Histopathology 2014, 64, 567-576. DOI: 10.1111/his.12283
Distinguishing adrenal cortical carcinomas and adenomas: a study of clinicopathological features and biomarkers
Cuiping Wang, Yang Sun, Huanwen Wu, Dachun Zhao & Jie Chen
Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tsinghua University, Beijing, China
Date of submission 17 May 2013 Accepted for publication 9 September 2013 Published online Article Accepted 14 September 2013
Wang C, Sun Y, Wu H, Zhao D & Chen J (2014) Histopathology 64, 567-576
Distinguishing adrenal cortical carcinomas and adenomas: a study of clinicopathological features and biomarkers
Aims: To determine clinicopathological criteria and molecular markers helpful in distinguishing adreno- cortical carcinomas (ACCs) from adrenocortical ade- nomas (ACAs).
Methods and results: We analysed retrospectively the clinical and pathological features of 50 adrenal corti- cal tumours, and tested the expression of miR483-3p by in-situ hybridization as well as the expression of IGF2 and Smad4 by immunohistochemistry. We found that tumour size, tumour weight, hormonal function and the Weiss system are all high-efficacy criteria for differentiating malignant from benign tumours (P < 0.001). MiR483-3p was overexpressed in 68% (17 of 25) of ACCs compared to 12% (three
of 25) of ACAs (P < 0.05). Using a combination of miR483-3p and Smad4 improved diagnostic accu- racy. Molecular markers were then tested in an inde- pendent set of 15 borderline tumours. We confirmed that the combined use of miR483-3p and Smad4 immunochemistry can complement the Weiss score in the diagnosis of ACC in cases that display border- line histology.
Conclusions: Tumour size, tumour weight, hormonal function and the Weiss system are useful clinicopath- ological criteria that can result in accurate diagnosis of most ACCs and ACAs. In challenging cases, miR483-3p and Smad4 expression may help in dis- tinguishing these two entities.
Keywords: adrenal cortical tumours, clinicopathological features, differentiation, diagnosis, hsa-mir-483-3p, in-situ hybridization
Introduction
Adrenal masses are among the most frequent tumours in humans.1 A vast majority of these tumours are benign, and only a small subset of adrenal masses are malignant adrenocortical carcinomas (ACCs). ACC is a rare but very aggressive cancer. It affects one to two people per million per year and accounts for 0.02- 0.2% of all cancer deaths.2-4 An accurate diagnosis is critical, because the prognosis, follow-up and thera-
peutic strategy for ACC are very different to those for a benign tumour. However, it is difficult in some cases to distinguish malignant from benign cortical tumours accurately through clinical characteristics or histologi- cal criteria. The Weiss system, including nine-point histopathological criteria, is currently the most com- monly used method for assessing malignancy. The ori- ginal threshold for malignancy, established in 1984, was the presence of at least four criteria5; but this was reduced to three or more criteria in 1989 after it was observed that some tumours with a score of 3 had recurred.6 In 2002, Aubert et al.7 proposed a modifi- cation of the Weiss system; the two systems correlated significantly, and in both the threshold for malignancy is a total score of 3. However, the Weiss system was found to be less specific, as several studies noted that
Address for correspondence: J Chen MD, PhD, Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Shuai Fu Yuan Hu Tong, Beijing 100730, China. e-mail: xhblk@163.com
some tumours with a Weiss score of 3 had a clinical benign course,7-9 and that individual tumours may behave in a malignant manner despite initially receiv- ing a Weiss score of 2.10,11 Therefore, there is a need for a comprehensive understanding of the clinical and morphological characteristics of adrenocortical tumours, and the identification of reliable biomarkers as an adjunct to routine pathological analysis.
MicroRNAs (miRNAs) are endogenous, non-coding RNA of approximately 22 nucleotides that regulate gene expression by controlling target mRNA transla- tion or degradation.12 Angelo et al.13 reported that miR483-3p is overexpressed in human breast, colon and liver cancers, suggesting a wide involvement of this miRNA in human tumorigenesis. However, its expression and the mechanisms underlying this expression in adrenocortical tumours are unclear.
