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Prognostic significance of immunohistochemical markers in adrenocortical carcinoma
Loncar Zlatibor, Ivan Paunovic, Vladan Zivaljevic, Dusko Dundjerovic, Svetislav Tatic & Vladimir Djukic
To cite this article: Loncar Zlatibor, Ivan Paunovic, Vladan Zivaljevic, Dusko Dundjerovic, Svetislav Tatic & Vladimir Djukic (2018): Prognostic significance of immunohistochemical markers in adrenocortical carcinoma, Acta Chirurgica Belgica, DOI: 10.1080/00015458.2018.1543822
To link to this article: https://doi.org/10.1080/00015458.2018.1543822
Published online: 30 Nov 2018.
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Prognostic significance of immunohistochemical markers in adrenocortical carcinoma
Loncar Zlatiborª, Ivan Paunovicb, Vladan Zivaljevich, Dusko Dundjerovic“, Svetislav Tatic“ and Vladimir Djukicª
1Emergency Centre, Clinical Centre of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia;2Centre for Endocrine Surgery, Clinical Centre of Serbia, Faculty of Medicine, University of Belgrade, Belgrade, Serbia;3Faculty of Medicine, Institute of Pathology, University of Belgrade, Belgrade, Serbia
ABSTRACT
Background: To present basic demographic and clinical characteristics of patients with adre- nocortical carcinoma (ACC), to determine the overall survival rate and to analyze the results of immunohistochemical staining and its correlation with the length of survival.
Material and methods: The study was conducted during the period between 1996 and 2010 and included 30 patients with ACC. Immunohistochemical staining (MMP9, melan A, inhibin, caltretinin, D2-40, synaptophysin and Ki-67) was performed.
Results: ACC was diagnosed in 19 females and 11 men (1.7:1). The average age was 50.1 years. The median tumor size was 10 cm, the median weight 400 g. Majority of subjects had positive immunohistochemical staining for the markers of interest. Patients with any nega- tive staining had shorter cancer-specific survival than ones with positive staining. According to the log-rank test results as well as according to the results of the univariate Cox analysis, negative staining for inhibin, D2-40 and synaptophysin and Ki-67 expression >7% were asso- ciated with poorer prognosis.
Conclusions: The results of our study suggest that the absence of staining for some immu- nohistochemical markers and increased expression of Ki-67 are associated with a poorer prognosis and shorter survival of patients with ACC. Immunohistochemical markers may serve as a prognostic factor for ACC.
ARTICLE HISTORY
Received 26 October 2017 Accepted 29 October 2018
KEYWORDS
Adrenal gland; survival; melan A; inhibin; calretinin; synaptophysin
Introduction
Tumors of the adrenal glands, especially incidental tumors, are being diagnosed more frequently due to the easier accessibility of CT and NMR diagnos- tics. Adrenocortical carcinoma (ACC) is rare tumors of the endocrine system, with an incidence of 1-2 per million and are responsible for approximately 0.2% of all cancer deaths [1,2]. Adrenal incidentalo- mas have small, but significant risk of malig- nancy [3].
Technologically-advanced diagnostics should enable the detection of these cancers when smaller in size and in an earlier stage, but the prognosis is poor. ACC is rarely detected in stage I (<6% of cases) [4,5]. Unfortunately, ACC is diag- nosed when the tumor is extensive, so the average size is approximately 10 cm [1,4]. ACC is very aggressive and the aggressiveness is indicated by the locally infiltrative growth and large propensity to metastasize to regional lymph nodes and
distant organs [5]. The 5-year survival rate of these patients is generally <50% [1,4].
Similar to benign tumors, ACC can be hormo- nally active or inactive. Approximately 70% of ACC is hormonally active and >50% of the cases are associated with Cushing’s syndrome [6].
The histopathologic diagnosis of an ACC is established on the basis of nine parameters com- prising the Weiss scoring system [7]. Histopathologic and immunohistological prognos- tic factors of ACC have not been fully established due to tumor’s rarity [8].
