon 3	GATTGCCAAGGCCAACTTT
		GGGACAAGCCAAGCTCACTA
	Exon 4	CTGGAGGATTCCAGACAGGT
		GGGTATGCCAGCATTTCAGA
	Exon 5	GATAGCCCATGTGCCGCTTA
		GCTGTGAGGCTCCATGTTGC
	Exon 6	GTCTGCCTGTCCCAGTGAAT
		CCTTCTAGGTCTTGGGCACC
	Exon 7	GCTGTGCAGAACTCCTTGG
		GAGTGTTCCCAAGCCTGCG
	Exon 8a	GCCTCTGACACCAGTATGCT
		CTACCACAGAGTCACTGGAG
	Exon 8b	GCTTCAGCTGCTATCACGGC
		GTCCTGGGCAAATGGTCGG
	Exon 8c	TGGCAGCAGCACCTTGTTC
		GGCCAGGCTGAGGAGTAACC
	Exon 9	TTGCCTGGTCCCATGTGTGG
		TGGGTTGACAAGGAGGGCTC

References

1 Xing, Y., Clements, W. K., Kimelman, D. & Xu, W. Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes & development 17, 2753- 2764, doi:10.1101/gad.1142603 (2003).

2 Gottardi, C. J. & Peifer, M. Terminal regions of beta-catenin come into view. Structure 16, 336- 338, doi:10.1016/j.str.2008.02.005 (2008).