Journal of Pediatric Surgery xxx (2016) xxx-xxx

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Journal of Pediatric Surgery

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Journal of Pediatric Surgery

Weineke criteria, Ki-67 index and p53 status to study pediatric adrenocortical tumors: Is there a correlation?

Soutrik Das ª, Moumita Sengupta ª, Nelofar Islam ª, Paromita Roy b, Chhanda Datta ª, Prafulla Kumar Mishra ”, Sugato Banerjee ª, Manoj Kumar Chaudhuri ª, Uttara Chatterjee ª,*

a Department of Pathology, IPGME&R, Kolkata, India

b Department of Pathology, TMC, Kolkata, India

” Department of Pediatric Surgery, IPGME&R, Kolkata, India

d Department of Pediatric Surgery, Park Clinic, Kolkata, India

ARTICLE INFO

Article history: Received 29 February 2016 Received in revised form 21 June 2016 Accepted 26 July 2016 Available online xxxx

Key words:

Pediatric adrenocortical tumors Wieneke criteria p53 status Ki-67 labeling index

ABSTRACT

Purpose: Adrenocortical tumors (ACTs) are rare in pediatric age group. Pediatric ACTs behave differently from their histologically similar adult counterparts and Weiss criteria often cannot accurately predict their clinical be- havior. Wieneke et al. proposed a set of 9 macroscopic and microscopic criteria for diagnosis of malignancy in pe- diatric ACTs. The aim of the present study was to validate the Wieneke criteria in pediatric ACTs and to correlate Ki-67 labeling index and p53 expression with the Wieneke score.

Methods: Our study comprised 17 cases of pediatric ACTs more than 11 years, from January 2005 to December 2015. Relevant clinical features were obtained from records. Comprehensive analysis of gross and microscopic features was performed, according to the criteria proposed by Wieneke et al. Each tumor was categorized as be- nign, intermediate for malignancy or malignant. Ki-67 and p53 immunostaining was done in all cases. The pa- tients were followed-up over a period of 6 months to 60 months.

Results: Applying Wieneke criteria, there were 9 benign and 7 malignant cases, and 1 case was assigned as inter- mediate for malignancy. The most significant markers in favor of malignancy were capsular and venous invasion, followed by the presence of mitotic figures >15/20 HPF. p53 was over-expressed in 86% of the carcinomas. We found a significant correlation between Ki-67 index and Wieneke scoring system. All cases of adenoma achieved complete remission, while 3 patients with carcinoma died.

Conclusion: Our study validates the utility of Wieneke criteria in differentiating adrenocortical carcinomas from adenomas in pediatric age group. Moreover, Ki-67 index and p53 status can be used as supplementary tools in distinguishing adrenocortical carcinomas from adenomas.

Adrenocortical tumors (ACTs) are a heterogeneous group of neo- plasms arising from the adrenal cortex. They constitute about 0.2% of all pediatric tumors and 5-6% of all adrenal tumors, with a reported in- cidence of 0.2-0.3 new cases per 1 million children per year. There is a geographical variation in incidence, as evident by its significantly in- creased occurrence in Southern Brazil [1-4], probably because of high prevalence of a specific germ-line mutation of p53 gene in their popula- tion [5]. However, the p53 status of pediatric ACTs in our population is not known.

* Corresponding author at: 57/11A, Ballygunje Circular Road, Kolkata 700019, West Bengal, India. Tel .: +91 9433763528.

E-mail addresses: drsoutrikdas2010@gmail.com (S. Das),

moumitasengupta83@gmail.com (M. Sengupta), nelofarislam88@gmail.com (N. Islam),

paromita.roy@tmckolkata.com (P. Roy), drcdatta2012@gmail.com (C. Datta),

mishraprafulla_k@rediffmail.com (P.K. Mishra), sugmala@yahoo.co.in (S. Banerjee),

manojchaudhuri@yahoo.co.in (M.K. Chaudhuri), uttarac1@gmail.com (U. Chatterjee).