The gene encoding Smad4/DPC4, a critical effector in the TGF-ß signalling pathway,14 has been reported to be a target for miR483-3p in some studies,15 but the role of this effector in adrenal cortical tumours has not been established definitively. MiR483 is expressed from intron 2 of the IGF2 gene, and several studies have iden- tified that up-regulation of IGF2 expression is the domi- nant change in malignant adrenocortical tumours.16,1
In this study, we analysed clinical and pathological features of adrenal cortical tumours and investigated the expression of miR483-3p and its association with IGF2 and Smad4 in ACCs, in order to develop a theo- retical basis for differential diagnosis, prognostic eval- uation and molecular target therapy.
Materials and methods
PATIENTS AND TUMOUR SAMPLES
Samples of adrenal tumour were collected from patients undergoing adrenalectomy at Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, from July 2001 to July 2012. This study was approved by the Peking Union Medical College Hospital Ethics Committee. Twenty-five malignant tumours were available and used as experimental samples, and 25 benign tumours selected randomly from 1564 patients with adrenocortical adenomas (ACAs) were used as control samples. All 50 tumours had full clinical and pathological data. Diagnoses were based on clinical, biochemical and morphologi- cal data. The follow-up period was 6-123 months.
The Weiss system was applied to all 50 adrenal cortical tumours separately by two independent observers (H.W. and J.Z.) blinded to the clinical courses of the patients. A consensual Weiss score was
established on a multiheaded light microscope. The nine Weiss histopathological criteria of malignancy were defined according to Aubert et al.7
An independent set of 15 borderline adrenal corti- cal tumours (Weiss score = 2 or 3) were used to vali- date further the diagnostic accuracy of the molecular markers. The morphological features of the 15 cases are summarized in Table S1.
IN-SITU HYBRIDIZATION
A miRCURY LNA detection probe for human mature miR483-3p (5’- AAGACGGGAGGAGAGGAGTGA-3’), U6-positive control and the scrambled negative con- trol, which were all 5’-digoxigenin- and 3’-digoxige- nin-labelled, were purchased from Exiqon (Vedbaek, Denmark). Detection of miR483-3p by in-situ hybrid- ization using the oligonucleotide probes was performed according to the manufacturer’s instructions. Briefly, human tissues were deparaffinized, treated with pro- teinase K, washed in sterile diethylpyrocarbonate-trea- ted phosphate-buffered saline (PBS), and subsequently fixed with 4% paraformaldehyde. After prehybridiza- tion at room temperature followed by incubation at 61℃ separately for 1 h, hybridization was performed at 61℃ for 21 h, followed by a wash in ×2 standard saline citrate and 3 × 30-min washes at 61℃ in x1 standard saline citrate and 50% formamide. After blocking (2% sheep serum and 2 mg/ml bovine serum albumin in PBS with Tween-20) at room temperature for 1 h, the blocking buffer was replaced with blocking buffer containing anti-digoxigenin-alkaline phospha- tase (Cat. no. 11 093274910,150U, 200 ul, 1:500; Roche, Basel, Switzerland), and all slides were then placed into a ddH2O box and incubated at 4℃ over- night. The next day, the slides were washed in PBS with Tween-20 for 7 x 5 min at room temperature, equilibrated, NBT/BCIP added (Cat. no. 11 681 451 001, 8 ml, 1:500, Roche), and incubated for 3 h in the dark at room temperature for visualization.
The slides were then scored independently by two pathologists (H.W. and Y.S.) as negative (-), weak or focally positive (1+) or strongly positive (2+).18
IMMUNOHISTOCHEMISTRY
Immunohistochemical detection of IGF2, Smad4 and MIB-1 was performed on standard sections of paraffin- embedded tissue, which were cut at a 4-um thickness and mounted on positively charged slides (SuperFrost Plus; Fisher Scientific, Loughborough, UK). After being conventionally deparaffinized and rehydrated, the sec- tions were treated separately in 10 mM citrate buffer
(pH 6.0) for 5 min for IGF2 and MIB-1, and in EDTA buffer (pH 8.0) for 10 min for Smad4, at 121℃ in an autoclave for antigen retrieval. The sections were then immersed in ddH2O containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, followed by incubation with 10% goat serum to block non-specific antibody binding sites. Sections were incu- bated at 4℃ overnight with anti-IGF2 goat polyclonal antibody (sc-1415; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted at 1:100, anti-MIB-1 monoclonal antibody working liquid (ZM-0167, ZSGB- BIO, China), or anti-Smad4 rabbit monoclonal anti- body (ab40759; Abcam, Cambridge, MA, USA) diluted at 1:100. Appropriate positive and negative controls were employed. Scoring of IGF2 staining was graded semiquantitatively from 0 to 4,19,20 and Smad4 stain- ing assessed as ‘diffusely positive’, ‘focally positive’ or ‘negative’, independently by two observers (H.W. and Y.S.).21 Discordant cases were reviewed on a multi- headed light microscope for discussion and consensus.
