|Year : 2015 | Volume
| Issue : 2 | Page : 175-180
|Histopathological study of adrenocortical masses with special references to Weiss score, Ki-67 index and p53 status
Gautam Mukherjee1, Chhanda Datta1, Uttara Chatterjee1, Moumita Sengupta1, Gaurav Chatterjee1, Malay Bera2, Subhankar Chowdhury3
1 Department of Pathology, Institute of PostGraduate Medical Education and Research, Kolkata, West Bengal, India
2 Department of Urology, Institute of PostGraduate Medical Education and Research, Kolkata, West Bengal, India
3 Department of Endrocrinology, Institute of PostGraduate Medical Education and Research, Kolkata, West Bengal, India
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|Date of Web Publication||17-Apr-2015|
| Abstract|| |
Background: Adrenal mass present with wide morphological spectrum and clinical manifestation, which can give rise to diagnostic confusion. Proper categorization is essential for individualized treatment. Aim and Objective: (1) Application of Weiss criteria to differentiate between benign and malignant adrenocortical neoplasm (2) co-relation of Ki-67 and p53 expression with the Weiss score. Materials and Methods: A prospective, observational study was conducted in the Department of Pathology in collaboration with department of Endocrinology and Urology of a tertiary care hospital including 19 patients presented with clinically symptomatic or radiologically detected adrenocortical mass. Tissue for histopathological study was obtained in the form of the postoperative material. Histopathological categorization was done, and Weiss score was calculated in all tumors. Ki-67 and p53 immunohistochemistry were performed. Result: A total 19 cases of adrenal mass lesions were included. Cushing syndrome was the presenting symptoms in 26.3% adrenocortical masses. All patients with tumors with Weiss's score <3 (Group 1) were alive after 24 months of follow-up compared to 20% of those with Weiss's score >3 (Group 2). Statistically significance difference was noted in average weights and size of the tumors. Distribution of Ki-67 and p53 expression between Group 1 and Group 2 were compared and found statistically highly significant with Fisher's two-tailed P < 0.001. Conclusion: The combination of the meticulous evaluation of clinical, morphological and immunohistochemical profile helps in proper categorization of adrenocortical mass.
Keywords: Adrenocortical mass, Ki-67, p53, Weiss criteria
|How to cite this article:|
Mukherjee G, Datta C, Chatterjee U, Sengupta M, Chatterjee G, Bera M, Chowdhury S. Histopathological study of adrenocortical masses with special references to Weiss score, Ki-67 index and p53 status. Indian J Pathol Microbiol 2015;58:175-80
|How to cite this URL:|
Mukherjee G, Datta C, Chatterjee U, Sengupta M, Chatterjee G, Bera M, Chowdhury S. Histopathological study of adrenocortical masses with special references to Weiss score, Ki-67 index and p53 status. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Feb 20];58:175-80. Available from: http://www.ijpmonline.org/text.asp?2015/58/2/175/155308
| Introduction|| |
Adrenal masses are a heterogeneous group of benign and malignant neoplasms arise from either cortex or medulla. Most of the tumors are nonpalpable even if they are palpable, the idea of their size and the extent of the lesion is not possible. Adrenocortical tumors present either with clinical manifestation of cortical hyperfunction such as Cushing's syndrome, virilization or with symptoms of mass effect. With the advent of high-resolution imaging techniques (ultrasonography, computerized tomography [CT] and magnetic resonance imaging [MRI]), small asymptomatic adrenal nodules (incidentalomas) can also be detected in patients during workup for unrelated indication.  The prevalence of these so-called "incidentally" detected adrenal lesions are at least 3% in those over the age of 50 years. Surgery is the mainstay of treatment for adrenal masses. 
Differentiation between adrenal adenoma and carcinoma gives rise to the diagnostic challenge. Weiss et al. proposed a multifactorial scoring system represent a major step toward standardization of reporting.  They are:
- Nuclear grade,
- Mitotic rate,
- Atypical mitotic figures,
- Diffuse architecture,
- Venous invasion,
- Sinusoid invasion and
- Invasion of tumor capsule.
Each Weiss criterion is scored 0 when absent and 1 when present. Thus, each tumor was graded from 0 to 9. The presence of three or more criteria indicates malignant potential.
p53 plays a significant role in cell-cycle progression and apoptosis pathway. Malignant transformation of the cell is caused by accumulation of the mutated form of p53, which can be expressed by immunohistochemical method.  On the other hand tumor, aggressiveness and prognosis can be detected monoclonal antibody Ki-67, which indicates toward cell proliferation rate.
| Materials and Methods|| |
A prospective, observational, single institution-based study was conducted in the department of Pathology in collaboration with the department of Endocrinology and Urology of a tertiary care hospital. 19 patients presented with clinically symptomatic or radiologically detected adrenocortical mass underwent surgery. Clinical and biochemical evaluation was done, and radiological findings were noted in all cases. Both size and appearance of the tumors on CT and MRI were noted.