Like their adult counterparts, it is always difficult to distinguish adre- nocortical adenomas (ACA) from adrenocortical carcinoma (ACC). This is especially difficult in pediatric cases as these behave in a remarkably different manner than their histologically similar adult counterparts [1]. Applying the standard Weiss criteria used to distinguish ACAs from ACCs in adults, these childhood ACTs are often misdiagnosed as carcinomas [6,7]. No single pathologic feature alone can predict malig- nancy, and a definitive set of pathologic criteria separating pediatric ACCs from ACAs remains elusive [8-11]. This is due, in part, to the rarity of these tumors which precludes large series. Previous studies have sug- gested that the clinical features including the age at diagnosis and pre- senting symptoms, especially endocrine manifestations are important prognostic parameters [12,13]. Wieneke et al. [10] proposed a set of 9 macroscopic and microscopic criteria for diagnosis of malignancy in pe- diatric ACTs. According to this scoring system, pediatric ACTs can be cat- egorized into “benign”, “intermediate for malignancy” and “malignant”. Although it has not yet gained widespread acceptance and popularity

among practicing pathologists, this system has been found quite easy to use in daily practice. In our previous study, the usefulness of Wieneke criteria in the assessment of the morphological characteristics and prog- nosis of pediatric ACTs was compared to that of Weiss criteria [14].

Although there are several studies on molecular pathogenesis and assessment of role of molecular markers (p53 and Ki-67) in prognostic categorization of adult ACTs [15], only a few studies are available on the role of these markers as supplementary tools in the diagnosis of pe- diatric ACTs.

The aim of the present study was to validate the Wieneke criteria in pediatric ACTs and to correlate Ki-67 index and p53 expression with the Wieneke score. We also compared the overall survival in the benign and malignant groups of ACTs using the Wieneke classification.

1. Materials and methods

This was a prospective, observational study comprising 17 cases of pediatric ACTs over the last 11 years from January 2005 to December 2015. Relevant clinical features such as age at presentation, sex, present- ing symptoms and signs and any relevant past history were obtained from records. A patient was considered to have Cushing’s syndrome if he or she had moon facies, weight gain, centripetal distribution of fat, plethora, hypertension, striae and easy bruising. Virilization was consid- ered to be present in case of a girl, if she had clitorimegaly, hirsutism, deepened voice or male musculature, and in case of a boy, if he had pre- cocious puberty. Radiological findings (CT or MRI), operative and gross pathologic findings including tumor weight and size were recorded.

A comprehensive analysis of macroscopic and microscopic patholog- ical features was done. According to the criteria proposed by Wieneke et al. [10] following features were carefully assessed and recorded:

1. Tumor weight more than 400 g

2. Tumor size more than 10.5 cm (largest dimension)

3. Extension into the peri-adrenal soft tissue and/or adjacent organs

4. Invasion into inferior vena cava

5. Venous invasion - considered to be present if tumor cells invaded the endothelial-lined vessels with smooth muscle in the wall

6. Capsular invasion - considered to be present when tumor nests or cords extended into or through the capsule with a correspond- ing stromal reaction

7. Tumor necrosis, usually occurring in confluent nests of cells

8. >15 mitosis/20 high power fields (Magnification of 40x with a 10× objective lens) - mitosis was evaluated by counting 10 ran- dom high power fields in the area of maximum number of mito- ses in a given slide

9. Atypical mitotic figures - defined as abnormal chromosomal spread, tripolar/quadripolar/circular forms or indescribably bi- zarre forms.

Tumors with two or less than two of the above mentioned features were classified as benign, the presence of four or more criteria indicated malignancy, whereas tumor with three features was categorized as in- termediate for malignancy.

The post-surgical staging was performed according to the modified staging system used in the International Pediatric Adrenocortical Tumor Registry (IPACTR) study [12]:

ST I - tumor completely excised with negative margins, tumor weight ≤200 g and absence of metastasis.