SCORING FOR IN-SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY
For the purposes of our analysis, we dichotomized staining scores as negative/low versus elevated. For miR483-3p and Smad4, elevated included only ‘strongly positive’ or ‘diffusely positive’ cases. For IGF2, elevated was regarded as an immunoscore ≥3.
STATISTICAL ANALYSES
Comparisons of frequencies and correlations between clinicopathological features, cellular proliferative activity and miR483-3p, IGF2 or Smad4 expression were undertaken using Pearson’s x2 test or Fisher’s exact test. Comparisons of the means were performed using Wilcoxon’s rank sum tests. Survival curves were plotted using the Kaplan-Meier method and P-values were calculated using the log-rank test. The length of overall survival (OS) was defined as the per- iod from the date of surgery to the date of death as a result of the tumour or the latest follow-up. All statis- tical analyses were performed with spss software (SPSS version 17.0; SPSS, Inc., Chicago, IL, USA). A P-value < 0.05 was considered statistically significant.
Results
CLINICAL FEATURES
The clinical data for both malignant and benign adre- nocortical tumours are summarized in Table 1. There
| Malignant (n = 25) | Benign (n = 25) | P value | |
|---|---|---|---|
| Sex | 11 | 11 | 1.000 |
| Male | |||
| Female | 14 | 14 | |
| Tumor location | 13 | ||
| Right | 13 | 0.879 | |
| Left | 12 | 11 | |
| Bilateral | 0 | 1 | |
| Endocrinic syndrome Cushing syndrome | 7 | 3 | – |
| Primary aldosterone syndrome | 1 | 12 | <0.001* |
| Sex characteristics abnormality | 3 | 0 | – |
| Non-functioning | 14 | 10 | – |
| Age (years) | |||
| Mean | 44.4 | 48.6 | 0.229 |
| Range | 18-75 | 34-69 | |
| Tumor size (cm) | |||
| Mean | 11.5 | 2.4 | <0.001 |
| >6.5 | 17 | 0 | <0.001 |
| Tumor weight (g) | |||
| Mean | 558.0 | 6.7 | <0.001 |
| >50 | 21 | 0 | <0.001 |
* P value for comparisons of frequencies between malignant and benign tumors presenting with primary aldosterone syndrome.
was no statistical difference in sex, age and tumour location between the patients with malignant and benign tumours. However, malignant tumours were significantly larger and heavier than benign tumours (P < 0.001). Moreover, patients presenting with pri- mary aldosterone syndrome were more likely to have a benign tumour (P < 0.001; Table 1).
Among the patients with a malignant tumour, 13 recurred (local recurrence and/or metachronous dis- tant metastasis) during follow-up. Moreover, 14 died from the cancer (mean OS: 23.1 months), two continue to live with the disease, three were alive and disease-
free at their last follow-up and six were lost to follow up. Among patients with a benign tumour, eight were lost to follow up and 17 were alive and disease-free after their operations (mean follow-up: 57 months).
PATHOLOGICAL FEATURES
The Weiss scores for the 25 benign tumours were all equal to or less than 2. No case of malignancy scored less than 4. The mean Weiss score was sig- nificantly higher for malignant tumours than benign tumours (P < 0.001). Each of the Weiss criteria was observed more frequently in malignant tumours (each P < 0.05). In our study, the diagnostic efficacy of Weiss scores ≥3 was high for malig- nancy, with 100% specificity and 100% sensitivity overall. The most sensitive criterion was ‘mitotic rate >5/50 HPF’ (100%), and the least sensitive cri- teria were ‘≤25% clear cells’ or ‘sinusoid invasion’ (36.0%). The most specific criteria were ‘mitotic rate >5/50 HPF’ ‘necrosis’, ‘venous invasion’, ‘sinusoid invasion’ and ‘capsular invasion’ (100%), and the least was ‘Führman nuclear grade III/IV’ (68%; Table 2).
