The Role of Immunohistochemistry in Histopathological Diagnostics of Clinically “Silent” Incidentally Detected Adrenal Masses
Authors
A. Babinska1, K. Sworczak1, P. Wisniewski1, A. Nałecz2, K. Jaskiewicz2
Affiliations
1 Department of Endocrinology and Internal Medicine, Medical University of Gdansk, Poland
2 Department of Pathology, Medical University of Gdansk, Poland
Key words
adrenal incidentalomas · expression of p53 >p21
· PCNA
Ki67
· immunohistochemistry
Abstract
&
Background: The detectability of adrenal inci- dentalomas (incidentally found adrenal tumours) in the whole population is estimated at 0.1%; 0.42% in non-endocrine patients and at 4.3% in oncologically diagnosed ones. Even up to 16% of incidentalomas of adrenal glands can be malig- nant lesions. The issue of crucial importance is the histopathological differentiation between benign lesions and malignant tumours of the adrenal cortex and medulla.
Objectives: To evaluate whether the immuno- histochemical analysis of the expression of p53, p21, PCNA and Ki67 in the tumour’s tissue can be useful in the histopathological diagnostics of adrenal incidentalomas and whether it is impor- tant for prognosis.
Material and methods: Our series consisted of 74 tumour samples from 164 patients oper- ated for incidentalomas. There were 43 cortical adenomas, 11 cortical adrenocarcinomas and
20 PHEOs (including 5 malignant lesions). Using monoclonal antibodies, the expression of p53, p21, PCNA and Ki67 was evaluated.
Results: We found a statistically significant correlation between the expression of p53, p21, Ki67 and the differential diagnosis of adrenal cor- tical adenoma and adrenocortical carcinoma (for proteins: p53 p=0.010, for p21 p=0.010, for Ki67 p<0.001). The statistical significant correlation between PCNA protein and diagnosis of adrenal cortical adenoma and adrenocortical carcinoma was not found. The statistically significant cor- relation between p21, PCNA proteins and the diagnosis of benign and malignant PHEOs was not estimated. There was no expression of Ki67 or p53 protein above the assumed level in benign and malignant pheochromocytomas. The statis- tically significant correlation between p53, p21, PCNA or Ki67 and the occurrence of metastases in adrenocarcinoma and malignant PHEOs was not found.
| received | 09.05.2007 |
| first decision | 16.07.2007 |
| accepted | 30.10.2007 |
Bibliography
DOI 10.1055/s-2007-993164 Exp Clin Endocrinol Diabetes 2008; 116: 246-251
@ J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart . New York ISSN 0947-7349
Correspondence A. Babinska
Department of Endocrinology Medical University Debinki 7 80-952 Gdansk Poland Tel .: + 58/349 28 40
Fax: + 58/349 28 41 a.mail@wp.pl
Introduction
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The detectability of adrenal incidentalomas in the whole population is estimated at 0.1 %; 0.42% in non-endocrine patients and at 4.3% in patients examined for the presence of a neoplastic dis- ease. It is worth mentioning that there has been a steady increase in the number of detected dis- eases for many years [8]. Based on the autopsy and imaging studies, an estimated 1-3% of per- sons older than 50 years show adrenal tumours. Most authors in their studies tell about 4-7% of adrenal carcinoma in the adrenal incidentaloma group. There are only single reports pointing at higher percentage of carcinomas, but these inves- tigation were performed on small groups and there is no confirmation on large groups studies [12].
Benign as well as malignant adrenal masses occur in a so-called hereditary cancer susceptibility syndrome: Beckwith Wiedemann syndrome, Li- Fraumeni syndrome, multiple endocrine neopla- sia syndromes (MEN-1,MEN-2), McCune-Albright syndrome and finally Carney syndrome [4, 10]. In the research of carcinogenesis of sporadic adreno- cortical masses in patients without inborn genetic tendencies, two groups of genes were recognized. The mutations of these genes result in the above mentioned neoplasia. These genes are classified as follows: protooncogenes, which regulate a pace of proliferation and differentia- tion of cells, and suppressor genes, which regu- late the cell cycle. The suppressor genes are: TP53, P57KIP2, H19 and CDKN2A. The TP53 gene is localized on the 17 chromosome in the region 17p13. This gene produces p53 protein. Abnor-
mal activity of the p53 protein more than its inactivity promotes a higher incidence of different types of organ carcinomas. Recent studies have shown that compared to benign lesions there is a statistically significant TP53 overexpression in adrenocortical cancers (ACC) [3]. The constitutional heterozygosis of the TP53 gene was identified in over 80% of pediatric patients with adre- nal carcinoma, however, neither patients nor their families ful- filled the criteria of Li-Fraumeni syndrome [10].
