- Email: [email protected]
Metallothionein protein and minichromosome maintenance protein-2 expression in adrenocortical tumors
G Model
ARTICLE IN PRESS
ANDO-1142; No. of Pages 5
Annales d’Endocrinologie xxx (2019) xxx–xxx
Disponible en ligne sur
ScienceDirect www.sciencedirect.com
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 Saiegh a,∗ , Mohammad Sheikh-Ahmad a , Carmela Shechner a , Maria Reut a , Yusef Darawsha a , Sagit Zolotov b , Hila Shefer c , Ilan Bejar c , Jacob Bejar c a
Endocrinology Department, Bnai-Zion Medical Center, Haifa, Israel Institute of Endocrinology, Diabetes, and Metabolism, Rambam Health Care Campus, Haifa, Israel c Pathology department, Bnai-Zion Medical Center, Haifa, Israel b
a r t i c l e
i n f o
Keywords: Adrenal Carcinoma Immunohistochemistry Metallothioneins Minichromosome maintenance protein-2 Prognosis
a b s t r a c t 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 minichromosome 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 correlation 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.
r é s u m é Mots clés : Glandes surrénales Carcinome Immuno-histochimie Protéines métallothionéines Protéines minichomosome maintenace-2 Pronostic
Objectif. – Certaines tumeurs corticosurrénales isolées réséquées se métastasent tandis que d’autres non. Cette étude a été conc¸ue 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 corticosurrénaliens, 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: [email protected] (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
G Model ANDO-1142; No. of Pages 5
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
2
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 corticosurrénaliens 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 fac¸on 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 fac¸on significative avec le développement des métastases. ´ ´ © 2019 Elsevier Masson SAS. Tous droits reserv es.
1. Introduction Adrenocortical carcinoma (ACC) is a rare and a highly aggressive malignancy with an annual incidence of 0.7–2.0 cases per million 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 generally 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 compose 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 correlation 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 malignancy 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 aggressiveness 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 candidates 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 proteins 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 development and progression [9,10]. MT-1 and MT-2 are MT isoforms that are produced in a trigger of several metals and inflammatory factors [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 expression 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 isoforms 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 MCM2 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 (ClinicalTrials.gov identifier NCT02747355). Adrenal specimens were collected from patients who had undergone adrenalectomy between the years 2003 and 2016. Tissues were fixed in 4% paraformaldehyde, processed routinely, embedded in paraffin and stored in the archives of the pathology department. The sample of the study comprised 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.
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
G Model
ARTICLE IN PRESS
ANDO-1142; No. of Pages 5
L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx Table 1 Clinical characteristics, histologic and IHC studies.
3
3. Results
Type of tumor
ACC
ACA
No. of patients Age (year) ± SD (range)** Sex (male, female) Side (right, left) Diameter (cm) ± SD (range)* Weight (gr) ± SD (range)* WS ± SD (range)* WRS ± SD (range)* MCM-2 staining % (range, median) * MT staining, 0-3 scale (range, median) *
14 48.5 ± 18.7 (22–77) (4, 10) (11, 3) 10.1 ± 3.4 (5–17) 284.0 ± 228.1 (56–913) 5.6 ± 1.2 (4–8) 5 ± 1 (3–6) 23.8 ± 16.7 (3–50, 29)
15 51.7 ± 13.9 (19–70) (4,11) (7, 8) 4.8 ± 0.8 (3.5–6) 39.9 ± 22.2 (13–80) 0.3 ± 0.6 (0–2) 0.4 ± 0.8 (0–2) 0.8 ± 0.7 (0–2, 1)
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 classification [2]. For study specimens, histologic and IHC staining studies were performed on coded 3 m-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 histopathologic 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 invasion) [3]. WRS score was also determined, receiving a numeric score ranging between 0 and 7 as follows: 2 × clear cells comprising 25% or less of the tumor + 2 × 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 primary antibody incubation time for all assays was 32 minutes after antigen retrieval in Tris-based buffer (36-64 minutes at 95–100 ◦ C). 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 (760091 Ventana Systems, Phoenix, AZ) was used for reaction detection according to manufacturer-recommended protocol, and hematoxylin 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 version 21 software. Mann–Whitney U-test was used to examine differences in staining of the studied markers, and Pearson’s correlation 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.