The aim of our study was to present basic demographic and clinical characteristics of patients with ACC, to determine the overall survival rate, to analyze the results of immunohistochemical stain- ing for MMP9, melan A, inhibin, caltretinin, D2-40, synaptophysin and the tumor proliferation marker, Ki-67 and to determine the correlation between the markers and the survival length.
| Number | % | |
|---|---|---|
| Gender | ||
| Women | 19 | 63.3 |
| Men | 11 | 36.7 |
| Age | ||
| ≤20 | 1 | 3.3 |
| 21-30 | 2 | 6.7 |
| 31-40 | 2 | 6.7 |
| 41-50 | 10 | 33.3 |
| 51-60 | 8 | 26.7 |
| 61-70 | 6 | 20.0 |
| >70 | 1 | 3.3 |
| Stage | ||
| I | 0 | 0 |
| II | 15 | 50.0 |
| III | 9 | 30.0 |
| IV | 6 | 20.0 |
| Tumor size | ||
| ≤10 cm | 19 | 63.3 |
| >10 cm | 11 | 36.7 |
| Tumor weight | ||
| ≤400 g | 21 | 70.0 |
| >400 g | 9 | 30.0 |
| Lymphatic metastasis | ||
| Yes | 3 | 10.0 |
| No | 27 | 90/0 |
| Distant metastases | ||
| Yes | 4 | 13.3 |
| No | 26 | 86.7 |
| Locally infiltrative tumor | ||
| Yes | 10 | 33.3 |
| No | 20 | 66.7 |
| Surgical approach | ||
| Laparotomia | 25 | 83.3 |
| Lumbotomia transdorsalis sec. | 5 | 16.7 |
| Young laparoscopy | 0 | 0 |
| Tumor localization | ||
| Left side | 16 | 53.3 |
| Right side | 14 | 46.7 |
| Type of the procedure | ||
| Biopsy | 1 | 3.3 |
| Tumor reduction | 2 | 6.7 |
| Adrenalectomy | 21 | 70.0 |
| Extended adrenalectomy | 6 | 20.0 |
| Mitotane therapy | ||
| Yes | 17 | 56.7 |
| No | 13 | 43.3 |
| Hormonal activity | ||
| Functional | 10 | 33.3 |
| Nonfunctional | 20 | 66.7 |
Material and methods
We analyzed the data from 30 ACC patients, treated at the Clinical Center of Serbia in Belgrade between 1996 and 2014. The definitive diagnosis of ACC was established based on histopathologic examination after surgical treatment.
The data were collected based on medical his- tory and other medical records, which contained basic demographic and clinical characteristics regarding the stage of the disease by MacFarlane and Sullivan [9,10], the size and weight of the tumor, regional lymph node metastases, distant metastases, tumor localization, local infiltration of adjacent organs, type of surgical procedure, radi- ation therapy, chemotherapy, hormonal activity of (functional vs. non-functional tumors) and type of hormonal hypersecretion in the active tumor.
The data were obtained by contact with patients or their family members. Patients who did not succumb to the carcinoma, or a medical condi- tion associated with ACC were not included in the survival calculation. The median follow-up period was 48 months.
Representative paraffin molds and correspond- ing hematoxylin and eosin (H&E)-stained slides were extracted from the archives of the Endocrine Pathology Cabinet at the Clinical Center of Serbia for immunohistochemical analysis. Construction of tissue microarrays (TMA), immunohistochemical analyses, photographing of sectional TMA and the software analysis were carried out at the Institute of Pathology of the Faculty of Medicine, Belgrade.