A lot of studies have been focusing on the immunohistochemical p53 protein analysis. The p53 protein hinders a cell cycle by the activation of p21 protein and the regulation of PCNA activity. This process enables the repair of a DNA strand [6,22]. The cor- relation between the expression of the above mentioned pro- teins and clinical symptoms as well as histopathological features of malignancy is the matter under investigation. The variation of protein expression seems to be helpful in the differentiation of a benign process and malignant one.
The proliferating activity of an adrenal mass can be also esti- mated on the basis of Ki67 expression. It is a proliferating marker most commonly preferred by researchers. Its role is to improve the gravity of morphological criteria in prognosis which are applied both to adrenal and medullar masses [13]. The immuno- histochemical overexpression of the proliferating markers can inform us in all probability of the character of masses, and ena- bles us to identify those adrenal carcinoma patients with a poor prognosis [18,20,23,28,30].
Similar diagnostic difficulties refer to distinguishing between benign and malignant PHEO forms. Not until there are metas- tases invading distant chromaffinless organs i.e. liver, kidneys, bones, lymph nodes, omentum and brain, is an appropriate diag- nosis of a malignant PHEO possible [5, 13, 16]. The markers which enable us to distinguish effectively benign types from malignant ones are under intensive investigation. Some researchers claim that the levels of neuron specific enolase (NSE), neuropeptide Y (NYP) and chromogranin (CgA) notably increases in the blood serum of patients with malignant PHEOs [15]. Similarly, a high dihydroxyphenylalanine concentration (DOPA) in the blood may imply a malignant type of tumour [16]. Also, flow cytometry may help to estimate the contents of DNA in a PHEO cell’s nucleus. The aneuploidal or polyploidal amount of DNA may be associated with malignancy [11].
The tissue of pheochromocytoma mass was searched for indica- tion of its malignancy by marking immunohistochemically markers of proliferation i.e. PCNA, Ki67, topoisomerasis Ilo, E- cadherin, p53 protein, bcl-2 protein, Rb gene product and others. The studies on receptors in ErbB family showed a connection between their detectable expression and the incidence of malig- nant or family PHEO types. However, the results of this research is still ambiguous [24,25].
Objectives
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We tried to scrutinize whether the expression of p53, p21, PCNA and Ki67, which are examined immunohistochemically in mass tissue, can be useful in a histopathological diagnostics of acci- dentally detected adrenal carcinomas and, whether it is impor- tant for the prognosis.
Materials and Methods
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This work was approved by the Research Ethics Committee at the Medical University in Gdansk, Poland.
Taking into consideration such factors as tumour size, its sub- clinical hormonal activity, or radiologically estimated risk of malignancy, 164 patients underwent surgical tumour removal in 1992-2004. Because of retrospective study, histopatological material of only 74 patients was recovered to immunohistichem- ical analysis. In all the patients diagnosis was based on the ultra- sound and CT imaging of the abdomen performed for other reason than the pathology of adrenals. In all the individuals the set of screening hormonal tests determining the pituitary - an adrenal axis function - was evaluated. The clinical symptoms of hormonal activity suggesting adrenal hyperfunction in all 74 patients were absent.
People with PHEO were a large group in our material with adre- nal incidentalomas. Most of PHEO patients had hypertension but stable and longstanding. Other typical symptoms as tachy- cardia, sweating were absent. PHEO tumors had various hormo- nal activity. Large tumors, most common in our material, had lower hormonal activity and were less symptomatic. This phe- nomenon may be related to ability of degradation and secretion to circulation inactive katecholamines. In our study interested is a strong negative correlation between tumor size and level of katecholamines in daily urine collection. Although it was no sta- tistical significance further examination may confirm above mentioned relationship.
All primary adrenocortical carcinoma reviewed as part of this study demonstrated three (vessels and capsule infiltration, high mitotic activity) or more of histopathologic criteria needed for the diagnosis of ACC as defined by Weiss et al. [19]. In all malig- nant PHEO’s cases a manifestation of metastasis in organs which do not contain chromaffin tissue has been detected.