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 disease 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 staining 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 MCM2 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 staining and the presence of histologic feature mitotic rate (R = 0.515, P = 0.033), but no significant correlation was found between MCM2 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 normal 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 correlation 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 purposes. 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 prognostic 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
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
G Model ANDO-1142; No. of Pages 5 4
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
Fig. 1. MT and MCM-2 staining in ACC and ACA (IHC). Staining of MT in ACA showing weak staining in cytoplasm and no staining in nucleus x200 (A); ACC stage II showing intense cytoplasmic and nuclear MT staining x200 (B); MCM-2 in ACC stage III showing relatively low nuclear staining (10%) × 200 (C). High nuclear MCM-2 staining (50%) in ACC stage IV × 100 (D).
they used Helsinki score (3 × mitotic rate + 5 × necrosis + Ki-67%), a cutoff value of 8.5 diagnosed a metastatic ACC with 100% sensitivity 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 diseaserelated 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, studies 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 hypermethylation, 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 correlated 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 completed 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 negative 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 correlation 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 malignancies [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 Ki67 [34]. MCM-2 was demonstrated to be over expressed in salivary gland malignant tumors [35] and in oral squamous cell carcinomas [36]. In gliomas, it was also shown to be correlated with poor outcome [37]. In our study, we observed that MCM-2 expression was significantly 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 prognosis. 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 Ki67 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 evaluation. In accordance with some other studies, we did not find a correlation 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 seldom behave in follow-up as ACCs [25]. As we showed that MT
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
G Model ANDO-1142; No. of Pages 5
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
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. Additionally, 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 unconventional 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 Institutional Review Board. Disclosure of interest The authors declare that they have no competing interest. References [1] Kebebew E, Reiff E, Duh QY, et al. Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress? World J Surg 2006;30:872–8. [2] Fassnacht M, Arlt W, Bancos I, et al. Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2016;175:G1–34. [3] Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163–9. [4] Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clinicopathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol 2002;26:1612–9. [5] Fassnacht M, Dekkers O, Else T, et al. European Society of Endocrinology Clinical Practice Guidelines on the Management of Adrenocortical Carcinoma in Adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2018;179:G1–46. [6] Libé R, Borget I, Ronchi CL, et al. Prognostic factors in stage III–IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study. Ann Oncol 2015;26:2119–25. [7] Pennanen M, Heiskanen I, Sane T, et al. Helsinki score-a novel model for prediction of metastases in adrenocortical carcinomas. Hum Pathol 2015;46:404–10. [8] Henkel G, Krebs B, Metallothioneins:. zinc, cadmium, mercury, and copper thiolates and selenolates mimicking protein active site features–structural aspects and biological implications. Chem Rev 2004;104:801–24. [9] Jasani B, Schmid KW. Significance of metallothionein overexpression in human tumours. Histopathology 1997;31:211–4. [10] Nagel WW, Vallee BL. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc Natl Acad Sci U S A 1995;92:579–83. [11] Coyle P, Philcox JC, Carey LC, et al. Metallothionein: the multipurpose protein. Cell Mol Life Sci 2002;59:627–47.
