28801349

C-myc expression in adrenocortical tumours

Mirkka Pennanen,1 Jaana Hagström,1 Ilkka Heiskanen,2 Timo Sane,3 Harri Mustonen,2 Johanna Arola,1 Caj Haglund2,4

1Department of Pathology, University of Helsinki and HUSLAB, Helsinki, Finland 2Department of Surgery, Helsinki University Hospital, and University of Helsinki, Helsinki, Finland

3Division of Endocrinology, Department of Medicine, Helsinki University Hospital, and University of Helsinki, Helsinki, Finland AResearch Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland

Correspondence to

Dr Mirkka Pennanen, Department of Pathology, Haartman Institute, University of Helsink, Helsinki, Finland; mirkka.pennanen@hus.fi

JA and CH contributed equally.

Received 9 April 2017 Revised 29 May 2017 Accepted 8 June 2017

CrossMark

To cite: Pennanen M, Hagström J, Heiskanen I, et al. J Clin Pathol Published Online First: [please include Day Month Year]. doi: 10.1136/ jclinpath-2017-204503

ABSTRACT

Aims Widespread use of high-resolution imaging techniques and thus increased prevalence of adrenal lesions has made diagnostics of adrenocortical tumours an increasingly important clinical issue. In non-metastatic tumours, diagnosis is based on histology. New or enhanced information for clinicopathological diagnosis, revealing the malignant potential of the tumour, could emerge by means of biomarkers. The connection of proto-oncogene c-myc to adrenocortical neoplasias is poorly known, although the Wnt/beta-catenin pathway, one of the signalling pathways leading to induction of c-myc expression, has been connected to development of adrenocortical neoplasias. We studied c-myc expression in adrenocortical tumours and investigated molecules associated with the signalling pathway of c-myc, including cell cycle-related proteins p27, cyclin E and cyclin D1.

Methods We studied 195 consecutive adult patients with 197 primary adrenocortical tumours. Histopathological diagnosis was determined by Weiss score and the novel Helsinki score. C-myc, cyclin D1, cyclin E and p27 expressions were determined by immunohistochemistry.

Results Benign adenomas showed prominent nuclear c-myc expression comparable to that of normal adrenocortical cells, whereas carcinomas showed increased cytoplasmic expression. Strong cytoplasmic and weak nuclear c-myc expressions associated with malignancy and adverse outcome. C-myc staining did not correlate with cyclin E. Cyclin D1 correlated with cytoplasmic c-myc expression and to a lesser extent with nuclear c-myc. P27 correlated with cytoplasmic c-myc, but not with nuclear c-myc. P27 correlated with cyclin E.

Conclusions Strong cytoplasmic c-myc expression and weak nuclear expression in adrenocortical tumours associated with malignancy and shorter survival.

INTRODUCTION

Most adrenocortical tumours are benign adenomas, whereas adrenocortical carcinomas are rare and highly aggressive. The therapeutic strategy for adrenocortical carcinomas differs from that for adenomas, which makes accurate diagnosis of adre- nocortical neoplasms imperative.

In non-metastatic tumours, diagnosis is based on histology. Various histopathological criteria differentiate adrenocortical carcinomas from adenomas; the most common is the Weiss scoring system.1 Recently, we described the Helsinki score, a more objective and accurate tool to evaluate these tumours’ metastatic potential.2 Biomarkers reflecting the malignant potential of the tumour

could further contribute to clinicopathological diagnosis.

The proto-oncogene c-myc, first detected in Burkitt lymphoma,3 has since been connected to many cancers. It is a transcription factor that induces diverse functions in cells like proliferation, cell growth, loss of differentiation and apoptosis. Under physiological conditions, the c-myc protein level is precisely regulated. Both overexpression of c-myc and deregulation of its expression or func- tion can lead to a cell’s malignant transformation.