In the histopathological examinations of all operated patients, we used tissue material which was fixed in 4% formaldehyde (formalin) and embedded in low-fusible paraffin. Additionally, we used 74 out of the 164 samples in the immunohistochemical study. In 20 cases it was the tissue of PHEO masses (including 5 malignant lesions), in 11 cases - the tissue of ACC and in 43 cases that of adrenal cortical adenomas. Using stained hematoxylin- eosin preparations (H&E) and excluding sections with necrosis or blood effusions some samples of masses with a representa- tive fiber pattern were chosen. The other four-micrometer sec- tions were used for the immunohistochemical studies with mouse monoclonal antibodies applied against:
PCNA antigene - PC10 clone
Ki67 antigene - KiS5 clone p21 antigene - Sx118 clone
p53 antigene - DO-7 clone
The four-micrometer samples were placed on slides coated with a 3% silane solution. After removing the paraffin and hydration, the sections were submitted to demasking in a water bath of a citrate buffer (pH -6.0) at 99℃ for 40 minutes. Endogenous per- oxidase was inactivated through incubating the sections in a 3% hydrogen peroxide solution (H2O2) for ten minutes. The incuba- tion with primary antibodies lasted for 30 minutes, then with secondary antibodies (biotinylated anti-mouse/anti-rabbit) for the next 30 minutes, and finally in a streptavidin-biotin complex coupled with horseradish peroxidase also for 30 minutes. The colour reaction was obtained by incubating the samples in a diaminebenzidine solution (DAB) for 5 minutes. Subsequently,
| Type of tumour | Ki67>5% | p21>10% | p53>5% | PCNA>10% |
|---|---|---|---|---|
| cortical adenoma n=43 | 1 | 3 | 3 | 35 |
| 2.3% | 7.0% | 7.0% | 81.4% | |
| cortical carcinoma | 6 | 4 | 4 | 8 |
| n=11 | 54.6% | 36.4% | 36.4% | 72.7% |
| p | <0.001 | 0.010 | 0.010 | 0.524 |
| PHEO | 0 | 1 | 0 | 11 |
| n= 15 | 6.7% | 73.3% | ||
| PHEO malignant | 0 | 1 | 0 | 2 |
| n=5 | 20.0% | 40.0% | ||
| p | – | 0.389 | – | 0.176 |
the sections were dyed with Mayer hematoxylin and embedded in Canadian balm. Between the stages of the procedure the tis- sue arrays were rinsed in PBS solution. Only the PCNA antigen of the PC10 clone was not submitted to a thermal treatment in a water bath.
The expression of the analyzed markers was estimated on the basis of the proportion of stained nuclei of tumour cells in 10 high power fields (hpf) with the use of the XC Olympus micro- scope. The studies were led autonomously by two pathologists from the Department of Pathomorphology at the Medical Acad- emy in Gdansk. Both pathologists conducting immunohis- topathological studies were unaware of the clinical diagnosis.
The cutoff values for tumour cell staining used in this study were defined as follows: high Ki67 proliferative index if more than 5% tumour nuclei stained; p53 nuclear overexpression if more than 5% tumour nuclei stained; p21 nuclear overexpression if more than 10% tumour nuclei stained and PCNA nuclear overexpres- sion if more than 10% tumour nuclei stained. The cutoff values for tumour cell staining used in this study were defined on the basis of the previously established cutoff values for different solid tumours [9] and were modified for ACC studying [21]. Tumours were then grouped into the binary categories defined as follows: negative expression (neoplasms below the defined cutoff value of immunoreactivity) and positive expression (neo- plastic tissues above the defined cutoff values of immunoreac- tivity).
Statistical analysis employed in the study
The researchers examined the correspondence between the dis- tribution of each variable with continuous value and a normal distribution with the Kolmogrov-Smirnov compatibility test and by analyzing histograms.
For variables with normal or close to normal distribution as a measure of central tendency and diffusion, the arithmetical mean and standard deviation were employed. The data with dis- tribution far from average was described with a median and range.
The t-Student test was used to compare measures of central ten- dencies for the data with a normal distribution, but after we used a logarithmical transformation, and the U Mann-Whitney test for the rest of the data.
Depending on the group size, the correlations between nominal variables and contingency tables were studied with the x2 Pear- son test and the Fisher exact test. For comparisons in which the expected group size equaled 0-5 the Fisher exact test was used and, for the rest of the cases, the Pearson test.
The value of a confidence coefficient p<0.05 was considered to be statistically significant. The calculations were performed with
the Statistica, 6.0 software by StatSoft, Inc., company, licensed for the Medical Academy in Gdansk.
Results
&
We examined the correlation between the expression of the fol- lowing proteins: Ki67, p21, p53, PCNA and the histopathological diagnosis of adrenal cortical adenoma, adrenal carcinoma and PHEO or its malignant form. We showed that the correlation between the expression of the analyzed proteins and the diag- nosis of adrenal adenoma and adrenal carcinoma exists: for Ki67p<0.001, for p21 p=0.010, for p53 p=0.010, for PCNA p=0.524. The obtained results for Ki67, p53 and p21 are statisti- cally significant as shown in Table 1. On the other hand, we did not find a statistically significant correlation between p21 and the PCNA expression or a diagnosis of PHEO and its malignant form. In the investigated groups we did not recognize Ki67 and p53 expression above the established level. The results are pre- sented in Table 1.