5
[12] Palmiter RD. The elusive function of metallothioneins. Proc Natl Acad Sci U S A 1998;95:8428–30. [13] Felizola SJ, Nakamura Y, Arata Y, et al. Metallothionein-3 (MT-3) in the human adrenal cortex and its disorders. Endocr Pathol 2014;25:229–35. [14] Hishikawa Y, Koji T, Dhar DK, et al. Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus. Br J Cancer 1999;81:712–20. [15] Weinlich G, Eisendle K, Hassler E, et al. Metallothionein - overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients. Br J Cancer 2006;94:835–41. [16] Maine GT, Sinha P, Tye BK. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics 1984;106:365–85. [17] Johnson EM, Kinoshita Y, Daniel DC. A new member of the MCM protein family encoded by the humanMCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13. Nucleic Acids Res 2003;31:2915–25. [18] Hunt DP, Freeman A, Morris LS, et al. Early recurrence of benign meningioma correlates with expression of mini-chromosome maintenance-2 protein. Br J Neurosurg 2002;16:10–5. [19] Tan DF, Huberman JA, Hyland A, et al. MCM2–a promising marker for premalignant lesions of the lung: a cohort study. BMC Cancer 2001;1:6. [20] Rodins K, Cheale M, Coleman N, et al. Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility. Clin Cancer Res 2002;8:1075–81. [21] Tokuyasu N, Shomori K, Nishihara K, et al. Minichromosome maintenance 2 (MCM2) immunoreactivity in stage III human gastric carcinoma: clinicopathological significance. Gastric Cancer 2008;11:37–46. [22] Giaginis C, Georgiadou M, Dimakopoulou K, et al. Clinical significance of MCM2 and MCM-5 expression in colon cancer: association with clinicopathological parameters and tumor proliferative capacity. Dig Dis Sci 2009;54:282–91. [23] Szajerka A, Dziegiel P, Szajerka T, et al. Immunohistochemical evaluation of metallothionein. Mcm-2 and Ki-67 antigen expression in tumors of the adrenal cortex. Anticancer Res 2008;28:2959–65. [24] van’t Sant HP, Bouvy ND, Kazemier G, et al. The prognostic value of two different histopathological scoring systems for adrenocortical carcinomas. Histopathology 2007;51:239–45. [25] Papotti M, Libè R, Duregon E, et al. The Weiss score and beyond–histopathology for adrenocortical carcinoma. Horm Cancer 2011;2:333–40. [26] Beuschlein F, Weigel J, Saeger W, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma aftercomplete resection. J Clin Endocrinol Metab 2015;100:841–9. [27] Assié G, Antoni G, Tissier F, et al. Prognostic parameters of metastatic adrenocortical carcinoma. J Clin Endocrinol Metab 2007;92:148–54. [28] Duregon E, Cappellesso R, Maffeis V, et al. Validation of the prognostic role of the “Helsinki Score” in 225 cases of adrenocortical carcinoma. Hum Pathol 2017;62:1–7. [29] Mete O, Asa SL, Giordano TJ, et al. Immunohistochemical Biomarkers of Adrenal Cortical Neoplasms. Endocr Pathol 2018;29:137–49. [30] Jouinot A, Assie G, Libe R, et al. DNA Methylation Is an Independent Prognostic Marker of Survival in Adrenocortical Cancer. J Clin Endocrinol Metab 2017;102:923–32. [31] Pastuszewski W, Dziegiel P, Krecicki T, et al. Prognostic significance of metallothionein, p53 protein and Ki-67 antigen expression in laryngeal cancer. Anticancer Res 2007;27:335–42. [32] Peng D, Hu TL, Jiang A, et al. Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas. PLoS One 2011;6:e22009. [33] Gonzalez MA, Tachibana KE, Laskey RA, et al. Control of DNA replication and its potential clinical exploitation. Nat Rev Cancer 2005;5:135–41. [34] Dudderidge TJ, Stoeber K, Loddo M, et al. Mcm2 Geminin, and KI67 define proliferative state and are prognostic markers in renal cell carcinoma. Clin Cancer Res 2005;11:2510–7. [35] Vargas PA, Cheng Y, Barrett AW, et al. Expression of Mcm-2, Ki-67 and geminin in benign and malignant salivary gland tumours. J Oral Pathol Med 2008;37:309–18. [36] Razavi SM, Jafari M, Heidarpoor M, et al. Minichromosome maintenance-2 (MCM2) expression differentiates oral squamous cell carcinoma from precancerous lesions. Malays J Pathol 2015;37:253–8. [37] Hua C, Zhao G, Li Y, et al. Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 2014;14:526. [38] Volante M, Bollito E, Sperone P, et al. Clinicopathological study of a series of 92 adrenocortical carcinomas: from a proposal of simplified diagnostic algorithm to prognostic stratification. Histopathology 2009;55:535–43. [39] Barnett CC, Varma DG, El-Naggar AK, et al. Limitations of size as a criterion in the evaluation of adrenal tumors. Surgery 2000;128:973–83. [40] Huimin Li, Zhikun Lin, Yuxin Bai, et al. Sinomenine inhibits ovarian cancer cell growth and metastasis by mediating the Wnt/b-catenin pathway via targeting MCM2. RSC Adv 2017;7:50017–26.