Construction of TMA
After the selection of a paraffin mold was formed (one mold = one case; ‘donor molds’), H&E-stained preparations were made, reviewed by two endo- crine pathologists. The interest zones were identi- fied and transferred from the slides to the appropriate mold. Using a hollow 1.2 mm diameter needle, three cylinders of the tissue were obtained from the pre-labeled donor molds and transferred to the recipient molds. External positive controls were included in the recipient molds and similarly transferred. All cases in which at least one tissue cylinder was present in all tested sections were included in the study.
Immunohistochemical staining
Each TMA mold was cut into 4 um sections, labeled with primary antibodies to Ki-67 (Clone-MIB1, 1:100 dilution; DAKO), synaptophysin (Clone-DAK- SYNAP, 1:20; DAKO), calretinin (Clone-DAK-Calre 1, 1:50; DAKO), inhibin (Clone-AlphaAB-1, 1:20; Lab Vision), melan A (Clone-A103, 1:50; Novocastra), D2-40 (Clone-D2-40, 1:200; DAKO) and MMP9 (Clone-15W2, Novocastra).
For external positive controls, tissues of the adrenal gland, mesothelial and appendiceal tissues (glandular epithelium and germinal centers) were used. Immunohistochemical staining was per- formed manually in accordance with the man- ufacturer’s instructions for each antibody. Visualization of positive staining was based on the use of the streptavidin-biotin technique (UltraVision Detection System; Lab Vision).
The following patterns of staining were consid- ered positive: cytoplasmic staining for synaptophi- syn, calretinin, inhibin, melan A, D2-40 and MMP9;
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and nuclear staining for Ki-67. Costs of additional immunohistochemical staining were 10 to 15 euros for each marker, or circa 100 euros per patients.
Immunoreactivity was considered positive if at least 6% of the tumor cells were positive for syn- aptophisyn, calretinin, inhibin, melan A, D2-40 and MMP9.
Sections of each TMA cylinder were photo- graphed with an Olympus DP70 camera (magnifi- cation of 200 times). Using the manual method in the ImageJ program (1.48V), the number of posi- tive tumor cell nuclei was determined and divided by the total number of tumor cells (also counted manually) and multiplied by 100. The cut-off value of Ki-67 was set at ≥7%.
Statistical analysis
Statistical analysis of the data was performed using Kaplan-Meier survival curves and a log-rank test to determine the overall survival rate and specific sur- vival rates with respect to each of the analyzed immunohistochemical parameters. The Cox hazard regression model was applied to determine which variables were significantly related to cancer- specific survival.
Results
In the observed series, there were more women than men (1.7: 1) (Table 1). The youngest patient was 19 and the oldest 72 years old (mean 50.1 years). No patients had stage I of the disease. Tumor size ranged from 45 to 210 mm (mean
=99mm) and the average weight was 400g; the largest tumor weighed 2300g. At the time of diag- nosis, locally infiltrative tumor was detected in 10 patients (33.3%), lymphatic metastases in 3 patients (10.0%), and distant metastases in 4 patients (13.3%). In only 3 patients (10.0%), biopsy or tumor reduction was performed, while in all patients a potentially radical procedure was per- formed. In more than half of the patients, mitotane therapy was administered post-operatively.
The 1-year survival was 56.7% and the 5-year survival was 44.8%; none of the patients with ACC had a 10-year cancer-specific survival (Figure 1). The median survival was 48 months and the over- all survival was 56.8 months.
The median survival was 10 times longer in the group with positive MMP9 staining. This difference, however, was not statistically significant (Tables 2 and 3).
One ACC patient had negative melan A staining and died 6 months after surgery and diagnosis. The average survival rate and median duration of survival in the group of ACC patients with positive immunohistochemical staining for melan A was eight times longer than patients with negative. This difference was not statistically significant because there was only one patient with nega- tive staining.
