G Model ANDO-1142; No. of Pages 5
Annales d’Endocrinologie xxx (2019) xxx-xxx
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Original article
Metallothionein protein and minichromosome maintenance protein-2 expression in adrenocortical tumors
L’expression des protéines métallothionéines et des protéines MCM (minichromosome maintenance) dans les tumeurs corticosurrénales
Leonard Saiegha,*, Mohammad Sheikh-Ahmada, Carmela Shechnerª, Maria Reutª, Yusef Darawsha ª, Sagit Zolotovb, Hila Sheferc, Ilan Bejar℃, Jacob Bejarc
a Endocrinology Department, Bnai-Zion Medical Center, Haifa, Israel
b Institute of Endocrinology, Diabetes, and Metabolism, Rambam Health Care Campus, Haifa, Israel ” Pathology department, Bnai-Zion Medical Center, Haifa, Israel
ARTICLE INFO
Keywords: Adrenal Carcinoma Immunohistochemistry Metallothioneins Minichromosome maintenance protein-2 Prognosis
ABSTRACT
Aim. - Some resected adrenal-confined adrenocortical carcinomas metastasize and others not. The present study was designed to evaluate the expression of metallothionein protein (MT) and minichromo- some maintenance protein-2 (MCM2) in adrenocortical carcinomas and adrenocortical adenomas, and to test the correlation between this and adrenocortical carcinoma aggressiveness.
Materials and methods. - The study comprised 14 patients operated on for adrenocortical carcinoma, 15 operated on for adrenocortical adenoma and 2 with normal adrenals. Hematoxylin-eosin staining was used for histological evaluation under light microscopy, and sequential sections were used for MCM2 and MT staining.
Results. - In normal adrenals, positive staining was weak for MT and zero for MCM2. Rates of positive staining for MT and MCM2 were significantly higher in adrenocortical carcinomas than in adrenocortical adenomas (P=0.008 and P<0.001, respectively). In adrenocortical carcinomas, a significant positive cor- relation was found between MCM2 staining and Weiss revisited score (P=0.022) but not for Weiss score, and a significant positive correlation was found between MCM2 and mitotic rate on histology (P=0.033). MCM2 but not MT staining was also shown to correlate significantly with stage IV carcinoma (P=0.008 and P=0.165, respectively).
Conclusion. - MCM2 and MT are overexpressed in adrenocortical carcinoma, and MCM2 expression correlates significantly with metastatic disease.
@ 2019 Elsevier Masson SAS. All rights reserved.
Mots clés : Glandes surrénales Carcinome Immuno-histochimie Protéines métallothionéines Protéines minichomosome maintenace-2 Pronostic
RÉSUMÉ
Objectif. - Certaines tumeurs corticosurrénales isolées réséquées se métastasent tandis que d’autres non. Cette étude a été conçue afin d’évaluer à la fois l’expression des protéines métallothionéines (MT) et des protéines minichromosome maintenance-2 (MCM2) dans les carcinomes et les adénomes corticosurré- naliens, et pour évaluer la corrélation entre l’expression de ces protéines et l’agressivité des carcinomes corticosurrénaliens.
Matériel et méthode. - L’étude comprenait 14 patients opérés pour un carcinome corticosurrénalien, 15 patients opérés pour un adénome corticosurrénalien et 2 patients présentant des glandes surrénales saines. L’examen histologique par microscopie optique a été réalisé avec une coloration à l’hématoxyline et l’éosine, avec des coupes séquentielles pour l’étude du taux de coloration en MCM2 et en MT.
Abbreviations: ACA, Adrenocortical Adenoma; ACC, Adrenocortical Carcinoma; ENSAT, European Network for the Study of Adrenal Tumors; IHC, Immunohistochemistry; WRS, Weiss revisited score; WS, Weiss score; MT, Metallothionein; MCMs, Minichromosome maintenance proteins.
* Corresponding author.
E-mail address: leonard.saiegh@gmail.com (L. Saiegh).
https://doi.org/10.1016/j.ando.2019.09.003
0003-4266/ 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Saiegh L, et al. Metallothionein protein and minichromosome maintenance protein-2 expression in adrenocortical tumors. Ann Endocrinol (Paris) (2019), https://doi.org/10.1016/j.ando.2019.09.003
Résultats. - Sur les glandes surrénales saines, le taux de coloration en MT était faible et inexistant en MCM2. Les taux de coloration en MT et en MCM2 étaient significativement plus élevés pour les carcinomes corti- cosurrénaliens que pour les adénomes corticosurrénaliens (respectivement, p=0,008 et p<0,001). Dans les carcinomes corticosurrénaliens, une corrélation positive significative a été trouvée entre la coloration en MCM2 et le score de Weiss (revisited) (p=0,022), mais pas pour le score de Weiss. Une corrélation significative a également été trouvée entre MCM2 et l’indice mitotique suite à l’examen histologique (p=0,033). La coloration en MCM2, mais pas la coloration en MT, corrélait de façon significative avec un carcinome de stade IV (respectivement p = 0,008 et p =0,165).