In adrenocortical neoplasias, no significant losses or gains occur in chromosome 8q24 harbouring the c-myc gene, nor any amplification nor rearrange- ment of the gene. 45 However, the Wnt/beta-catenin pathway, one of the signalling pathways leading to induction of c-myc expression, has been connected to development of adrenocortical neoplasias.6-12 Expression of the c-myc protein in adrenocortical tumours has thus far been reported in one study of 15 tumours. Suzuki et al13 observed immunohisto- chemical expression of c-myc protein in the nuclei of all tumours. In carcinomas, c-myc staining also occurred in the cytoplasm.

Our aim was to study c-myc expression in adre- nocortical tumours, and furthermore to investigate molecules associated with the signalling pathway of c-myc, including the cell cycle-related proteins p27, cyclin E and cyclin D1.

MATERIALS AND METHODS

Clinical data and tumour material

We identified all adult patients who underwent surgery for a primary adrenocortical tumour at the Department of Surgery, Helsinki University Hospital between 1990 and 2003. The series comprised 195 patients with 197 tumours, 2 patients with two separate tumours. Tumour specimens were stored in the archives of the Department of Pathology, and clinical data came from patient records. The crite- rion for clinically malignant disease was metastasis. Survival data and cause of death came from the Population Register Centre and Statistics Finland. The study was approved by the Ethics Committee of Helsinki University Hospital and the National Supervisory Authority for Welfare and Health. For clinical characteristics, see table 1.

Follow-up time of the patients ranged from 0.03 to 21.65 years (median 10.17; mean 10.71). Among patients with local disease, 36 patients died of causes other than adrenocortical tumour. Their survival times ranged from 0.03 to 18.51 years (median 4.62; mean 5.82). Follow-up for patients alive was 7.84-21.83 years (median 11.27; mean 12.64). Of the 195 patients, 14 had metastatic disease and all

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Table 1 Clinical and histological characteristics of tumours and distribution of immunohistochemical stainings
		All tumours	Local tumours	Metastatic tumours	p Value
Tumours (N)		197	183	14
Age (years)	Range	24-82	24-82	32-76
	Mean	54.2	54.3	53.5	0.777
	Median	55	54	55.5
Gender*	Male	66	60	6	0.560
	Female	129	121	8
Side	Right	101	93	8	0.784
	Left	96	90	6
Size (cm)	Range	0.5-28	0.5-28	4-22
	Mean	3.4	2.8	12.1	<0.001
	Median	2	2	12.5
Hormonal secretion
Aldosterone	N	96	94	2
	%	49	51	14	0.010
Cortisol	N	74	67	7
	%	38	37	50	0.394
Androgens	N	11	7	4	0.004
	%	6	4	29
Inactive	N	29	24	5	0.038
	%	15	13	36
Weiss score	Range	0-9	0-9	5-9
	Mean	1.7	1.2	7.8	<0.001
	Median	1	1	8
Helsinki scoret	Range	0-62.2	0-11.3	8.9-62.2
	Mean	3.1	1.3	24.2	<0.001
	Median	0.8	0.8	15
C-myc
Cytoplasmic score 0-3	Range	0-3	0-3	1-3
	Mean	1.4	1.3	2.6	<0.001
	Median	1.0	1.0	3.0
Nuclear score 0-4	Range	0-4	0-4	0-4
	Mean	3.1	3.2	2.1	0.004
	Median	4.0	4.0	1.5
Cytoplasmic-to-nuclear ratio	≤0.75	142	141	1	<0.001
	>0.75	50	37	13
p27 %	Range	0-100	0-100	20-100
	Mean	53.8	53.0	63.6	0.307
	Median	70.0	70.0	75.0
Cyclin E %	Range	0-80	0-80	0-60
	Mean	8.7	7.7	21.4	0.001
	Median	5.0	5.0	15.0
Cyclin D1 %	Range	0-90	0-90	0-50
	Mean	12.6	12.1	18.6	0.227
	Median	9.0	9.0	16.5

*One man and one woman had two separate local tumours.

tHelsinki score was determined for 194 tumours.

succumbed to their disease 0.30-6.84 years (median 2.05; mean 2.97) from surgery.