EXPRESSION OF MIR 483-3P, IGF2 AND SMAD4
MiR483-3p was overexpressed significantly in 68% (17 of 25) of ACCs compared to 12% (3 of 25) of ACAs (P < 0.05; Table 3, Figure 1A,D). Moreover, there was a positive correlation between expression of miR483-3p and IGF2 (P < 0.05; Figure 1A,B,E, Table S2). Although Smad4 showed statistically significantly lower expression in ACCs than in ACAs (P < 0.05; Table 3, Figure 1C,F), there was no nega- tive relationship between expression of miR483-3p and Smad4 (P > 0.05; Table S2). Smad4 negative/ low expression was sensitive in differentiating ACCs from ACAs (sensitivity = 92%), but with a low specificity of only 40%. However, the combination of negative/low Smad4 and elevated miR483- 3p expression provided a diagnostic specificity of 92.8%.
CORRELATION BETWEEN THE EXPRESSION OF MIR483-3P, IGF2 OR SMAD4 AND CLINICOPATHOLOGICAL VARIABLES IN ADRENOCORTICAL CARCINOMAS
We evaluated the relationship between the three markers and clinicopathological variables in ACCs. MiR483-3p overexpression was not associated with
| Malignant (n = 25) | Benign (n = 25) | P value | |
|---|---|---|---|
| Nuclear grade Fu"rhman III/IV | 21 | 8 | <0.001 |
| Mitotic rate >5/50 HPF | 25 | 0 | <0.001 |
| Abnormal mitoses | 23 | 2 | <0.001 |
| ≤25% clear cells | 9 | 3 | 0.047 |
| >1/3 diffuse architecture | 10 | 1 | 0.002 |
| Necrosis | 21 | 0 | <0.001 |
| Venous invasion | 11 | 0 | <0.001 |
| Sinusoid invasion | 9 | 0 | 0.002 |
| Capsular invasion | 18 | 0 | <0.001 |
| Total Weiss score (mean) | 5.76 | 0.56 | <0.001 |
age, tumour size, tumour weight, hormonal func- tion, recurrence, metastasis or Weiss scores, but there was a relationship with gender, as male patients were statistically more likely to overexpress miR483-3p than were females (P < 0.05; Table S3). For IGF2, there was no relationship with any clini- copathological variables (Table S3). Regarding Smad4, there were only two patients with elevated Smad4 expression, and there were no specific corre- lations with the clinicopathological features (Table S4).
CORRELATION BETWEEN MIR 483-3P, IGF 2 OR SMAD4 AND CELLULAR PROLIFERATIVE ACTIVITY IN ADRENOCORTICAL CARCINOMAS
Because the MIB-I labelling index has been shown to possess diagnostic and prognostic value in several studies,20,22-25 we tested MIB-1 expression and anal- ysed its relationship with miR483-3p, IGF2 and Smad4 expression using the same study cohort. According to previous studies,24,25 a proliferation index <5% was considered to be low and a prolifera- tion index >5% was considered to be high. Our results showed that MIB-1 was highly expressed in 64% (16 of 25) ACCs, compared to 4% (one of 25) of ACAs. However, the MIB-1 expression level was not associated with miR483-3p, IGF2 or Smad4 expres- sion in ACCs. (Table 4).
| Malignant (n = 25) | Benign (n = 25) | P value | |||
|---|---|---|---|---|---|
| Elevated | Negative/low | Elevated | Negative/low | ||
| miR483-3p | 17 | 8 | 3 | 22 | <0.001 |
| IGF2 | 16 | 9 | 7 | 18 | 0.011 |
| Smad4 | 2 | 23 | 10 | 15 | 0.008 |
THE DIFFERENTIAL DIAGNOSTIC EFFICACY OF THE MOLLECULAR MARKERS IN BORDERLINE TUMOURS
To validate further the differential diagnostic efficacy of the molecular markers, their expression levels were evaluated in the 15 borderline tumours (Weiss score = 2 or 3). The results and the relationship with clinicopathological data are summarized in Table 5. The follow-up period was 42-103 months. Among the 15 borderline cases, three relapsed (local recur- rence and/or metastasis) during follow-up, four were lost to follow up and the other eight were all alive and disease-free after their surgery. We found that none of the four markers alone could predict the malignant potential accurately. However, there are only four cases with both elevated miR483-3p and negative/low Smad4 expression. Notably, among these four cases, three recurred and died of disease (mean survival: 29.7 months) and one patient was lost to follow-up. Together, these results indicated that the combination of miR483-3p and Smad4 is useful in assisting the Weiss system in establishing a diagnosis in borderline cases.