Conclusion. - Les protéines MCM2 and MT sont surexprimées dans les carcinomes corticosurrénaliens et l’expression des protéines MCM2 est corrélée de façon significative avec le développement des métastases.
@ 2019 Elsevier Masson SAS. Tous droits réservés.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare and a highly aggressive malignancy with an annual incidence of 0.7-2.0 cases per mil- lion population [1]. In contrast, adrenocortical adenoma (ACA) is a common finding, which may be incidentally found in up to 10% of healthy population [2]. When an adrenal mass is suspected to be malignant, patients usually undergo unilateral adrenalectomy. Adrenal gland biopsy is frequently unhelpful. Moreover, it is gener- ally not recommended due to a probable risk for procedure-related tumor spread [2].
ACC staging is commonly determined according to the 2008 European Network for the Study of Adrenal Tumors (ENSAT) group staging system [2]. This system defines ACC stage I and stage II as tumors localized to the adrenal with a size of ≤5.0 and >5.0 cm, respectively. Stage III ACCs are defined on the basis of penetration to the surrounding tissue, involving regional lymph nodes, or by tumor thrombus in the vena cava and/or the renal vein, whereas stage IV tumors are defined by the presence of distant metastasis [2]. Diagnosis of ACC relies on 9 histopathological criteria that com- pose a scoring system called the Weiss score (WS) [3]. In order to improve WS inter and intra-observer variations, a scoring system of 7 histopathological criteria was later developed. This system is referred to as the Weiss revisited score (WRS) [4]. While scoring methods are useful in diagnosing ACC, they have a limited cor- relation with tumor stage and a limited value in predicting ACC aggressiveness. Namely, some totally resected adrenal-confined ACCs with a high score may metastasize while others may not, challenging clinicians in selecting patients who may gain long-term benefit from adjuvant therapy aimed to reduce ACC recurrence risk [5]. Mitotane is currently the cornerstone adjuvant medical therapy for completely resected ACC, aiming to reduce the risk of malig- nancy recurrence [5]. However, mitotane treatment comes with significant toxicity; including dizziness, vertigo, gastrointestinal symptoms, and eventually most mitotane treated patients develop adrenal insufficiency [5].
In order to efficiently identify patients with a high recurrence risk, several studies have been conducted to assess tumor aggres- siveness using different histopathological proliferative markers.
Ki-67 is one of the most studied proliferative markers in adrenal tumors, and has been shown to be positively correlated with tumor aggressiveness, making it highly valued in predicting survival in ACC patients [6]. Consequently, Ki-67 was proposed as an indicator assisting to select high risk patients suitable for mitotane therapy [6]. Completely resected tumors with Ki-67 expression in > 10% of cells are usually considered aggressive and may be the best can- didates for treatment [6]. At the same time, some studies have shown that tumors with low Ki-67 index might also metastasize, while high index tumors may not [7]. Subsequently, vigorous efforts seek nowadays to identify possible histopathological, clinical and
immunohistochemistry (IHC) markers that might act as a surrogate for Ki-67 in predicting tumor behavior and aggressiveness.
One of the well-studied IHC markers is the Metallothionein (MT) family of proteins. MTs are intracellular low molecular weight pro- teins that have been shown to act as scavengers of intracellular reactive oxygen species and as a buffer of toxic heavy metals ions [8]. MTs are over expressed in various human tumors and their expression may be related to different stages of tumor develop- ment and progression [9,10]. MT-1 and MT-2 are MT isoforms that are produced in a trigger of several metals and inflammatory fac- tors [11]. MT-3 isoform has been found to function as a cell growth inhibitory factor in the nervous system and it has been shown to be expressed in adrenal aldosterone producing adenomas and in ACCs, whereas MT-4 isoform has been shown to be expressed mainly in epithelial cells [12,13]. In multiple neoplasms, MTs over expres- sion was correlated with high mitotic rate and has been considered a potential prognostic factor in squamous cell carcinomas [14] and in melanomas [15].