Histopathology

The histopathological diagnosis according to the Weiss criteria1 14 for each tumour was reviewed from H&E-stained sections by two pathologists (MP and JA). The novel Helsinki score introduced by the authors2 was also determined. The Helsinki score includes 3 x mitotic rate (>5/50 high-power field)+5 x presence of necrosis + proliferation index (Ki67/mib-1). A score >8.5 iden- tifies metastatic carcinoma with 100% sensitivity.

The 14 metastatic tumours had Weiss score (WS) 5-9 and Helsinki score (HS) 8.9-62.2, so they were correctly diagnosed as malignant according to both scoring systems. Of 183 local tumours, 166 had WS 0-2, that is, were considered benign, and 17 local tumours received a malignant score of WS 3-9. Of the 17 patients, 2 died of other causes, while the other 15 patients were alive at the end of follow-up (7.92-20.33 years). Of the 183 local tumours, 181 had a benign HS (0-8.5). Only two local tumours had a malignant diagnosis according to HS. These were locally invasive tumours that were radically excised. The patients are alive having follow-up times of 15 years (HS 8.7) and 7.92

Table 2 Tumours with malignant potential according to Weiss score (≥3) or Helsinki score (>8.5)

Helsinki score	Weiss score	Local/Metastatic	Hormonal activity	C-myc nuclear score	C-myc cytoplasmic score	C-myc cytoplasmic- to-nuclear ratio
62.2	8	Metastatic	Cortisol	1	3	1.5
59.5	9	Metastatic	Inactive	1	1	0.5
47.3	8	Metastatic	Cortisol	1	3	1.5
33.8	9	Metastatic	Cortisol, testosterone	1	3	1.5
32	8	Metastatic	Cortisol, testosterone	0	3	3.0
16.2	6	Metastatic	Inactive	3	3	0.75
15.7	9	Metastatic	Cortisol, testosterone	3	3	0.75
14.3	7	Metastatic	Inactive	1	1	0.5
11.3	9	Local	Inactive	1	3	1.5
11.2	9	Metastatic	Inactive	4	3	0.6
9.6	6	Metastatic	Aldosterone	4	3	0.6
9.3	9	Metastatic	Cortisol	4	3	0.6
9.3	7	Metastatic	Aldosterone	2	3	1.0
9.1	5	Metastatic	Inactive	4	2	0.4
8.9	9	Metastatic	Cortisol, testosterone	0	3	3.0
8.7	8	Local	Cortisol	1	3	1.5
8.3	8	Local	Cortisol	4	3	0.6
8	9	Local	Inactive	2	3	1.0
8	3	Local	Aldosterone	3	2	0.5
7.4	6	Local	Inactive	4	1	0.2
5.5	4	Local	Inactive	1	3	1.5
4.8	3	Local	Cortisol	2	3	1.0
4.1	3	Local	Aldosterone	4	2	0.4
3.1	5	Local	Cortisol	3	2	0.5
1.9	3	Local	Cortisol	4	1	0.2
1.4	3	Local	Cortisol	4	3	0.6
1.1	3	Local	Cortisol	3	1	0.25
1.0	3	Local	Cortisol	3	2	0.5
0.9	3	Local	Testosterone	3	2	0.5
0.8	3	Local	Cortisol	4	2	0.4
0.3	3	Local	Cortisol	4	3	0.6

years (HS 11.3). All tumours with malignant potential according to either WS (≥3) or HS (>8.5) are presented with their meta- static status in table 2.