ST II - tumor completely excised with negative margins, tumor weight >200 g or microscopic residual tumor and absence of metastasis.

ST III - macroscopic residual or inoperable tumor.

ST IV - hematogenous metastasis at presentation.

Patients with recurrent or metastatic disease were considered clini- cally malignant. Children with advanced stage disease received

radiotherapy and multidrug chemotherapy (cisplatin, doxorubicin, etoposide, mitotane) in addition to surgery.

Ki-67 and p53 immunostaining was done after antigen retrieval by heat induced epitope retrieval technique using Cell Marques Trilogy in conjunction with a pressure cooker. Primary antibodies used were p53 and Ki-67 diluted in phosphate buffer saline at pH 7.4, with protein base and preserved in sodium azide (Cell Marques). Positive staining in both cases was observed as brown nuclear staining. Nuclear overex- pression of p53 was considered if >5% tumor nuclei were stained [16,17]. Proliferation index was evaluated by counting Ki-67-positive cells in five high power fields (400x magnification). This was then expressed as a percentage of total cell count for each field and the re- sults were expressed as the mean of percentages in five independent fields. The immunostaining was scored semi-quantitatively by means of a modified histoscore method, taking into account the staining inten- sity and the percentage of positive tumor cells. Ki-67 proliferative index was considered as high if >5% tumor cell nuclei were stained [16,17].

The patients were followed-up over a period of 6 months to 60 months. Complete follow-up data was available for 14 patients in- cluding tumor stage, treatment methods used and current disease sta- tus/metastasis. Three children were lost to follow-up.

1.1. Statistical analysis

Numerical variables were compared between groups by unpaired t test and categorical variables were compared by Fisher’s exact test. The statistical tests were performed and analyzed using the software Graph Pad, Graph Pad Prism version 5 and Vassarstats. Kaplan-Meier analyses were performed using MedCalc version 11.6 (Mariakerke, Belgium: MedCalc Software 2011). P value <0.05 was considered statis- tically significant.

2. Results

2.1. Clinical findings

A total of 17 cases of pediatric ACTs were diagnosed over a period of 11 years. Majority of the patients (82%) presented with signs and symp- toms of endocrine dysfunction. Cushing’s syndrome, with or without virilization, was the most common presentation (53%). Most common presentation in adenomas was Cushing’s syndrome, while virilization was the commonest presentation in malignant cases. An infant girl with ACC presented with features of feminization with vaginal bleeding. Non-functional cases presented with a mass in the abdomen or abdom- inal pain. No case was associated with Beckwith-Wiedemann or any other named syndrome.

The patients were followed-up over a period of 6 months to 60 months. All the 9 cases of adenomas achieved complete remission which included 7 infants. Two children with histological features of ma- lignancy were lost to follow-up. Among the remaining five patients of ACCs, 3 died and 2 were alive with disease. Kaplan-Meier survival plots were drawn to compare the survival between the two groups (adenoma and carcinoma) (Fig. 1). The case assigned as ‘intermediate for malignancy’, showed a malignant clinical course with development of liver metastasis.

The clinicopathological features of the cases are summarized in Table 1.

2.2. Pathological findings

Using the Weineke criteria, 9 cases were classified as adenomas, 7 as carcinomas and 1 case as intermediate for malignancy. There were a total of 11 girls and 6 boys. Out of 11 girls, 7 had features of ACA and 4 of them presented with ACC. Among 6 boys, 3 had features of ACA, 2 presented with ACC and one had intermediate features. The mean age at presentation was 2.5 years with age ranging from 4 months to

Please cite this article as: Das S, et al, Weineke criteria, Ki-67 index and p53 status to study pediatric adrenocortical tumors: Is there a correlation?, J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.07.014

Fig. 1. Kaplan-Meier survival plots comparing survival in the two groups - benign/ adenoma (solid line) versus malignant/carcinoma (stippled line).