Other IHC markers that have been previously studied are Minichromosome maintenance proteins (MCMs) [16]. MCM iso- forms activation by cyclin-dependent kinases leads to initiation of DNA synthesis [17], and over expression of MCM isoform-2 protein (MCM-2) enabled identification of cycling cells and also non-cycling cells with a proliferative potential [18,19]. Several studies have confirmed MCM-2 over expression in neoplastic cells from different anatomical sites, such as kidney, stomach, and colon [20-22].
A previous study demonstrated that expression of MTs and MCM-2 was significantly higher in ACCs than ACAs. However, the sample of the study was small and the correlation between marker expression and tumor stage was not studied [23].
The present study was designed to evaluate MT and MCM- 2 expression in ACCs and ACAs, and to correlate between their expression and ACC aggressiveness.
2. Materials and methods
The current retrospective study was performed on specimens of patients that have been treated in Bnai-Zion medical center. Approval by the local ethics committee and authorization by The National Institute of Health for the study were obtained (ClinicalTri- als.gov identifier NCT02747355). Adrenal specimens were collected from patients who had undergone adrenalectomy between the years 2003 and 2016. Tissues were fixed in 4% paraformalde- hyde, processed routinely, embedded in paraffin and stored in the archives of the pathology department. The sample of the study com- prised 31 patients; 14 patients operated on for ACC, 15 patients operated on for ACA, and 2 patients with normal adrenals operated on for kidney cancer. Clinical characteristics of ACC and ACA cases are presented in Table 1.
| Type of tumor | ACC | ACA |
|---|---|---|
| No. of patients | 14 | 15 |
| Age (year) ± SD (range) ** | 48.5± 18.7 (22-77) | 51.7 ± 13.9 (19-70) |
| Sex (male, female) | (4, 10) | (4,11) |
| Side (right, left) | (11,3) | (7,8) |
| Diameter (cm) ± SD (range)* | 10.1±3.4(5-17) | 4.8±0.8(3.5-6) |
| Weight (gr)±SD (range)* | 284.0±228.1 (56-913) | 39.9±22.2 (13-80) |
| WS ± SD (range)* | 5.6±1.2(4-8) | 0.3± 0.6 (0-2) |
| WRS ±SD (range)* | 5±1(3-6) | 0.4±0.8(0-2) |
| MCM-2 staining % (range, | 23.8 ± 16.7(3-50,29) | 0.8±0.7(0-2,1) |
| median) * | ||
| MT staining, 0-3 scale (range, median) * | 2.3±1.2 (0-3,3) | 1.2±0.6(1-3,1) |
*P<0.05; ** P> 0.05.
2.1. Tissue sampling
ACC staging was determined according to 2008 ENSAT clas- sification [2]. For study specimens, histologic and IHC staining studies were performed on coded 3 pm-thick sections mounted on super-frost slides. Hematoxylin and eosin staining was used for histological evaluation under light microscope and sequential sections were used for MCM-2 and MT staining. For each tumor, WS score was determined, receiving a numeric score ranging between 0 and 9. WS included the nine following histopatho- logic features: presence of necrosis, diffuse architecture, high nuclear grade, atypical mitosis, clear cells comprising 25% or less of the tumor, mitotic rate (greater than 5 per 50 high-power fields), and tumor invasion (sinusoidal, venous and capsular inva- sion) [3]. WRS score was also determined, receiving a numeric score ranging between 0 and 7 as follows: 2 x clear cells com- prising 25% or less of the tumor +2 x mitotic rate + abnormal mitoses + necrosis + capsular invasion [4].
2.2. IHC staining and analysis
The IHC staining studies were performed on an automated stainer (Benchmark Ultra; Ventana Systems, Phoenix, AZ). The pri- mary antibody incubation time for all assays was 32 minutes after antigen retrieval in Tris-based buffer (36-64 minutes at 95-100 ℃). We used Rabbit anti-Human MCM-2 polyclonal antibody (ab4461, Abcam; diluted 1:600) and Mouse anti-Human MT antibody clone E9 (M0639, DAKO; diluted 1:200). iVIEW DAB detection kit (760- 091 Ventana Systems, Phoenix, AZ) was used for reaction detection according to manufacturer-recommended protocol, and hema- toxylin counterstain was used for color development. Negative control was conducted by omitting primary antibody from IHC reaction. MCM-2 and MT staining was evaluated in selected hot spots of the specimen sections. The evaluation of MCM-2 staining was recorded as a numeric percentage of cells with a nuclear color reaction, while MT staining was recorded on a 4-point scale, with 0, 1,2 and 3 points representing negative, weak, moderate and intense staining patterns, respectively.
2.3. Statistical analysis
Statistical analyses were performed by IBM-SPSS statistics ver- sion 21 software. Mann-Whitney U-test was used to examine differences in staining of the studied markers, and Pearson’s cor- relation to examine the correlation of marker staining with other tumor characteristics. Student t test was used for data comparison between groups, two-tailed tests were used and all results were considered statistically significant for P<0.05. Data are presented as mean ± SD.