Tissue microarray construction

Tissue microarray (TMA) blocks were constructed from archived surgical formalin-fixed, paraffin-embedded specimens. Repre- sentative areas of each tumour were chosen from H&E-stained slides. From histologically benign (WS 0-2) tumours, three 1 mm cores, and from histologically malignant (WS 3-9) tumours, six cores were obtained with a semiautomatic TMA instrument (Beecher Instruments, Silver Spring, Maryland, USA) with two cores also from normal adrenal cortex.

Immunohistochemistry

Sections from TMA blocks 4 um thick were deparaffinised in xylene and rehydrated through a graded alcohol series. Immu- nostaining was by incubation for 1 hour for c-myc and cyclin E, 44 min for cyclin D1 and overnight for p27. Antibody clones, dilutions, manufacturers, pretreatment and endogenous peroxi- dase-blocking procedures are in table 3.

Interpretation of immunohistochemical stainings

C-myc, cyclin E, p27 and cyclin D1 expressions were determined for 192-196 tumours.

Stainings were scored independently by two experienced pathologists (MP and JH). Any discrepancy between evaluations called for a consensus.

Table 3 Detailed information on the immunohistochemical stainings
	C-myc	Cyclin D1	Cyclin E	p27
Antibody	Mouse	Rabbit	Mouse	Mouse
	monoclonal	monoclonal	monoclonal	monoclonal
	9E10	SP4-R	IgG2B 713573	lgG, Kip1 AA 1-197
Laboratory	Santa Cruz, UK	Roche, Tucson, Arizona, USA	R&D Systems, Minnesota, USA	Transduction Laboratories, Kentucky, USA
Dilution	1:400	rtu	1:600	1:100
Detection	Dako REAL	ultraView DAB	Dako REAL	Vectastain
	EnVision/ HRP, DAB+		EnVision/ HRP, DAB+	ABC Kit
	Chromogen		Chromogen
Pretreatment	Tris-HCL	Standard CC1	Tris-EDTA	Citrate
Endogenous	0.3%	According to manufacturer	0.3%	0.5% H2O2
peroxidase	Dako REAL		Dako REAL
blocking	Peroxidase-		Peroxidase-
	Blocking		Blocking
	Solution		Solution

Original article

For c-myc, the percentage of positive tumour cell nuclei was evaluated and scored from 0 to 4. Positivity up to 30% was scored as 1, 30%-50% as 2, 50%-80% as 3 and over 80% as 4. The intensity of cytoplasmic staining of c-myc was scored on a scale from 0 to 3. Negative cytoplasmic staining was scored as 0, weakly positive as 1, moderately strong positivity or focally strong positivity as 2, and homogeneously strong positivity as 3.

Assessment of cyclin D1 staining was conducted with inter- net-based, free image analysis software, ImmunoRatio, used for quantitative assessment of any nuclear markers.13 Image capture was performed with a Nikon Eclipse 80i light micro- scope (40x objective). From every core biopsy, one digital image was captured from the area of highest positivity. Of all images available from each tumour, the highest count was the one recorded, at a precision of 1%. Any positivity between 0% and 1% was recorded as 0.5%. For cyclin E and p27 we quanti- fied the proportion of positively stained tumour cell nuclei, at a precision of 10%. Any positivity between 0% and 10% was 5%.

Statistical analysis

Results are given as mean, median and range, or number of patients and proportion of patients. The Mann-Whitney U test served to analyse differences between patient groups in contin- uous and ordinal variables. Fisher’s exact test analysed differ- ences in dichotomous variables. Spearman’s r correlation coef- ficient was calculated between different scores. Survival rates were estimated by the Kaplan-Meier method and the log-rank test compared survival curves. p Value <0.05 was considered statistically significant. Two-tailed tests were used. Statistical calculations were with SPSS V.22.