100

Survival probability (%)

Time (month)

16 years. There were a total of 8 infants in our series. Seven of them had features of ACA and 1 fell into the category of intermediate for malig- nancy. The girls presented at an older age than the boys (3.1 years vs 15 months). The mean age of presentation for children with ACC was 59 months, compared to 10 months in children with ACA (P = 0.055).

The weight of these tumors ranged from 150 to 700 g with a mean of 382.4 g. One out of 9 ACAs (11%) and 5 out of 7 ACCs (71%) weighed more than 400 g. ACAs had a mean tumor weight of 275 g whereas the ACCs had a mean tumor weight of 510.7 g (P value = 0.0056, significant). The ACTs ranged in size from 3 cm to 15 cm with a mean of 7.47 cm. The average size of ACAs (5.6 cm) was significantly less than that of ACCs (10 cm) (P value = 0.0124). Only one ACA (11%) and 3 (out of 7; 43%) ACCs measured more than 10.5 cm in the largest dimension.

Capsular and venous invasion were present in 6 cases of carcinomas (86%), but not in the adenomas (P value < 0.001). Extension into periadrenal soft tissue/adjacent organs was identified in 4 out of 7 ACCs (57%), but none of the adenomas showed this feature (Pvalue = 0.0192). Only one case of ACC showed macroscopic invasion into inferior vena cava.

A total of 5 ACCs (71%) and 3 ACAs (33%) showed presence of tumor necrosis, which was not statistically significant (P value = 0.1262). All of the ACCs and 2 of 9 ACAs (22%) had >15 mitoses/20 HPF

Table 1 Parameters of Wieneke criteria: statistical significance.
Wieneke criteria		Number of cases (%)		P value
Wieneke criteria		Carcinoma	Adenoma	P value
Weight >400 g	Present	5	1	0.0056
Weight >400 g	Absent	2	8
Size >10.5 cm	Present	3	1	0.0124
Size >10.5 cm	Absent	4	8
Periadrenal extension	Present	4 (25%)	0 (0%)	0.0192
Periadrenal extension	Absent	3(19%)	9 (56%)
Capsular invasion	Present	6 (38%)	0 (0%)	0.0009
Capsular invasion	Absent	1 (6%)	9 (56%)
Vascular invasion	Present	6 (38%)	0 (0%)	0.0009
Vascular invasion	Absent	1 (6%)	9 (56%)
Vena caval extension	Present	1 (6%)	0 (0%)	0.4375
Vena caval extension	Absent	6 (38%)	9 (56%)
Mitotic count >15/20 HPF	Present	7 (44%)	2 (13%)	0.0032
Mitotic count >15/20 HPF	Absent	0 (0%)	7 (44%)
Atypical mitotic figures	Present	3 (19%)	0 (0%)	0.0625
Atypical mitotic figures	Absent	4 (25%)	9 (56%)
Necrosis	Present	5 (31%)	2(13%)	0.1262
Necrosis	Absent	2 (13%)	7(44%)

Statistically significant values are shown in bold.

(P value = 0.0032) and this was found to be significant. Three of the 7 ACCs (43%) showed presence of atypical mitotic figures (Fig. 2).

Applying this scoring system proposed by Wieneke et al. [10] 9 tu- mors were classified as benign, as they had 2 or less than 2 unfavorable criteria and 7 tumors with 4 or more than 4 of the above criteria were categorized as malignant. There was one case with 3 unfavorable fea- tures and was classified as intermediate for malignancy (Table 2).

Distribution of p53 expression between cases of adenoma (group 1) and carcinoma (group 2) was also evaluated and found to be statistically significant (P value < 0.001). In Group 1, all masses demonstrated neg- ative expression (<5% nuclear expression) but 86% cases of Group 2 showed positive p53 expression (>5% nuclear positivity).