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
ARTICLE IN PRESS
ANDO-1142; No. of Pages 5
Annales d’Endocrinologie xxx (2019) xxx–xxx
Disponible en ligne sur
ScienceDirect www.sciencedirect.com
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 Saiegh a,∗ , Mohammad Sheikh-Ahmad a , Carmela Shechner a , Maria Reut a , Yusef Darawsha a , Sagit Zolotov b , Hila Shefer c , Ilan Bejar c , Jacob Bejar c a
Endocrinology Department, Bnai-Zion Medical Center, Haifa, Israel Institute of Endocrinology, Diabetes, and Metabolism, Rambam Health Care Campus, Haifa, Israel c Pathology department, Bnai-Zion Medical Center, Haifa, Israel b
a r t i c l e
i n f o
Keywords: Adrenal Carcinoma Immunohistochemistry Metallothioneins Minichromosome maintenance protein-2 Prognosis
a b s t r a c t 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 minichromosome 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 correlation 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.
r é s u m é Mots clés : Glandes surrénales Carcinome Immuno-histochimie Protéines métallothionéines Protéines minichomosome maintenace-2 Pronostic
Objectif. – Certaines tumeurs corticosurrénales isolées réséquées se métastasent tandis que d’autres non. Cette étude a été conc¸ue 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 corticosurrénaliens, 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: [email protected] (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
G Model ANDO-1142; No. of Pages 5
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
2
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 corticosurrénaliens 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 fac¸on 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 fac¸on significative avec le développement des métastases. ´ ´ © 2019 Elsevier Masson SAS. Tous droits reserv es.
1. Introduction Adrenocortical carcinoma (ACC) is a rare and a highly aggressive malignancy with an annual incidence of 0.7–2.0 cases per million 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 generally 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 compose 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 correlation 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 malignancy 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 aggressiveness 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 candidates 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 proteins 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 development and progression [9,10]. MT-1 and MT-2 are MT isoforms that are produced in a trigger of several metals and inflammatory factors [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 expression 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 isoforms 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 MCM2 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 (ClinicalTrials.gov identifier NCT02747355). Adrenal specimens were collected from patients who had undergone adrenalectomy between the years 2003 and 2016. Tissues were fixed in 4% paraformaldehyde, processed routinely, embedded in paraffin and stored in the archives of the pathology department. The sample of the study comprised 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.
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
G Model
ARTICLE IN PRESS
ANDO-1142; No. of Pages 5
L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx Table 1 Clinical characteristics, histologic and IHC studies.
3
3. Results
Type of tumor
ACC
ACA
No. of patients Age (year) ± SD (range)** Sex (male, female) Side (right, left) Diameter (cm) ± SD (range)* Weight (gr) ± SD (range)* WS ± SD (range)* WRS ± SD (range)* MCM-2 staining % (range, median) * MT staining, 0-3 scale (range, median) *
14 48.5 ± 18.7 (22–77) (4, 10) (11, 3) 10.1 ± 3.4 (5–17) 284.0 ± 228.1 (56–913) 5.6 ± 1.2 (4–8) 5 ± 1 (3–6) 23.8 ± 16.7 (3–50, 29)
15 51.7 ± 13.9 (19–70) (4,11) (7, 8) 4.8 ± 0.8 (3.5–6) 39.9 ± 22.2 (13–80) 0.3 ± 0.6 (0–2) 0.4 ± 0.8 (0–2) 0.8 ± 0.7 (0–2, 1)
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 classification [2]. For study specimens, histologic and IHC staining studies were performed on coded 3 m-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 histopathologic 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 invasion) [3]. WRS score was also determined, receiving a numeric score ranging between 0 and 7 as follows: 2 × clear cells comprising 25% or less of the tumor + 2 × 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 primary antibody incubation time for all assays was 32 minutes after antigen retrieval in Tris-based buffer (36-64 minutes at 95–100 ◦ C). 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 (760091 Ventana Systems, Phoenix, AZ) was used for reaction detection according to manufacturer-recommended protocol, and hematoxylin 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 version 21 software. Mann–Whitney U-test was used to examine differences in staining of the studied markers, and Pearson’s correlation 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.