RESULTS

C-myc staining

The staining pattern of c-myc is illustrated in figure 1. Staining differed between local and metastatic tumours. In benign adenomas, cytoplasm did not stain or stained weakly (0-1), whereas nuclear staining was moderate to strong (3-4). This was also the case in normal adrenal cortex (data not shown). In meta- static carcinomas, cytoplasmic staining was typically strong and

Figure 1 Staining pattern of c-myc in adrenocortical tumours: (A) cytoplasmic staining score 0, nuclear score 4; (B) cytoplasmic score 1, nuclear score 3; (C) cytoplasmic score 2, nuclear score 2; and (D) cytoplasmic score 3, nuclear score 0. All immunohistochemistry images ×400.

AC

B

C

D

Figure 2 Survival according to c-myc staining and cytoplasmic-to- nuclear ratio for all tumours and for tumours with Weiss score ≥3.

All patients

Weiss ≥ 3

100

100

Cumulative Survival (%)

80

80

60

60

40

0-0.75

40

0-0.75

20

>0.75

20

>0.75

0

0

0

5

10

15

20

0

5

10

15

20

Number at risk

Time (years)

0-0.75

141

128

76

29

4

10

8

4

2

0

>0.75

50

35

22

15

4

22

13

7

4

1

nuclear staining weaker than in adenomas. The difference both in cytoplasmic and in nuclear staining of c-myc between local and metastatic tumours was significant (p<0.001 and p=0.004) (table 1).

The optimal cut-off value for cytoplasmic and nuclear c-myc staining to distinguish local tumours from metastatic ones was 3 at p<0.001 and p=0.004. To further investigate in these tumours the importance of the localisation of c-myc protein, we calculated the ratio of cytoplasmic to nuclear c-myc in each tumour. In order to avoid dividing by 0, we increased every score of nuclear c-myc by 1. The difference in this new parameter between local and metastatic tumours was significant (p<0.001). The optimal cut-off value for the cytoplasmic-to-nuclear c-myc ratio distinguishing local tumours from metastatic ones was 0.75 (p<0.001).

Correlation of c-myc with Weiss and Helsinki scores

Cytoplasmic c-myc staining correlated with the total WS of the tumour, R=0.531, p<0.001, and with the HS, R=0.283, p<0.001. The cytoplasmic-to-nuclear c-myc ratio also correlated with the WS (R =0.398, p<0.001) and the HS (R =0.193, p=0.008). Nuclear c-myc staining correlated neither with the WS (p=0.102) nor the HS (p=0.133).

Prognostic significance of c-myc

Kaplan-Meier survival analysis showed an association between both cytoplasmic and nuclear staining and patient survival (log-rank test, p<0.001 and p=0.008). The Kaplan-Meier curves according to cytoplasmic-to-nuclear c-myc ratio with cut-off value of 0.75 for all tumours and for tumours with WS ≥3 are shown in figure 2.

C-myc-related markers

Distributions of nuclear cyclin D1, p27 and cyclin E stainings are shown in table 1. There was no significant difference in nuclear staining of cyclin D1 or p27 between local and metastatic tumours (p=0.227 and p=0.307), but the difference in nuclear cyclin E staining was significant using the optimal cut-off value of 35 (p=0.001). Cytoplasmic c-myc staining correlated with p27 (R=0.303, p<0.001) and cyclin D1 (R=0.260, p<0.001), but not with cyclin E (p=0.130). Nuclear c-myc staining did not correlate with p27 nor cyclin E staining, but did correlate with cyclin D1 (R =0.148, p=0.040).

P27 correlated with cyclin E (R =0.143, p=0.046) and cyclin D1 (R =0.388, p<0.001). Also, cyclin E and cyclin D1 correlated (R=0.197, p=0.006).

DISCUSSION

Adrenocortical neoplasias express c-myc protein. Benign adenomas showed prominent nuclear c-myc expression compa- rable to that of normal adrenocortical cells, whereas in carci- nomas the increased c-myc expression was cytoplasmic and nuclear staining vanished. Strong cytoplasmic c-myc and weak nuclear c-myc expression thus associated with malignancy and adverse outcome.