Four times as many patients had positive inhibin staining than negative (24:6). Among the patients with negative inhibin staining, one-half died during the first 6 months and only one of six patients lived longer than 1 year and that
| Immunohistochemical staining | Average period of survival (in weeks) | Standard error | 95% confidence interval | Median (in weeks) | Standard error | 95% confidence interval | Log-rank test | |
|---|---|---|---|---|---|---|---|---|
| MMP9 | No (n = 3) | 39.0 | 34.5 | 0-106.6 | 6.0 | 2.5 | 1.2-10.8 | p =. 40 |
| Yes (n = 27) | 59.2 | 10.1 | 39.4-78.9 | 60.0 | 27.8 | 5.4-114.6 | ||
| Melan A | No (n = 1) | 6.0 | 6.0 | — | 6.0 | — | — | p =. 12 |
| Yes (n = 29) | 58.6 | 9.4 | 38.4-75.2 | 48.0 | 37.4 | 0-121.3 | ||
| Inhibin | No (n = 6) | 11.5 | 5.3 | 1.2-21.8 | 6.0 | 3.7 | 0-13.2 | p =. 00 |
| Yes (n =24) | 68.2 | 10.5 | 47.6-88.7 | 84.0 | 14.1 | 56.3-111.7 | ||
| Calretinin | No (n= 10) | 35.3 | 13.5 | 8.8-61.7 | 12.0 | 3.1 | 5.9-18.1 | p =. 07 |
| Yes (n =20) | 68.2 | 12.1 | 44.4-92.0 | 84.0 | 23.7 | 37.5-130.5 | ||
| D2-40 | No (n=11) | 27.7 | 9.5 | 9.1-46.4 | 12.0 | 3.2 | 5.7-18.2 | p =. 03 |
| Yes (n = 19) | 71.4 | 11.6 | 48.1-94.6 | 108.0 | 19.8 | 69.2-146.8 | ||
| Synaptophysin | No (n= 4) | 5.3 | 2.6 | 0.3-10.3 | 3.0 | 3.0 | 0-8.8 | p =. 00 |
| Yes (n = 26) | 64.8 | 9.9 | 45.2-84.3 | 84.0 | 32.0 | 21.3-146.7 | ||
| Ki67 | <7% (n = 19) | 70.3 | 11.1 | 48.5-91.9 | 84.0 | 15.7 | 53.2-114.8 | p =. 04 |
| ≥7% (n=11) | 26.8 | 10.6 | 5.9-47.5 | 8.0 | 3.7 | 0.7-15.3 |
difference in survival length was highly statistically significant.
Patients with positive calretinin staining lived longer than patients with negative staining; how- ever, the difference was not statistically significant. Patients with positive D2-40 staining had a lon- ger survival length; the median survival was nine times longer in the group with positive D2-40 staining than the group of patients with negative D2-40 staining, and this difference was statistically significant.
Of the four patients with negative synaptophy- sin staining, only one lived >6 months; 1-year sur- vival was not observed in this group of patients. The median and average survival were significantly longer in patients with ACC and positive synapto- physin staining.
In the group of patients with higher Ki-67 expression, the overall and median survival were significantly shorter than in the group of patients with ACC in which the Ki-67 was <7%.
Discussion
Endocrine tumors produce hormones, as well as normal glandular tissue, thus the overproduction of hormones can occur from the primary tumor, as well from metastases. If the hormone produced in excess by the endocrine tumor is known, then this hormone can be monitored and used as a tumor marker in the post-operative period. Based on its value, the success of surgical treatment can be assessed or the timing to detect recurrence can be estimated [11].
Because these tumors retain the characteristics of the tissue from which the tumor originates, it is not easy to histopathologically distinguish benign from malignant tumors of the endocrine system. Tumor size, tumor weight, hormonal function and the Weiss system are all high-efficacy criteria for differentiating malignant from benign adrenal tumors. In challenging cases, immunohistochemi- cal markers may help in distinguishing these two entities [8,12].