3. Results
Clinical and pathological characteristics of ACC and ACA cases are presented in Table 1. At diagnosis, one of the ACC patients was with ENSAT stage I, 6 with stage II, 2 with stage III and 5 with stage IV. Patients with local disease (stage I, II and III) had follow-up times from 42 to 186 months (80.7 ±44.8), all were treated with mitotane with no other chemotherapy, and none had tumor stage progression during study follow-up. All patients with stage IV dis- ease eventually succumbed to their disease. All ACC cases had the conventional morphological variant, WS and WRS score > 2, and all ACA cases had WS and WRS score ≤2. ACCs had significantly greater diameter and weight (Table 1).
In normal adrenals, weak cytoplasmic MT and no MCM-2 stain- ing was observed. MT staining was significantly more intense in ACCs compared to ACAs (R=0.797, P=0.008) (Table 1, Fig. 1). In ACAs, weak MT staining was observed to be limited to cytoplasm, while in ACCs MT staining was observed to be intense in both nuclei and cytoplasm (Fig. 1). MCM-2 nuclear staining was observed in both ACAs and ACCs, but was found to be significantly higher in ACCs (R=0.716, P<0.001) (Table 1).
In ACAs, no significant correlation was found between MCM- 2 and MT staining or between their staining and tumor diameter or weight. In ACCs, a significant correlation was found between MCM-2 staining with WRS (R=0.604, P=0.022) but not with WS. No significant correlation was found in ACCs between MT staining neither with WS nor with WRS, and no significant correlation was found between MCM-2 and MT staining. In addition, no correlation was found between MCM-2 staining and diameter or weight of ACC tumors. However, an inverse correlation was found between MT staining and carcinoma diameter (R= - 0.628, P=0.016). In ACCs, a significant positive correlation was found between MCM-2 stain- ing and the presence of histologic feature mitotic rate (R=0.515, P=0.033), but no significant correlation was found between MCM- 2 staining with the other WS histologic features, and no significant correlation was found between MT staining with any of the WS histologic features.
In ACCs, MCM-2 but not MT staining was shown to be highly correlated with stage IV carcinoma (R =0.765, P=0.008) (Fig. 1). No correlation was found between WS, WRS, tumor diameter or tumor weight and stage IV ACC.
4. Discussion
In this study, we introduce MCM-2 and MT as two proliferative markers that are overexpressed in ACC compared to ACA and to nor- mal adrenal; we also show that MCM-2 expression is significantly correlated with metastatic (stage IV) disease.
Nowadays, major and well-established clinical determinants of ACC patient outcome are tumor stage and complete tumor resection. Neither WS nor WRS constantly show a satisfactory cor- relation with malignancy behavior, tumor stage or survival, mainly in cases with borderline histological features [24,25].
Ki-67 protein expression is widely used for prognostic pur- poses. In two independent cohorts of 569 patients with completely resected ACCs, proliferative index Ki-67 emerged as the single most important factor predicting tumor recurrence [26]. However, though Ki-67 is the most widely used index for predicting ACC behavior, this index has its own limitations, as some low Ki-67 ACCs may metastasize and high index tumors may not [7]. Given that “mitotic rate” correlates with survival, it may be helpful in the prog- nostic stratification of ACCs, even though it has been occasionally found not to correlate with Ki-67 index [27].
In 2015, Pennanen et al. developed the Helsinki score, using an equation composed of Ki-67, mitotic rate and necrosis [7]. When
A
B
C
D
they used Helsinki score (3 x mitotic rate +5 x necrosis + Ki-67%), a cutoff value of 8.5 diagnosed a metastatic ACC with 100% sensi- tivity and 99.4% specificity [7]. The main limitation of this study is that most of the cases that were considered ACCs according to WS might had actually been ACA, as their WRS was below 3. A more recent study that validated Helsinki score in predicting disease- related death on 225 ACC patients showed a good performance using Helsinki score threshold of 28.5 points, and Ki-67 threshold of 20.5% [28].
In order to improve the ability to predict tumor behavior, stud- ies have been conducted to search for surrogate prognostic tumor markers [29]. Two common molecular alterations observed in ACCs are overexpression of insulin-like growth factor II and constitutive activation of the catenin pathway [25]. Furthermore, Steroidogenic factor 1 increases proliferation in human adrenocortical tumor cells and its high expression in ACCs has been shown to be associated with poor survival [25]. Moreover, a recent study by Jouinot et al. demonstrated a strong positive correlation between DNA hyper- methylation, ACC recurrence and death [30]. While these molecular markers may turn out to be helpful in diagnosing high-risk tumors in the future, none of them has yet been widely validated.