Underexpression of c-myc has been suggested as a pathogenic event in adrenocortical carcinomas.16 17 Decreased c-myc expres- sion has been suspected in pathway analysis of gene expression microarray and comparative genome hybridisation studies, and speculation is that it is connected to deletions of chromosome 8q24. In studies investigating c-myc gene expression by mRNA analysis, Liu et al showed c-myc mRNA to express abundantly in normal adrenocortical tissue. Also, hormonally inactive carci- nomas and cortisol-secreting or aldosterone-secreting adenomas showed similar amounts of c-myc mRNA as normal adrenocor- tical tissue, whereas hormonally active carcinomas and testos- terone producing adenomas presented with a decreased level of c-myc mRNA.18 19 The authors also stated a good correlation between mRNA expression and immunohistochemical staining. However, Liu et al only18 observed nuclear immunohistochem- ical staining of c-myc, no cytoplasmic staining, as we did. We did not observe any difference in c-myc staining between testos- terone-secreting adenomas and other adenomas, or hormonally active and inactive carcinomas.

The characteristic staining pattern of c-myc in benign adre- nocortical tumours was strong by nuclear and absent or weak by cytoplasmic, whereas in malignant tumours staining was the reverse: strong by cytoplasmic and weak by nuclear. The differ- ence in both cytoplasmic and nuclear c-myc staining between local and metastatic tumours was significant. A similar staining pattern has been characterised in a small study consisting of 15 adrenocortical tumours, 9 carcinomas and 6 adenomas.13 All these tumours presented nuclear c-myc staining, whereas cyto- plasmic staining only occurred in carcinomas. No correlation between c-myc staining and clinical outcome emerged. Nor did this study provide an explanation for the function of cytoplasmic c-myc staining.

Not surprising for a transcription factor, c-myc protein is usually evident in the nuclei of tumours presenting over-regu- lated or deregulated expression of the c-myc gene. Cytoplasmic c-myc has also been evident in various neoplasias,20-23 correlating with cancer aggressiveness and with chromosomal alterations involving c-myc gene. To our knowledge, no such correlation of cytoplasmic c-myc staining with malignancy has previously been observed, as in adrenocortical neoplasias. The role of cytoplasmic c-myc has been studied recently.2425 In 2010 Conacci-Sorrell et al 25 published their findings of a truncated, transcriptionally inac- tive cytoplasmic c-myc protein called myc-nick, which promotes cancer cell survival and augments cancer cell motility. They showed that myc-nick is expressed in a wide range of tumours and hypothesised that it would likely represent the cytoplasmic c-myc staining seen in previous studies. Myc-nick is generated in response to metabolic and cytotoxic stress by calpain-medi- ated proteolysis under which conditions the full-length c-myc would cause cell death by apoptosis. The cytoplasmic c-myc in adrenocortical tumours, however, does not seem to be myc-nick. The 9E10 antibody we used for immunohistochemistry binds to the C-terminus of the c-myc protein, which is truncated in the myc-nick protein. There is the possibility, though, that cyto- plasmic c-myc seen in adrenocortical tumours would represent

full-length c-myc protein retained in the cytoplasm under stress conditions, waiting for processing to myc-nick similar to what happens at the invasive front in colon cancer.24

In our study, strong cytoplasmic c-myc staining in adrenocor- tical carcinomapredicted shorter survival. For nuclear staining, the opposite correlation appeared: low nuclear expression predicted shorter survival. Cytoplasmic-to-nuclear c-myc ratio further illustrated the difference in survival according to c-myc localisation. These results are consistent with cytoplasmic c-myc staining as correlating with the total WS of the tumour and with HS, which both correlate with survival.