THE PROGNOSTIC VALUE OF MIR 483 - 3P AND SMAD4 IN ACCS
We further validated the prognostic value of the com- bined use of miR483-3p and Smad4 inmmunohisto- chemistry in adrenocortical carcinomas. Only patients with sufficient follow-up data were included (19 of 25). The trend of the OS indicated that ACC patients with tumours showing elevated expression of miR483-3p and negative/low Smad4 had a worse dis- ease outcome than other patients, but the association was not statistically significant (P = 0.053, Figure 2).
Discussion
Distinguishing malignant from benign adrenal corti- cal tumours represents a great challenge for both cli- nicians and pathologists. In 1974, Lewins et al.26
reported that the definitive evidence for malignancy was distant metastasis and/or local infiltration. How- ever, with the development of imaging techniques, a growing number of unexplained adrenal incidentalo- mas have been found, making the diagnosis of poten- tial malignancy more challenging in clinical practice. Therefore, the differential diagnosis of adrenal cortical carcinoma from adenoma requires comprehensive clinical characteristics, pathological morphological evaluation and a variety of auxiliary investigations.
Tumour weight and size have been important clues for malignancy. Tang and Grey27 demonstrated that tumour weights >95 g can be used to determine malignancy with 100% sensitivity. Aubert et al.7 determined that a tumour weight greater than 50 g predicted malignancy with 100% sensitivity and 90.9% specificity in a retrospective analysis of 49 adrenal masses, including 24 ACCs and 25 ACAs. Our study confirmed that malignant tumours had sig- nificantly larger sizes and greater weights than benign tumours. Furthermore, we determined that large tumours with a weight >50 g were malignant, with 100% specificity and 100% sensitivity. However, some studies have also reported tumours with a weight lower than 50 g following a malignant course.28,29 Tumour size is often underestimated by radiological investigation. Thresholds ranging from 3 to 6 are often used to predict the risk of malignancy, according to various studies.30,31 However, small thresholds are applied mainly to increase the preoper- ative radiological identification of non-functioning tumours. Aubert et al.7 proposed a threshold of larger than 6.5 cm for distinguishing malignancy with 100% sensitivity and 91.7% specificity. Somewhat differently, our retrospective study determined that a tumour size greater than 6.5 cm significantly pre- dicted malignancy, with 68% sensitivity and 100% specificity. Further studies with larger sample sizes are needed to confirm the role of tumour weight and size in assessing adrenocortical malignancy. More- over, in this retrospective study, patients presenting with primary aldosterone syndrome were found to be
ACCs
ACAS
A
D
miR483-3p
B
E
IGF2
C
F
Smad4
more likely to have a benign tumour than a malig- nant tumour.
Diagnosis of adrenal cortical carcinomas is not always straightforward. Lesions of intermediate size
with some suspicious features are often equivocal for diagnosis. At this point, a large number of histologi- cal factors can be taken into account and several multiparametric systems have been proposed for
| miR483-3p | IGF2 | P | Smad4 | P | |||||
|---|---|---|---|---|---|---|---|---|---|
| Elevated | Negative/Low | P | Elevated | Negative/Low | Elevated | Negative/Low | |||
| MIB1 | |||||||||
| >5% | 12 | 4 | 0.394 | 9 | 7 | 0.401 | 2 | 14 | 0.520 |
| <5% | 5 | 4 | 7 | 2 | 0 | 9 | |||
delineating malignancy. Among various histological criteria, the Weiss system is most commonly used. To evaluate its diagnostic efficacy further, this retrospec- tive study reviewed the Weiss criteria among all 50 samples, revealing that a total Weiss score of 3 or higher predicted malignancy significantly, with 100% specificity and 100% sensitivity. However, it has been reported previously that some tumours with three Weiss criteria maintain a clinical benign course,7-9 and individual tumours with two Weiss criteria have behaved in a malignant manner during follow- up.10,11 Thus, although the Weiss system can result in accurate diagnosis of most ACCs and ACAs, it is not applicable in some challenging cases.
To date, many approaches have been studied for the distinction of benign and malignant adrenocorti- cal tumours, such as genotyping methods, molecular profiling studies and most commonly immunohisto- chemical approaches.32 Numerous molecular mark- ers, including topoisomerase II alpha, MIB-1, p53, E-cadherin, HER-2, p27 and the IGF family members, have been validated as diagnostic and prognostic indicators.16,17,20,22-25 Among these, in most studies the MIB-1/Ki-67 labelling index was shown to possess diagnostic and prognostic value. diagnostic threshold used was not consistent, and diagnostic accuracy still requires confirmation in a larger sample size.