Tissue for histopathological study was obtained in the form of the postoperative material. The masses were weighed and measured. After proper processing of the representative sections, slides were stained with hematoxylin and eosin stain. Histopathological categorization was done according to WHO classification after examining the slides under the light microscope.
An average 12 slides were examined for determination of Weiss score. The Fuhrman grading system used in renal cell carcinomas was the basis of nuclear grading.  The areas of highest concentrations of mitotic figures were examined for calculation mitotic rate. The qualitative distribution of atypical mitotic figures was documented. In the case of cytoplasmic clearing, 25% was considered as cut-off value. Diffuse architecture was considered when tumor displayed patternless growth in >33% areas. Confluent tumor cell necrosis was noted. Presence of tumor cells within the venous or sinusoidal lumen was considered as venous or sinusoidal invasion. Presence of cord of tumor cells penetrating through the capsule was regarded as capsular invasion.
Ki-67 and p53 immunohistochemistry (IHC) were performed in cases of adrenocortical lesions. 5 μ sections of these blocks were cut and placed on charged polylysine-coated slides. These sections were used for IHC analysis. Method used was heat-induced epitope retrieval techniques using Cell Marques Trilogy™ in conjunction with a pressure cooker. Primary antibody used was p53 and monoclonal antibody Ki-67 diluted in phosphate buffer saline pH 7.4, with protein base and preserved with Sodium azide (Cell Marque). Positive staining was observed as brown nuclear staining. In smaller adrenals, Ki-67-positive cells were counted on the entire adrenal surface. In larger tumors, proliferation index was evaluated by counting Ki-67-positive cells in five high power field pictures (400 magnification) for each individual tumor. This was then expressed as a percentage of the total cell count for each field. For each individual adrenal, results were expressed as the mean of counts in five independent fields. The immunostaining was scored semi-quantitatively by means of a modified histoscore method, taking into account the staining intensity and the percentage of positive tumor cells. The cut-off values for tumor cell staining used in this study were defined as follows: High Ki-67 proliferative index if >5% tumor nuclei stained; p53 nuclear over expression if >5% tumor nuclei stained.
Mean, media and mode of the data were calculated. Data were analyzed by entering in Microsoft excel 2007 data sheets with Winstat and Analyze-it add on, IBM-SPSS Statistical Software (version 19), Medcale Software (version 11) and Visualstat Software.
| Result|| |
A total no of 19 cases were included in this study of which 9 patients were male and rest 10 patients were female. The age ranged was from 31 years to 60 years.
Clinical evidence of adrenal cortical hyperfunction was noted in 9 (47.4%) patients but rest of cases (10 cases; 52.6%) were nonfunctioning. Cushing syndrome was the presenting symptoms in 26.3% adrenocortical masses.
Of the 19 cases, 10.5% were adrenal hyperplasia, 63.2% cases were adrenal adenomas (Image 1) and rests (26.3%) were adrenocortical carcinoma (Image 2)
All adrenocortical mass lesions were weighed, measured and categorized according to Weiss criteria into broad groups (Group I = Weiss score <3 and Group II = Weiss score >3). 12 cases and 5 cases belonged to Group I and Group II, respectively. In the weight, [Figure 1] and size distribution [Figure 2] statistically significance difference were noted between two groups (P <0.001).
[Table 1]: Showing results of adrenocortical masses according to Weiss criteria
Venous, capsular, and sinusoidal invasion and necrosis was evident in 26.3%, 21.1%, 26.3%, and 26.3% of adrenocortical masses, respectively (Image 3). As per as mitotic rate was concerned, 5.3% cases displayed >5 mitosis per 50 high-power fields, and atypical mitoses was found in 26.3% of cases. Grade 4 nuclei were observed in 10.5% of adrenocortical masses.
Immunohistochemistry profiling of Ki-67 and p53
Distribution of Ki-67 expression between Weiss score ≤3 (Group 1) and >3 (Group 2) were assessed and it was found statistically highly significant with Fisher's two-tailed P < 0.001. Ki-67 expression in Group 1 ranged from 0.40% to 3.6% with a median value 1.45%. In Group 2, the median value of Ki-67 expression was 10.50%. Considering 5% as the cut-off value 26.3% adrenocortical masses showed positive expression [Figure 3].
Distribution of p53 expression between Weiss score ≤3 (Group 1) and >3 (Group 2) was also evaluated and found statistically significant (Fisher's two-tailed P = 0.010). In Group 1, all adrenocortical masses demonstrated negative expression but 60% cases of Group 2 showed positive p53 expression.