Distribution of Ki-67 expression between the two groups was assessed. The median Ki-67 labeling index was found to be 2% in adeno- mas and 12.5% in carcinomas. High Ki-67 index (>5% nuclear staining) was present in all 7 ACCs, but in no ACA (P value < 0.0001) (Fig. 3). All patients with ACAs (having low Ki-67 index) were alive at the end of the study, whereas only 2 patients with ACC with high Ki-67 index were alive with disease (P value = 0.0275). Among the 5 patients with ACCs whose survival data was available, the mean Ki-67 index was 7.8 for those who were doing well, and 17 for those whose outcome was poor.

3. Discussion

Pediatric ACTs are rare tumors. Very few studies have dealt with the clinicopathological features of these tumors. Till date, no single reliable morphologic parameter has been identified as a potential prognostic marker [18]. There is no established correlation between histopatholog- ical features and clinical behavior of these tumors. Previous studies have shown that the established criteria used to distinguish ACCs from ACAs in adults do not help in predicting the biological behavior of pediatric ACTs, and lead to overdiagnosis in favor of ACC. In some series, nearly 90% of ACTs have been diagnosed pathologically as ACCs [1]. Despite having ominous histological features, pediatric ACTs usually follow a more benign course than their histologically similar adult counterparts. This has led to the search for new pathologic parameters to accurately predict the clinical nature of these tumors [10,18-20]. In the present study, in addition to clinical and morphological features, p53 status and Ki-67 labeling index were analyzed to distinguish ACCs from ACAs.

The IPACTR study, conducted on 254 children, has been the largest study on pediatric ACTs till date [12]. However, no single consistent his- tologic criterion was found to be useful in classifying pediatric ACTs as benign or malignant. In our study, there was a significant association be- tween early age at presentation and benign nature of the tumor. There seemed to be a biphasic age distribution with 15 out of 17 cases present- ing at or earlier than 3 years of age. Only 2 children presented at the age of 10 years and 16 years. Among these two older children, one present- ed with isolated virilization and the other with asymptomatic abdomi- nal mass and both turned out to be cases of ACC. Pediatric ACTs are actually a heterogeneous group of tumors comprising those presenting at or below 4 years of age and those at or above 10 years [1,10]. The 2013 SEER study also concluded that age at presentation was a signifi- cant predictor of clinical outcome [13].

In our study, we had 11 girls (with 7 ACAs and 4 ACCs) and 6 boys (3 ACAs, 2 ACCs and 1 with intermediate features). We did not find any significant association between gender and clinical outcome, as sug- gested by Sbragia et al. [11] Most of the pediatric ACTs are hormonally functional. In our study, most of the cases presented with Cushing’s syn- drome (53%).

Among the macroscopic criteria, tumor weight and size have been well studied and have been a part of various proposed staging systems, although using different cut-offs [1,12]. The Wieneke scoring system uses 400 g and 10.5 cm as cut-offs, respectively for tumor weight and size [10]. In our study, 1 out of 9 ACAs and 5 out of 7 ACCs weighed more than 400 g. Only 3 out of 7 ACCs had a size of more than

Fig. 2. (a) Low power view of an adrenocortical carcinoma with broad fibrous bands and marked nuclear atypia of tumor cells (H&E, 100x); (b) high power view showing tumor cells with large, pleomorphic atypical nuclei, prominent nucleoli and atypical mitotic figures (H&E, 400x); (c) photomicrograph showing necrosis (H&E, 40x); (d) photomicrograph shows invasion of a capsular vessel by the tumor cells (H&E, 40x); (e) adrenocortical carcinoma with high Ki-67 labeling index (400x); (f) adrenocortical carcinoma with increased p53 expression (100x).