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 disease 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 staining 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 MCM2 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 staining and the presence of histologic feature mitotic rate (R = 0.515, P = 0.033), but no significant correlation was found between MCM2 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 normal 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 correlation 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 purposes. 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 prognostic 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
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
G Model ANDO-1142; No. of Pages 5 4
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
Fig. 1. MT and MCM-2 staining in ACC and ACA (IHC). Staining of MT in ACA showing weak staining in cytoplasm and no staining in nucleus x200 (A); ACC stage II showing intense cytoplasmic and nuclear MT staining x200 (B); MCM-2 in ACC stage III showing relatively low nuclear staining (10%) × 200 (C). High nuclear MCM-2 staining (50%) in ACC stage IV × 100 (D).
they used Helsinki score (3 × mitotic rate + 5 × necrosis + Ki-67%), a cutoff value of 8.5 diagnosed a metastatic ACC with 100% sensitivity 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 diseaserelated 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, studies 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 hypermethylation, 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 correlated 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 completed 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 negative 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 correlation 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 malignancies [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 Ki67 [34]. MCM-2 was demonstrated to be over expressed in salivary gland malignant tumors [35] and in oral squamous cell carcinomas [36]. In gliomas, it was also shown to be correlated with poor outcome [37]. In our study, we observed that MCM-2 expression was significantly 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 prognosis. 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 Ki67 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 evaluation. In accordance with some other studies, we did not find a correlation 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 seldom behave in follow-up as ACCs [25]. As we showed that MT
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
G Model ANDO-1142; No. of Pages 5
ARTICLE IN PRESS L. Saiegh et al. / Annales d’Endocrinologie xxx (2019) xxx–xxx
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. Additionally, 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 unconventional 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 Institutional Review Board. Disclosure of interest The authors declare that they have no competing interest. References [1] Kebebew E, Reiff E, Duh QY, et al. Extent of disease at presentation and outcome for adrenocortical carcinoma: have we made progress? World J Surg 2006;30:872–8. [2] Fassnacht M, Arlt W, Bancos I, et al. Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2016;175:G1–34. [3] Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8:163–9. [4] Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clinicopathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol 2002;26:1612–9. [5] Fassnacht M, Dekkers O, Else T, et al. European Society of Endocrinology Clinical Practice Guidelines on the Management of Adrenocortical Carcinoma in Adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2018;179:G1–46. [6] Libé R, Borget I, Ronchi CL, et al. Prognostic factors in stage III–IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study. Ann Oncol 2015;26:2119–25. [7] Pennanen M, Heiskanen I, Sane T, et al. Helsinki score-a novel model for prediction of metastases in adrenocortical carcinomas. Hum Pathol 2015;46:404–10. [8] Henkel G, Krebs B, Metallothioneins:. zinc, cadmium, mercury, and copper thiolates and selenolates mimicking protein active site features–structural aspects and biological implications. Chem Rev 2004;104:801–24. [9] Jasani B, Schmid KW. Significance of metallothionein overexpression in human tumours. Histopathology 1997;31:211–4. [10] Nagel WW, Vallee BL. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc Natl Acad Sci U S A 1995;92:579–83. [11] Coyle P, Philcox JC, Carey LC, et al. Metallothionein: the multipurpose protein. Cell Mol Life Sci 2002;59:627–47.