In the postoperative period, no death was noted. The patients were followed-up over a period of 4 months to 24 months. All patients with tumors with Weiss's score <3 (Group 1) were alive after 24 months of follow-up compared to 20% of those with Weiss's score >3 (Group 2) ([Figure 4]).
|Figure 4: Kaplan– Meier survival plots comparing survival in the two groups Weiss score ≤3 (Group 1) and >3 (Group 2)|
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| Discussion|| |
In the present study, clinic-morphological profile and histopathological examination was done in all cases.[Table 2]
We came across only 5 cases (14.3%) of adrenocortical carcinoma during our study period. According to different studies, adrenocortical carcinoma is a rare, but aggressive tumor with a prevalence of between 0.5 and 2/million. ,, It accounts for up to 2% of all malignancies. ,,
Regarding age distribution, we found a unimodal peak in adrenocortical carcinoma with 100% patient belonged to age group of 41-60 years. Ng and Libertino  postulated that there has been a bimodal age of incidence of adrenal carcinoma with most patients 40-50 years old. We saw slight female predominance in case of adrenocortical carcinoma, which in concordance with Jain et al. 
We classified adrenocortical masses as functional (47.4%) and nonfunctional (52.6%). Wooten and King  found 59% functional tumor and 41% nonfunctional tumors in their study. Among the functional tumors in the present study, Cushing syndrome was the presenting symptoms in 26.3% adrenocortical masses which were mostly adrenocortical adenoma. According to the literature, functional adenomas are most commonly associated with hyperaldosteronism and Cushing syndrome, whereas a virilizing neoplasm is more likely to be a carcinoma.  Our findings are similar with results of Takehara et al. study.
We did meticulous macroscopic examination of all specimens. Sasano et al.  emphasized that macroscopic examination is the first step towards diagnosis of adrenocortical malignancy and should include accurate measurement of weight and dimension of the specimens and description of the cut surface of the tumors. In our study, size and weight of adrenocortical mass revealed statistically significant correlation with Weiss score. Stojadinovic et al.  demonstrated 78% of primary tumors were larger than 10 cm in size. Jain et al. also postulated that the mean size and weight of the malignant tumors were found to be statistically significantly higher than that of the benign tumors. We found weight >100 g as the threshold of suspicious of malignancy. Hough et al.  also postulated similar opinion.
In our study, the multi-factorial scoring system proposed by Weiss et al.  played a critical role in differentiation of adrenocortical adenoma and carcinoma. We considered Weiss score 3 as the cut-off value. Jain et al.  found that all adrenocortical carcinomas possessed four or more of these criteria of malignancy. Nine independents variables displayed P < 0.001 in the present study, which in concordance with the study of Lucon et al. 
Immunohistochemistry profiling of the Ki-67 proliferative index showed absence of nuclear staining in all adrenal masses with Weiss score <3and high Ki-67 proliferative index was noted in 100% of adrenal carcinomas. In Takehara et al.  study, the mean Ki-67% of adrenal cortical carcinomas was markedly higher than that of adrenal cortical adenomas. Proliferative activity Ki-67 index >5% was seen only in carcinoma in different study, ,, but it should be noted that a low index does not define benign behavior.  Ki-67 correlates with mitotic activity and morphologic index in Stojadinovic et al. study. 
p53 overexpression was observed in only 60% of adrenal masses with Weiss score >3 and no adrenal masses with Weiss score demonstrated >5% nuclear staining for p53. Stojadinovic et al.  also found p53 overexpression only in adrenal carcinoma. Arola et al.  and McNicol et al.  demonstrated immunopositivity for p53 is seen in about 50% of carcinomas and rarely in adenomas. Barzon et al.  evaluated a subgroup of 48 tissues and concluded that mutations in TP53 gene are frequent in adrenocortical carcinomas and might be used as a marker of malignancy.
Statistical analysis of survival period was done in our study. All patients with tumors with Weiss's score <3 (Group 1) were alive after 24 months of follow-up compared to 20% of those with Weiss's score >3 (Group 2). Lucon et al.  found 100% of the patients with Weiss's scores of <3 were alive compared to 61.65% of those with Weiss's score of 4-8.
| Conclusion|| |
Correlation of clinical findings and lab investigation with histopathological reports helps in identification of the nature of adrenal tumor. Validity and reliability of Weiss in distinguishing adrenocortical adenoma and carcinoma was established.
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Dr. Moumita Sengupta
Flat No. 14, Adhar Apartment, 1907 Sreenagar Main Road, New Garia, Kolkata - 700 094, West Bengal
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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