MTs and MCMs were observed to be over expressed in tumors from multiple origins. Yet, not in all tumors their expression cor- related with poor prognosis. One study showed that expression of MTs was positively correlated with esophageal-tumor metastatic activity and was associated with chemotherapy resistance [14]. At the same time, in a study conducted on laryngeal cancer, intensity of MT expression was not related to poor prognosis [31]. A study by Felizola et al. demonstrated that MT-3 was over expressed in ACCs; nevertheless, its correlation with prognosis was not addressed [13].
In accordance with Szajerka et al.’s observation that was com- pleted on six ACCs cases, in the current study we observed that MT expression was significantly higher in ACCs than in ACAs [23]. However, unlike the observation of Szajerka et al., we found a neg- ative correlation between MT expression and ACC diameter. We do not have a clear explanation of the negative correlation between MT staining and carcinoma diameter. However, as MT staining was not correlated with prognosis, we do not find this negative cor- relation with tumor diameter as an awkward inspection. The high
cytoplasmic expression of MTs in ACCs indicates that MTs may play a role in the biological behavior of malignant cells, in parallel to what has been claimed regarding MT-3 expression in other malig- nancies [32]. Still, it is to be remembered that, in the current study, we used MT antibody clone which reacts with a conserved epitope shared only by human MT isoforms 1 and 2 and not shared by MT-3.
MCM-2 is a protein that plays an essential role in regulating cell proliferation [33], and it seems that in some tissues, detection of MCM-2 enables identifying more cycling cells in comparison to Ki- 67 [34]. MCM-2 was demonstrated to be over expressed in salivary gland malignant tumors [35] and in oral squamous cell carcino- mas [36]. In gliomas, it was also shown to be correlated with poor outcome [37].
In our study, we observed that MCM-2 expression was signif- icantly higher in ACCs than in ACAs, and we also demonstrated a significant correlation between MCM-2 expression and metastatic stage IV disease, a finding that has not been addressed before. Moreover, in agreement with previous studies, MCM-2 expression was found to be mainly nuclear, demonstrating its role in DNA replication [33]. We observed that MCM-2 expression was also in correlation with “mitotic rate”, a histologic feature that has been previously shown to be likewise correlated with poor prognosis [5,38].
The limitation of our study constitutes in its retrospective design in which potential confounding factors could affect patient prog- nosis. Specifically, the mitotane adjuvant treatment proposed to ACC patients could alter disease long-term behavior. Moreover, we had a small number of ACCs to study. Finally, we did not study Ki- 67 expression and, as a result, could not correlate it with the other studied indexes, and we did not have double reading of the slides as intra- and inter-observer variability are frequent in staining evalu- ation.
In accordance with some other studies, we did not find a correla- tion between WS, WRS, tumor diameter or weight with metastatic ACC [39]. These clinical data may be considered as important but not as perfect prognostic markers. In most cases, scoring methods like WS and WRS might make it possible to differentiate between ACC and ACA. Yet, “borderline” scored benign tumors may sel- dom behave in follow-up as ACCs [25]. As we showed that MT
G Model ANDO-1142; No. of Pages 5
and MCM-2 expression is more intense in ACCs than in ACAs, “borderline” scored benign tumors may be evaluated for MT and MCM-2 expression, and intense marker expression may support closer surveillance. Despite a significant correlation between MT staining and malignancy, we demonstrated an overlap in degree of MT staining between ACC and ACA groups. However, no overlap between groups in MCM-2 staining was detected, making MCM-2 more reliable in differentiating ACC from ACA.
Moreover, given that some ACCs may metastasize and other may not, and as MCM-2 expression is significantly correlated with metastatic disease, significant MCM-2 expression may support adjuvant treatment and a closer surveillance for ACCs. Addition- ally, as anti-cancer treatment in ovarian cancer showed to suppress tumor proliferation by modulating MCM-2 protein [40], targeted therapy may become appropriate in the future in selected ACC cases.
5. Conclusions
MCM-2 and MTs are over expressed in ACCs, and MCM-2 expression is significantly correlated with metastatic disease. More studies are needed to evaluate MT and MCM-2 expression as a potential diagnostic tool for “borderline” scored and for unconven- tional histology tumors, and to evaluate MCM-2 expression as a prognostic marker in ACCs.
Ethical approval
This study was approved by Bnai-Zion Medical Center Institu- tional Review Board.
Disclosure of interest
The authors declare that they have no competing interest.
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