· Fig. 1 We examined the correlation expression of the proteins (Ki67, p21, p53, PCNA) and tumor size. The obtained results are not statistically significant.
We analyzed the co-expression of Ki67 and p53 proteins depend- ing on a histopathological diagnosis of cortical adenoma or car- cinoma. All cases of high co-expression of these proteins were adrenocortical cancers. In the examined patients the low expres- sion of Ki67 and p53 was correlated 92.78% with diagnosis of cortical adenoma. It is a statistically essential difference p<0.001 as shown in Table 2.
We examined the correlation between the expression of Ki67, p21, p53 and PCNA in a primary adrenal tissue (ACC, PHEO) and the incidence of distant metastases. cases, expression of all pro- teins were not statistically important in all reported.
Discussion
&
The quick development of modern diagnostic techniques and easier access to imaging, especially ultrasonography, in the last decade has contributed to the higher detectability of inci- dental adrenal tumours. A lot of patients have qualified for adrenalectomy.
An important issue is histopathological distinguishing of benign masses from their malignant equivalents in the cases of cortical as well as medullar masses. The majority of researchers suggest that the vessels and capsule infiltration, together with high mitotic activity are the most essential markers of malignancy.
p53
p21
Ki67
Other characteristics, such as necrotic foci or pleomorphism of cell nuclei, are still discussed [13,29,31]. The immunohisto- chemical markers complementary to the morphological criteria have been still investigated.
Under physiological conditions, the p53 protein activates the p21 protein and regulates PCNA thus inhibiting cell replication enabling the restoration of a DNA strand. If the defect of the genetic material is too deep, the TP53 gene activates a cell apop- tosis pathway. The loss of the suppressive function by the TP53 gene as a result of its mutation, is the most common molecular reason of malignant carcinoma in human [1,7,13]. In normal cells, p21 is bound with Mdm2 protein in an inactive complex and generally does not affect the cell cycle. In the situation of
| Ki67>5%+p53>5% | ||
|---|---|---|
| no | yes | |
| cortical adenoma | 43 | 0 |
| n=43 | 92.8% | 0% |
| cortical carcinoma | 7 | 4 |
| n=11 | 7.2% | 100% |
p<0.001
cell stress, this complex disintegrates, releasing the active p53 protein which is involved in a cell cycle regulation. If cell dam- age is not severe and is reversible, a growing amount of p53 increases the amount of regulatory proteins therefore influenc- ing the blockage of the cell cycle and restoring DNA. However, if the damage to DNA is irreversible, the TP53 gene, together with bax protein, activates the pathway of programmed cell death. A mutated form of p53 loses its ability to activate p21 and bax pro- teins and in this way loses its suppressive function. Additionally, a p53 mutant can stimulate a cell proliferation by activating PCNA transcription. The immunohistochemical expression of p53, p21 and PCNA was analyzed in a series of human malignan- cies [1,7,13] Only few reports on p53 immunohistochemical expression in adrenocortical cancer exist. Linde, in his work [15], proved a minor importance of p53 in adrenocortical carcinoma diagnostics. The high expression of this protein was present in only 5% of all cases. Song [19] reported low proliferative indices and absence of p53 in immunohistochemical analysis of four cases of oncocytic adrenocortical cancer. He suggested that most oncocytic adrencortical cancers might be low-grade malignan- cies with less aggressive histological features compared to other carcinomas. Bernini [3] determined p53 expression in 16 adren- ocortical carcinomas. In 15/16 cases i.e. 94%, regardless of their hormonal activity, he proved overexpression of p53, moreover, in 10 tumours it was even as high as 50% of mass cells.
Another issue is the application of immunohistochemical diag- nostics for prognostic goals. There is no one specific and sensi- tive protein that 100% enables the differentiation between morphologically similar masses. Not until a set of some proteins is analyzed, is an apt diagnosis possible. Ki67 is one of the most commonly referred to proteins which can improve the effective- ness of morphological prognostic criteria. It is equally applicable to medullar and cortical neoplasms. Stojadinovic analyzed a five-year long survival in patients with cortical carcinoma by juxtaposing Ki67 expression in neoplastic tissue. Patients with a high Ki67 expression lived shorter in comparison to those with a low expression; 25% vs 48% [21]. Similarly Terzolo et al. [27], who by analyzing 37 patients with cortical adenomas, pointed out significantly higher Ki67 expression in a group with carcino- mas compared to a group with adenomas. A high Ki67 expres- sion tended to be present in disseminated adrenocortical cancer with a poor prognosis. Takehara et al. [26] also observed higher Ki67 staining index in adrenocortical cancer compared do corti- cal adenoma. In other studies [20] Ki67 overexpression was present in 36% of cases of adrenocortical carcinoma, but it was not detected in any adenomas. So the researchers confirm that the Ki67 protein is an important marker, which differentiates these two pathological lesions.