Biomarkers for accurate diagnosis, prognosis or molecular therapy are needed urgently. miRNA markers are ideal for this purpose because miRNAs seem to be more stable and less prone to degradation than mRNAs. Moreover, miRNAs are short, and can therefore be easily knocked down or overexpressed for therapy. In our study, locked nucleic acid in-situ hybridization was used to determine the expression of miR483-3p in both adrenal cortical carcinomas and adenomas. We found 17 cases of overexpression in 25 subjects with primary malignancies (68%) com- pared to three of 25 (12%) patients with benign tumours. In line with these results, Patterson et al.33 demonstrated that miR483-3p was highly expressed
in ACCs by real-time quantitative RT-PCR in 34 sam- ples (10 primary ACCs and 24 ACAs).
Previous studies have shown that altered expres- sion of specific miRNAs can contribute to the initia- tion and progression of cancer.34 Our results indicate that miR483-3p is highly expressed in primary ACCs. Therefore, it leads us to hypothesize that miR483-3p may promote the development of adrenal cortical car- cinomas. In addition, some experiments have demon- strated that miR483-3p can protect cells from apoptosis in liver and colorectal cancer cell lines. 13 Future functional studies are necessary to assess whether miR483-3p plays a similar role in ACCs.
MiR483-3p is located at intron 2 of IGF2, which has been reported to be one of the most commonly overex- pressed genes in ACCs.16,17,25 This suggests a high like- lihood of co-expression. To test this hypothesis, we measured the expression of IGF2 in all 50 samples by immunohistochemistry. We found high expression of IGF2 in 16 of 25 ACCs (64%). Furthermore, 14 patients showed simultaneous high expression of miR483-3p and IGF2. There was a positive correlation between the expression of IGF2 and miR483-3p. Thus, the IGF2 gene not only exerts mitogenic effects through interactions with the IGF-I receptor (IGF-IR)35; it may also exploit miR483-3p to regulate target gene expres- sion post-transcriptionally. However, a small subset of patients displayed divergent expression between miR483-3p and IGF2, thus revealing a mechanism in which IGF2 and miR483-3p are not co-regulated. This is an area that requires further investigation.
A recent study reported that the expression of Smad4/DPC4 was suppressed by miR483-3p in pan- creatic cancer.15 Moreover, it has been reported that approximately 55% of pancreatic adenocarcinomas show inactivation of the Smad4/DPC4 gene by one of two mechanisms: homozygous deletion and loss of heterozygosity.36-38 However, the expression status of Smad4 and its correlation with miR483-3p are still unknown in adrenal cortical tumours. Therefore, we also evaluated the expression of Smad4 in all 50 tumours. We found that ‘diffusely positive’ expression
| Tu-mor no. | Sex | Age (years) | Tumor size (cm) | Tumor weight (g) | Tumor Lacation | Clinical symptom | Weiss score | MiR483-3p | IGF2 | Smad-4 | MIB-1 | Follow-up |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | F | 55 | 6 | 82 | Left | No | 3 | E | N | N | L | Relapse, DOD after 47 m |
| 2 | M | 49 | 5 | 70 | Right | Cushing syndrome | 3 | E | E | E | L | NED after 90 m |
| 3 | F | 27 | 9 | 85 | Left | No | 3 | E | E | N | H | Metastases, DOD after 17 m |
| 4 | F | 26 | 5 | 33 | Right | No | 3 | E | E | E | L | NED after 58 m |
| 5 | F | 44 | 3 | 7 | Right | Cushing syndrome | 3 | E | E | N | L | Relapse, DOD after 25 m |
| 6 | F | 29 | 4 | 27 | Right | No | 3 | N | N | E | L | NED after 45 m |
| 7 | M | 40 | 2 | 2 | Right | No | 2 | N | E | N | L | NED after 100 m |
| 8 | M | 58 | 4.5 | 30 | Left | No | 2 | N | N | N | H | LTFU |
| 9 | M | 56 | 10 | 90 | Left | No | 2 | E | N | N | L | LTFU |
| 10 | F | 52 | 5 | 23 | Right | No | 2 | N | E | E | L | NED after 99 m |
| 11 | F | 23 | 3 | 11 | Left | Primary aldosterone sydrome | 2 | N | N | E | H | LTFU |
| 12 | F | 52 | 3 | 12 | Right | No | 2 | N | E | E | L | NED after 84 m |
| 13 | F | 59 | 7 | 125 | Left | No | 2 | N | N | N | L | NED after 61 m |
| 14 | F | 56 | 5 | 64 | Right | Primary aldosterone syndrome | 2 | E | N | E | H | LTFU |
| 15 | F | 45 | 3 | 20 | Left | No | 2 | N | N | E | L | NED after 53 m |
NED, No evidence of disease; DOD, dead of disease; LTFU, lost to follow-up; E, elevated; N, negative/low; H, high, >5%; L, low, <5%.