To further investigate the role of c-myc in adrenocortical neoplasias, we studied the association of c-myc expression with that of cell cycle-related proteins p27, cyclin E and cyclin D1. The association of c-myc and cyclin E expression in adre- nocortical tumours is interesting because cyclin E is a target gene of c-myc, and increased expression of cyclin E has been correlated with malignancy and poor prognosis of adrenocor- tical tumours.26 Our nuclear staining of cyclin E was higher in metastatic than in local tumours, but neither cytoplasmic nor nuclear c-myc staining correlated with cyclin E. Thus c-myc and cyclin E seem to play independent roles in adrenocortical tumour development, suggesting that cyclin E may not be the target of c-myc in ACC.

The association of cyclin D1 and c-myc expression is inter- esting, because both genes are target genes of the Wnt/beta-cat- enin pathway, which has been connected to adrenocortical tumour development. In colon carcinomas with an activated Wnt/beta-catenin pathway, both cyclin D1 and c-myc have acted as target genes contributing to malignant transformation.27 In our series, nuclear cyclin D1 staining was higher on average in metastatic tumours than in local tumours, but the difference was not significant. That cyclin D1 correlated with cytoplasmic c-myc expression and to a lesser extent with nuclear c-myc suggests a common inducer of the expression of both proteins, which may prove to be the Wnt/beta-catenin pathway.

Expression of the cell cycle inhibitor P27 (cyclin-dependent kinase inhibitor 1B) has been connected to good prognosis in several cancers.28 29 However, p27 plays a dual role in cancer, because elevated levels have been connected to metastatic poten- tial.3º C-myc is known to inhibit p27 expression. Our mean nuclear p27 staining was slightly higher in metastatic than in local tumours, but this difference was not significant; p27 correlated with cytoplasmic c-myc, but not with nuclear c-myc, and correlated with cyclin E. C-myc does not seem to inhibit p27 expression in adrenocortical tumours, but its correlation with cyclin E indicates a possible role in inhibiting the cell cycle-pro- moting function of overexpressed cyclin E.

A major advantage of our study is its large cohort size and the comprehensive follow-up data on all patients. A limitation is the small number of rare metastatic tumours.

We conclude that strong cytoplasmic c-myc expression and weak nuclear c-myc expression in adrenocortical tumours

Take home messages

Benign adrenocortical tumours show strong nuclear c-myc expression.

In carcinoma, nuclear c-myc vanishes and strong expression appears in cytoplasm.

Transition of c-myc expression correlates with malignancy and prognosis.

Original article

associate with malignancy and shorter survival. We hypothesise that transition of c-myc from nucleus to cytoplasm prevents c-myc from acting as a transcription factor and from inducing apoptosis, but further studies must uncover the mechanism of c-myc function in adrenocortical tumours.

Handling editor Cheok Soon Lee.

Acknowledgements We thank Eija Heiliö, Elina Aspiala and Päivi Peltomäki for technical support, Satu Remes for expert advice on immunohistochemical stainings and Olli Tynninen for his help with the images.

Contributors MP, IH, TS, JA and CH designed the study. MP, IH and TS collected clinical information of the patients, and IH also operated the majority of the tumours in this study. MP and JA assessed the tumours histologically. MP and JH assessed the immunohistochemical stainings. HM analysed the data statistically. MP and HM interpreted the data. MP drafted the manuscript. JA, CH and TS revised the manuscript. Both JA and CH were the supervisors of the study. All authors read and approved the final manuscript.

Funding This work was supported by grants from the Finnish Cancer Foundation, Sigrid Jusélius Foundation, Helsinki University Hospital Research Fund and Medicinska Understödsföreningen Liv och Hälsa. Funding is obtained from independent scientific foundations. We only have to report scientific results, that is, published papers.

Competing interests None declared.

Ethics approval Ethics Committee of Helsinki University Hospital and the National Supervisory Authority for Welfare and Health.

Provenance and peer review Not commissioned; externally peer reviewed.

@ Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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JCP	C-myc expression in adrenocortical tumours
	Mirkka Pennanen, Jaana Hagström, Ilkka Heiskanen, Timo Sane, Harri Mustonen, Johanna Arola and Caj Haglund J Clin Pathol published online August 11, 2017
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