Fig. 2a

Fig. 2b

Fig. 2c

Fig. 2d

Fig. 2e

Fig. 2f

10.5 cm. Wieneke et al. [10] and Magro et al. [21] have suggested that tumor weight and size cannot be used alone as a predictor of malignan- cy. But, according to other studies, tumor weight appeared to be the most important prognostic marker [1]. Michalkiewicz et al. found tumor size to be the most significant predictor of clinical outcome [12]. Microscopically, most tumors were composed of sheets of cells having abundant eosinophilic cytoplasm and varying nuclear pleomor- phism and atypia. Capsular invasion and venous invasion were present in most (6 out of 7) of the ACCs, and both these criteria were 100% spe- cific for ACCs. Five ACCs and 2 ACAs had tumor necrosis. All 7 ACCs had high mitotic activity (more than 15 mitoses/20 HPF), and 3 of them showed presence of atypical mitoses. Although presence of atypical mi- totic figures was absolutely specific for ACCs, 2 out of 7 ACAs showed necrosis and mitotic count >15/20 HPF.

In our study, the most significant markers in favor of malignancy were capsular and venous invasion, followed by the presence of mitotic figures >15/20 HPF. There are a few other pathologic features which are not included in Wieneke criteria. Papotti et al. [22] have re- ported the significance of stromal myxoid change in adult ACTs. Magro et al. observed focal myxoid change in most childhood ACCs in their study [19]. In our series, 2 out of 7 ACCs showed presence of

focal myxoid change, whereas one infant girl with ACA had extensive myxoid change [23].

A comprehensive grading method for ACTs, suitable for surgical pa- thology practice is yet to be agreed upon. In adult patients, among all the parameters included in the Weiss criteria, mitotic count has been shown to have the highest prognostic significance. According to some authors, mitotic grading of ACCs is the gold standard and essential to in- dividualize the treatment plan [24,25]. Weiss et al. proposed a 2-tier grading system for ACCs, based solely on mitotic count [7]. Besides mi- totic counts, grading of ACCs has been attempted using steroidogenic factor (SF-1) and other proliferation based scoring methods, mainly based on immunohistochemical analysis of Ki-67, p53 and ß-catenin.

Ki-67 is a proliferation marker which is often used as a prognostic in- dicator in different tumors. Takehara et al. observed that the mean Ki-67 labeling index (LI) of ACCs was markedly higher than that of ACAs in adults [26]. In our study, mean Ki-67 LI was 2% for adenomas, as com- pared to a value of 12.5% for carcinomas. Although various studies have shown Ki-67 index >5% only in carcinomas, it should be noted that a low index does not rule out malignancy [27]. Stojadinovic et al. established correlation between Ki-67 LI and morphologic index (Weiss) [16]. However, scanty literature is available on correlation of

S. Das et al. / Journal of Pediatric Surgery xxx (2016) xxx-xxx

Table 2 Application of Wieneke criteria and correlation with clinical outcome, p53 expression and Ki-67 index.
Case no.	Age at presentation (months)	Sex	Tumor weight (g)	Tumor size (cm)	Extension into peri- adrenal soft tissue and/or adjacent	Mitosis >15/20 HPF	Capsular invasion	Vascular invasion	Invasion into IVC	Necrosis	Atypical mitosis	Wieneke category	Follow-up	Follow-up months	p53 expression	Ki-67 expression	Ki 67 index (%)
Case no.	Age at presentation (months)		Tumor weight (g)		organs							Wieneke category	Follow-up	Follow-up months
1	6	F	300	5	−		-	−	−	+	−	Benign	Doing well	60	−	−	3.4
2	36	M	300	4.5	−	+	+	+	−	+	−	Malignant	Doing well on CT	60	+	+	6.2
3	14	M	500	10	+	+	+	+	+	−	+	Malignant	Died	18	+	+	16.2
4	6	F	425	12	−			−	−	−	−	Benign	Doing well	60	−	−	0.8
5	5	F	200	4	−			−	−	−	−	Benign	Doing well	60	−	−	1.6
6	12	F	600	12	+	+	+	+				Malignant	Died	6	+	+	10.2
7	6	M	450	7	−	−	+	−	−	+	−	Intermediate	lost to	NA	−	+	7.6
													follow-up
8	192	F	600	13	+	+	+	+	−	+	−	Malignant	Doing well on CT	60	+	+	9.4
9	24	F	350	6	−	+	-	-		-		Benign	doing well	60	−	−	2.4
10	24	F	200	7.5	−	+	+	+	−	+	+	Malignant	lost to follow-up	NA	+	+	12.5
10	24												lost to follow-up
11	120	F	700	8	−	+				+	+	Malignant	lost to follow-up	NA	+	+	18.4
11	120	F	700	8	−	+				+	+	Malignant	lost to follow-up	NA	+	+
12	9	F	200	5	−			−	−	−	−	Benign	doing well	60	−	−	0.4
13	4	M	300	5.5	−	+						Benign	doing well	60	−	−	2
14	8	F	350	5.5	−			−	−	−	−	Benign	doing well	60	−	−	4.1
15	14	M	675	15	+	+	+	+	−	+	−	Malignant	Died	12	−	+	24.5
16	18	M	200	4	−			−	−	+	−	Benign	doing well	60	−	−	0.5
17	11	F	150	3	−			−	−	−	−	Benign	doing well	60	−	−	2.8