In our work done on the basis of 74 tissue samples of resected incidentalomas, we analyzed the correlation between the expression of Ki67, p21, p53, PCNA and the histopathological
diagnosis of adrenal carcinoma, adrenal adenoma, PHEO and its malignant type. We demonstrated a higher than the assumed level of Ki67 expression in 54.6% of ACC in contrast to 2.3% expression of the same protein in adenomas. The difference was statistically significant. Similarly, p53 expression was much higher in adrenocortical carcinomas i.e. 36.4% in comparison to a group of adenomas i.e. 7.0% and this difference was also statis- tically significant. Similar results we obtained for p21. The statis- tical significant correlation between PCNA protein and diagnosis of adrenal cortical adenoma and adrenocortical carcinoma was not found, however due to the very high PCNA expression in cell nuclei of all masses, both malignant and benign ones, the pre- cise evaluation of PCNA was impaired. The rate of immunoposi- tivity among adrenal malignant tumours were highly variable, ranging from 5% to 94%. This may be related to the antibody selection, inability of the antibody to recognize a protein with altered configuration or retrieval methodology.
The high co-expression of Ki67 and p53 was always associated with histopathological diagnosis of ACC i.e. 100%, whereas if it was low with diagnosis of adenoma i.e. 92.8%. In 7.2% of cases of ACC the expression of p53 and Ki67 was low. The histopatho- logical analysis of PHEO masses is a highly difficult task for pathologists. Hyperchromatism and pleomorphism of cell nuclei only sometimes testify to a tumour malignancy. The only explicit criteria is a manifestation of metastasis in organs which do not contain chromaffin tissue [14,24]. In the examined series we found malignant PHEOs in 5 patients (5/21). The interpretation of data was limited due to the small size of the group. We did not find Ki67, p53 expression above the assumed level (>5%). Only p21, PCNA expression was analyzed, but any statistically signifi- cant correlation between protein expression and malignant or benign lesions was not found. Probably the smaller group of malignant PHEOs vs ACC in other study had an impact on the different results in both groups. The figures obtained by other researchers are contradictory. Some authors point out the increase of p53 expression in malignant PHEOs. However, others did not confirm these results in the bigger group of patients i.e. 22 patients [13].
All the patients from our study had been observed for 1-11 years after the initial operation. At the time of the last follow-up 6 patients died of adrenal malignant disease (4 cortical adreno- carcinomas and 2 malignant PHEOs), 1 died of other causes, 2 were alive with distant metastases (1 cortical adrenocarcinomas and 1 malignant PHEO), 2 had no evidence of disease, and 5 were lost to follow - up. In addition, we estimated the connec- tion between Ki67, p21, p53, PCNA expression and the occur- rence of remote metastases in patients with ACC and malignant PHEO. In neither of the groups were any statistically significant correlations between the ability to generate metastases and the high expression of the examined proteins proved. Nevertheless, if the expression of the examined proteins was low, in neither of the group were any metastases observed. Similar results were obtained by Stojadinovic et al. [20] Individual marker expression did not provide insights into metastatic potential of adrenal malignant tumours. There is a high probability that histopatho- logical features of a tumour are better predictors of its malig- nant potential than immunohistochemical characteristics. It seems that a mitotic index together with classical histopatho- logical criteria are the most essential factors in the differentia- tion between malignant and benign adrenocortical masses [15, 17,20,21,29,31]. Alongside this, some authors advise the complementation of histopathology with an immunochemical
examination of p53 and Ki67 expression therefore helping to identify malignancy of a detected lesion [14]. Additionally, p53 can be a useful marker recommended for highly malignant car- cinomas (ACC) [32].
Still pioneering immunohistochemical studies of adrenal tumour need further investigations in bigger size groups.
Conclusions
&
An immunohistochemical evaluation of the expression of p53, p21, and Ki67 helps to distinguish between adrenocortical carci- noma and adenoma, but is not useful in benign and malignant pheochromocytoma differentiation.
There is no significant correlation between the expression of p53, p21, PCNA and Ki67 and the occurrence of remote metas- tases in patients with adrenocortical carcinomas and malignant pheochromocytoma.
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