1.0
0.8
negative/low miR483-3p or elevated Smad4 expression
Cum survival
0.6
0.4
0.2
elevated miR483-3p and negative/low Smad4 expression
0.0
P= 0.053
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
Time (months)
of Smad4 occurred in only two of 25 ACCs, with 23 of 25 ACCs showing ‘focally positive’ or ‘negative’ staining. According to Wilentz et al.21 cases known to have at least one wild-type Smad4 gene were expected to show diffusely positive expression of Smad4. Immunohistochemical staining for Smad4 labelled as ‘negative/low’ indicated deregulation of Smad4, regardless of the mechanism (i.e. overexpres- sion of miR483-3p or the two types of gene inactiva- tion). In our study, negative/low expression of Smad4 occurred in 23 of 25 ACCs, compared to 10 of 25 ACAs, a statistically significant difference. This sug- gests that down-regulation of Smad4 expression may play roles in the carcinogenesis of adrenocortical car- cinoma. The mechanism underlying the role of miR483-3p and the down-regulation of Smad4 expression in the progression of adrenal cortical carci- nomas still requires further biological investigation.
Proliferative activity measured by MIB-1 staining was reported in several previous studies to be useful for the diagnosis and prognostic assessment of ACCs.20,22-25 However, diagnostic accuracy was not verified. We tested MIB-1 expression in the same study cohort, and analysed its relationship with miR483-3p, IGF2 and Smad4 expression in ACCs. We found that 64% (16 of 25) ACCs had high expression of MIB1, compared to 4% (one of 25) of ACAs. How- ever, the MIB1 expression level was not associated with miR483-3p, IGF2 or Smad4 expression in ACCs.
To validate further the differential diagnostic effi- cacy of the molecular markers and identify those that were most reliable, their expression was assessed in an independent set of 15 borderline tumours (Weiss score = 2 or 3), which were followed-up for 42- 103 months. The results showed that none of the
four markers alone could predict malignant potential accurately. However, the combination of elevated expression of miR483-3p and negative/low expression of Smad4 could differentiate the three tumours that followed a malignant course from others.
Based on our finding that miR483-3p and Smad4 expression together could predict clinically malignant behaviour of borderline tumours, we further analysed the prognostic value of the two markers in carcino- mas. The results indicated a trend towards ACC patients with tumours having elevated miR483-3p and negative/low Smad4 expression showing a worse disease outcome than other patients, although the association was not statistically significant. Thus, a larger sample size is needed to evaluate further the diagnostic and prognostic efficacy of the two markers.
In conclusion, our study confirmed that tumour size, tumour weight, hormonal function and the Weiss sys- tem can differentiate malignant from benign tumours in most cases. The combined use of miR483-3p and Smad4 immunohistochemistry provides a helpful addi- tional tool to the Weiss systems in borderline cases. However, larger sample sizes are needed to determine further the differential diagnostic and prognostic effi- cacy of miR483-3p and Smad4 expression. Moreover, the mechanisms through which miR483-3p modu- lates the development of adrenal cortical carcinoma remain to be clarified in future biological studies.
Acknowledgements
We are grateful to Dr Jing Zhang for helping to review the Weiss system. This study was supported by The National Nature Science Foundations of China (30973470 and 81172334).
Conflict of interest
The authors declare no conflicts of interest.
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Supporting information
Additional supporting information may be found in the online version of this article:
Table S1. The morphological features of the 15 borderline tumours.
Table S2. Relationship between expression of miR483-3p and IGF2, and miR483-3p and Smad4 in adrenocortical carcinomas.
Table S3. Correlations between clinicopathological variables and expression of miR483-3p and IGF2 in adrenocortical carcinomas.
Table S4. Clinicopathological features of the two adrenaocortical carcinoma patients with strong Smad4 staining.