Ki-67 with Wieneke criteria. Assessment of Ki-67 index is particularly useful while reviewing cases with a discrepancy in mitotic counts among pathologists or when mitotic figures cannot be identified easily, possibly because of suboptimal histologic preparation. The Ki-67 index is now regarded as the most informative grading method for ACCs. Duregon et al. demonstrated that the Ki-67 LI was the strongest predic- tor of overall survival in patients with ACCs [28]. However, its prognos- tic utility in pediatric ACTs is yet to be established. In our study, we found significant correlation between Ki-67 LI and Wieneke scoring system.

p53 plays a significant role in cell-cycle progression and apoptosis pathway. The studies on the molecular pathogenesis of ACTs have impli- cated the role of mutation in the p53 tumor suppressor gene in tumor development and progression. In our study, p53 was over-expressed in 86% of the carcinomas, but not in adenomas, and this was found to be significant. Germ-line mutation of the p53 gene has been found in 50-80% ACCs in children [29]. In an Italian series, mutations involving the common hot spots were found in 35% of tumors [30]. Wasserman et al. showed that germ-line mutations of the p53 gene result in broad

Fig. 3. Box and whiskers, vertical: distribution of Ki-67 labeling index in all cases of adrenocortical adenomas and carcinomas.

Ki67 LI

Adenoma

Carcinoma

spectrum of functional loss and do not essentially correspond to the conventional hotspot mutations [31].

Recently, several genomic, transcriptomic, microRNA-based and DNA methylation analysis of ACTs have been performed, the results of which seem to be promising in differentiating ACCs from ACAs [32]. However, most of these studies have been performed on adult ACTs. A recent study on childhood ACTs has identified recurrent alterations in loci comprising well-known oncogenes (MYC, MDM2, PDGFRA, KIT, MCL1, BCL2L1) and tumor suppressor genes (TP53, RB1, RPH3AL) [33]. Doghman et al. found 26 dysregulated miRNAs in a study comprising 25 childhood ACTs [34]. Their role in pathogenesis of pediatric ACTs is yet to be established.

However, in our study, because of limited number of cases, the prog- nostic utility of p53 status and Ki-67 index could not be evaluated. Moreover, genetic analysis to evaluate p53 mutation in our study popu- lation could provide further insights into the pathogenesis of these tumors.

4. Conclusion

Our study validates the utility of Wieneke criteria in differentiating ACCs from ACAs in pediatric age group. We also found significant corre- lation between Ki 67 labeling index as well as the p53 status and the Wieneke score. Therefore, Ki-67 LI and p53 expression could be used as supplementary tools in distinguishing ACCs from ACAs.

Acknowledgement

We are grateful to Prof. Avijit Hazra, Department of Pharmacology, I.P.G.M. E & R, Kolkata for statistical analysis.

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