The Weiss score is the most widely used system in the diagnosis of ACC; it ranges from 0 to 9 and the higher the score, the greater the risk of malig- nant tumors of the adrenal gland. However, diag- nostically challenging cases exist and pathologic evaluation would benefit from the availability of adjunctive molecular testing. Adrenal tumors with a score <3 are benign and a score >3 are malig- nant [13,14]. However, adrenal tumors with a score
| Marker | p value | OR | 95% confidence interval |
|---|---|---|---|
| MMP9 (positive vs. negative) | .45 | 0.61 | 0.17-2.23 |
| Melan A (positive vs. negative) | .17 | 0.23 | 0.03-1.87 |
| Inhibin (positive vs. negative) | .01 | 0.25 | 0.08-0.72 |
| Calretinin (positive vs. negative) | .11 | 0.48 | 0.19-1.18 |
| D2-40 (positive vs. negative) | .04 | 0.37 | 0.14-0.97 |
| Synaptophysin (positive vs. negative) | .01 | 0.20 | 0.06-0.67 |
| Ki 67 (≥7%) (yes vs. no) | .02 | 1.05 | 1.01-1.09 |
of 3 can be malignant and benign, so a Weiss score =3 requires additional immunohistochemical diagnostics. The combination of IGF2 and Ki-67 is highly accurate in distinguishing between the two groups and is particularly helpful in adrenocortical tumors with a Weiss score =3 [15]. An assessment of neoplastic cell proliferation using immunostains of cell cycle-associated nuclear antigen, such as Ki- 67, is the only useful auxiliary method of evaluat- ing malignancy in resected adrenocortical neo- plasms at present [16,17].
Ki-67 correlates with mitotic activity and the morphologic index [18]. The cut-off value for the tumor proliferation marker, Ki-67, was set at a level of 7% in the current study, as done by Morimoto et al. [17] in a study involving 17 ACC patients. These authors showed that the Ki-67 labeling index in ACC ranges from 1-26%, with an average of 7.9%. They reported that a Ki-67 ≥ 7% is associ- ated with a shorter disease-free survival, but not associated with overall survival. Ki-67 can predict recurrences and affects the reccurence-free period. Mc Nicol et al. [19] set a Ki-67 cut-off at 3% and did not find that Ki-67 impacts overall survival in patients with ACC, but only impacts the disease- free survival. In contrast, Stojadinovic et al. ana- lyzed 31 case of ACC and did not find that stratifi- cation of the Ki-67 labeling index to a cut-off value of 5% affects the disease-free survival [20].
Our results showed that a Ki-67 > 7% was sig- nificantly associated with shorter survival in patients with ACC according to the log-tank test (p =. 04) and univariate Cox model (p =. 02, HR =1.05, 95% CI =1.01-1.09).
Advani et al. [21] analyzed 11 patients with ACC, and showed that the median Ki-67 index was 19.0% (range, 3.7%-44.1%) and that the Ki-67 index was unrelated to survival. Indeed, an ele- vated Ki-67 index is a feature of ACC, although it does not appear to predict individual patient sur- vival. Iino et al. [22] reported that the Ki-67 label- ing index in patients with ACC and metastases (11.21 ± 3.15; n = 7) was significantly higher than in patients without metastasis (2.58 ±0.61; n =10). This finding indicates that an
immunohistochemical analysis of Ki-67 can help differentiate carcinoma from adenoma in resected adrenocortical neoplasms and might predict aggressive biologic behavior in carcinomas. The Ki- 67 labeling index in carcinomas is significantly higher than adenomas, as Nakazumi et al. [23] reported. Moreover, the Ki-67 labeling index in liv- ing patients with ACC tended to be lower than deceased patients with ACC.