5
[12] Palmiter RD. The elusive function of metallothioneins. Proc Natl Acad Sci U S A 1998;95:8428–30. [13] Felizola SJ, Nakamura Y, Arata Y, et al. Metallothionein-3 (MT-3) in the human adrenal cortex and its disorders. Endocr Pathol 2014;25:229–35. [14] Hishikawa Y, Koji T, Dhar DK, et al. Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus. Br J Cancer 1999;81:712–20. [15] Weinlich G, Eisendle K, Hassler E, et al. Metallothionein - overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients. Br J Cancer 2006;94:835–41. [16] Maine GT, Sinha P, Tye BK. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics 1984;106:365–85. [17] Johnson EM, Kinoshita Y, Daniel DC. A new member of the MCM protein family encoded by the humanMCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13. Nucleic Acids Res 2003;31:2915–25. [18] Hunt DP, Freeman A, Morris LS, et al. Early recurrence of benign meningioma correlates with expression of mini-chromosome maintenance-2 protein. Br J Neurosurg 2002;16:10–5. [19] Tan DF, Huberman JA, Hyland A, et al. MCM2–a promising marker for premalignant lesions of the lung: a cohort study. BMC Cancer 2001;1:6. [20] Rodins K, Cheale M, Coleman N, et al. Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility. Clin Cancer Res 2002;8:1075–81. [21] Tokuyasu N, Shomori K, Nishihara K, et al. Minichromosome maintenance 2 (MCM2) immunoreactivity in stage III human gastric carcinoma: clinicopathological significance. Gastric Cancer 2008;11:37–46. [22] Giaginis C, Georgiadou M, Dimakopoulou K, et al. Clinical significance of MCM2 and MCM-5 expression in colon cancer: association with clinicopathological parameters and tumor proliferative capacity. Dig Dis Sci 2009;54:282–91. [23] Szajerka A, Dziegiel P, Szajerka T, et al. Immunohistochemical evaluation of metallothionein. Mcm-2 and Ki-67 antigen expression in tumors of the adrenal cortex. Anticancer Res 2008;28:2959–65. [24] van’t Sant HP, Bouvy ND, Kazemier G, et al. The prognostic value of two different histopathological scoring systems for adrenocortical carcinomas. Histopathology 2007;51:239–45. [25] Papotti M, Libè R, Duregon E, et al. The Weiss score and beyond–histopathology for adrenocortical carcinoma. Horm Cancer 2011;2:333–40. [26] Beuschlein F, Weigel J, Saeger W, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma aftercomplete resection. J Clin Endocrinol Metab 2015;100:841–9. [27] Assié G, Antoni G, Tissier F, et al. Prognostic parameters of metastatic adrenocortical carcinoma. J Clin Endocrinol Metab 2007;92:148–54. [28] Duregon E, Cappellesso R, Maffeis V, et al. Validation of the prognostic role of the “Helsinki Score” in 225 cases of adrenocortical carcinoma. Hum Pathol 2017;62:1–7. [29] Mete O, Asa SL, Giordano TJ, et al. Immunohistochemical Biomarkers of Adrenal Cortical Neoplasms. Endocr Pathol 2018;29:137–49. [30] Jouinot A, Assie G, Libe R, et al. DNA Methylation Is an Independent Prognostic Marker of Survival in Adrenocortical Cancer. J Clin Endocrinol Metab 2017;102:923–32. [31] Pastuszewski W, Dziegiel P, Krecicki T, et al. Prognostic significance of metallothionein, p53 protein and Ki-67 antigen expression in laryngeal cancer. Anticancer Res 2007;27:335–42. [32] Peng D, Hu TL, Jiang A, et al. Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas. PLoS One 2011;6:e22009. [33] Gonzalez MA, Tachibana KE, Laskey RA, et al. Control of DNA replication and its potential clinical exploitation. Nat Rev Cancer 2005;5:135–41. [34] Dudderidge TJ, Stoeber K, Loddo M, et al. Mcm2 Geminin, and KI67 define proliferative state and are prognostic markers in renal cell carcinoma. Clin Cancer Res 2005;11:2510–7. [35] Vargas PA, Cheng Y, Barrett AW, et al. Expression of Mcm-2, Ki-67 and geminin in benign and malignant salivary gland tumours. J Oral Pathol Med 2008;37:309–18. [36] Razavi SM, Jafari M, Heidarpoor M, et al. Minichromosome maintenance-2 (MCM2) expression differentiates oral squamous cell carcinoma from precancerous lesions. Malays J Pathol 2015;37:253–8. [37] Hua C, Zhao G, Li Y, et al. Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 2014;14:526. [38] Volante M, Bollito E, Sperone P, et al. Clinicopathological study of a series of 92 adrenocortical carcinomas: from a proposal of simplified diagnostic algorithm to prognostic stratification. Histopathology 2009;55:535–43. [39] Barnett CC, Varma DG, El-Naggar AK, et al. Limitations of size as a criterion in the evaluation of adrenal tumors. Surgery 2000;128:973–83. [40] Huimin Li, Zhikun Lin, Yuxin Bai, et al. Sinomenine inhibits ovarian cancer cell growth and metastasis by mediating the Wnt/b-catenin pathway via targeting MCM2. RSC Adv 2017;7:50017–26.
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