We evaluated a panel of six markers for immu- nohistochemical staining with ACC (MMP9, melan A, inhibin, caltretinin, D2-40 and synaptophysin). Most patients with ACC had positive staining for all immunohistochemical markers evaluated in our study, as follows: MMP9 27/30; melan A 29/ 30; inhibin 24/30; calretinin 20/30; D2-40 19/30; and synaptophysin 26/30. However, in patients with negative staining, survival was less inde- pendent of the marker, and negative staining was associated with a poorer prognosis. Given that for some markers a small number of cases had nega- tive staining (melan A, n = 1; MMP9, n = 3 and cal- retinin, n=4), this difference was not always statistically significant. According to the log-rank test, negative staining for inhibin (p =. 001), D2-40 (p =. 030), and synaptophysin (p =. 001) was sig- nificantly associated with shorter survival. Negative staining for these markers was also asso- ciated with worse prognosis in patients with ACC according to the univariate Cox model, as follows: inhibin (p =. 010, HR =0.25, 95% CI =0.08-0.72); D2-40 (p =. 040, HR =0.37, 95% CI =0.14-0.97); and synaptophysin (p =. 010, HR =0.20, 95% CI =0.06-0.67).
Mondal et al. [24] reported that vimentin and synaptophysin were positive in eight, melan A in six, calretinin in four out of ten patients and Ki-67 was ≥20% in all of them. Inhibin was assessed in only two patients and both were positive.
Weissferdt et al. [25] reported that the adreno- cortical tumors were positive for steroid receptor cofactor 1, inhibin, melan A, calretinin, and synap- tophysin, but negative for high molecular weight cytokeratin, CAM5.2, and Pax8. Among 40 patients with ACC, Weissferdt and his co-authors [26]
reported that positive staining results were as fol- lows: SRC-1 39/40; inhibin-a 37/40; calretinin 32/ 40; synaptophysin 29/40; melan A 26/40; and CAM5.2 9/40. Rare cases were positive for chro- mogranin A 2/40 and Pax8 1/40.
According to Gaffey and associates [27] the immunostaining patterns of adrenocortical tumors are not clearly defined, primarily due to the incon- sistent expression of cytokeratins. Immunoreactivity for a-inhibin was detected in 67.5% of ACC, accord- ing to Pan [28].
D2-40, an antibody commonly used to highlight lymphatic endothelial cells, is consistently positive in the normal adrenal cortex. D2-40 is strongly and diffusely positive in the cells of the neoplastic and non-neoplastic adrenal cortex, but negative in renal cell carcinoma and pheochromocytomas. D2-40 may be a useful marker for distinguishing primary adrenal cortical neoplasms from both metastatic renal cell carcinoma and pheochromo- cytoma [29].
Ghorab et al. [30] found that 31 of 32 adreno- cortical neoplasms had strong and diffuse granular cytoplasmic staining for melan A. There were no differences in staining patterns between adreno- cortical adenomas and carcinomas.
The limitation of our study was the relatively small number of patients, although it belongs to a larger series which analyzed the results of immu- nohistochemical staining in ACC. This was reason why multivariate analysis was not performed. This is the first series that examined the correlation between immunohistochemical markers and length of survival in patients with ACC.
In conclusion, the results of our study show that the absence of staining on specific immunohisto- chemical markers and a higher Ki-67 are associated with a poorer prognosis and shorter survival in patients with ACC and this indicates a weaker dif- ferentiation of these tumors and the greater malig- nant potential. These results would be useful to verify a greater number of patients in a multicen- tric trial. Such a study involving a larger number of patients would verify whether or not those markers that were found to be factors associated with cancer-specific survival (inhibin, D-40, syanap- tophysin, and Ki-67 > 7%) were actually associated with the length of survival of ACC patients. Also, a larger study would show if markers, such as MMP9, melan A, and calretinin, which were not associated with the length of survival of ACC patients due the small number of patients, impacted survival. The main conclusion of our study was that use of
immunohistochemical markers may serve as prog- nostic factors in patients with ACC.
Ethical approval
All procedures performed in studies involving human par- ticipants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amend- ments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual partici- pants included in the study.
Disclosure statement
No potential conflict of interest was reported by the authors.
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