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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 2  |  Page : 129-134
Importance of P53, Ki-67 expression in the differential diagnosis of benign/malignant phyllodes tumors of the breast


Department of Pathology, Tepecik Research and Training Hospital, Izmir, Turkey

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Date of Web Publication23-Sep-2013
 

   Abstract 

Background: Conventionally growth pattern, stromal overgrowth, stromal cellularity and stromal mitotic activity are the main parameters in the grading of phyllodes tumors (PTs). Recent studies revealed that both p53 and Ki-67 expressions are correlated with grade of PTs of the breast. Expression of hormone receptors and overexpression/amplification of HER2 has been studied in PTs to discover the roles of these markers as new treatment modalities. Materials and Method: We studied 26 PT cases. Seventeen benign and nine malignant PTs were re-evaluated as regards stromal cellularity mitotic activity, p53/Ki-67 expression rates and the relation between these parameters. Estrogen receptor and progesterone receptor (ER, PR) positivity were determined by counting nuclear staining in five high-power fields. Also, the presence of any HER2 staining and staining patterns were documanted. Results: Stromal cellularity, mitotic rate, p53 and Ki-67 expression rates were all correlated with benign and malignant histologic subgroups (P = 0.000-0.001). Ki-67 and p53 expressions were statistically significantly correlated with histologic subgroups, stromal cellularity and mitotic rate (P < 0.005). ER and PR expressions in the epithelial component were not statistically significant between the two groups. HER2 showed different staining patterns in the epithelial component, and there was no staining in the stromal component. Conclusion: Ki-67 and p53 expression rates were statistically significantly correlated with grade of mammary PTs; therefore, they can be used in the determination of tumor grade, especially for the differential diagnosis of benign and malignant tumors. Malignant and benign tumors did not differ significantly in terms of hormone receptor and HER2 expression. HER2 expression showed different patterns in the epithelial component of the PTs.

Keywords: Breast, estrogen, HER2, phyllodes, progesterone

How to cite this article:
Kucuk U, Bayol U, Pala EE, Cumurcu S. Importance of P53, Ki-67 expression in the differential diagnosis of benign/malignant phyllodes tumors of the breast. Indian J Pathol Microbiol 2013;56:129-34

How to cite this URL:
Kucuk U, Bayol U, Pala EE, Cumurcu S. Importance of P53, Ki-67 expression in the differential diagnosis of benign/malignant phyllodes tumors of the breast. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Feb 26];56:129-34. Available from: http://www.ijpmonline.org/text.asp?2013/56/2/129/118690



   Introduction Top


Phyllodes tumor (PT) is a benign biphasic breast tumor that is composed of cellular spindle stroma with epithelial elements. It constitutes 0.3-1% of all breast tumors and 2.5% of fibroepithelial lesions of the breast. [1] The neoplastic component of the tumor is the stroma, which determines its behavior. [2] Although there is no grading scheme showing the accurate tumor behavior, histological features correlate with the biological attitude. [3] Today, PTs are divided into three subgroups; benign, borderline and malignant on the basis of histological criteria that include the degree of stromal cellularity, stromal cytological atypia, mitotic activitiy, stromal overgrowth and the type of tumor margins (infiltrating/pushing). [4],[5],[6] Literature data reveals that increasing tumor grade is correlated with the increase of p53 and Ki-67 expressions in the stromal component. [7] In our study, we investigated the role of Ki-67 and p53 expressions in the grading of PT. We also documented the ER, PR and HER2 status in benign and malignant PTs to analyze the differencess in their expression patterns.


   Materials and Methods Top


A total of 26 PT cases that were diagnosed on the basis of conventional histological parameters were included in this study. The ages of the patients, tumor locations and the relevant details were obtained from the pathology reports. All cases were histologically re-graded on the basis of stromal cytologic atypia, stromal hypercellularity, mitotic count, stromal overgrowth, tumor necrosis and tumor margins.

Stromal atypia and hypercellularity were grouped as mild, moderate and severe. [7] The evaluation of stromal overgrowth was made on a low-power field within the areas containing only the stromal component. [8] Mitotic activity was counted in 10 high-power fields (HPF). According to these criteria, the tumors with mild to moderate stromal cellularity and stromal cellular atypia, low mitotic rate (0-4 mitosis/10 HPF), without stromal overgrowth and pushing margins were grouped as benign and the tumors with severe stromal cellularity and stromal atypia, high mitotic rate (>10 mitosis/10 HPF), stromal overgrowth and infiltrative tumor margins were grouped as malignant. [9]

All tumor specimens were fixed in 10% buffered formalin and embedded in paraffin according to standard procedures. Serial 4-μm-thick sections were placed on positively charged slides. Immunohistochemical detection of ER (IR084, DAKO), PR (IR068, DAKO), HER2 (A0485, DAKO), Ki-67 (IR626, DAKO) and p53 (IR616, DAKO) were performed. All immunohistochemical (IHC) stainings were made with Dako Flex ready to use primary antibodies on an Autostainer Link 48 (Dako, Denmark) by using the envision flex system.

Nuclear staining was evaluated as positive for Ki-67 and p53 and counted in five HPF within the stromal and epithelial cells, where 20% was considered as the threshold value for Ki-67 and p53 positivity. The positive groups were divided into three groups as <20%, 21-40% and >40%.

The presence of HER2 staining in ductal epithelial cells and staining patterns (membranous basolateral, basal granular) were also documanted.

Statistical evaluation was performed with Statistical Package for the Social Science (SPSS) version 15.0. The correlation between Ki-67/p53 and benign/malignant subgroups, stromal cellularity and mitotic index was assessed using the Chi-square test. The results were considered to be statistically significant when the P-values were less than 0.05.


   Results Top


Of the 26 PT cases, 17 were classified as benign and nine were classified as malignant. The mean age of the benign group was 31.88 years (min: 22 years, max: 44 years), while it was 48.00 years (min: 31 years, max: 74 years) in the malignant group. The correlation between age and benign/malignant subgroups was statistically significant (P < 0.002).

The median tumor size of the benign cases was 3.32 cm (min: 2 cm, max: 6 cm), while that in the malignant cases was found to be 11.56 cm (min: 3 cm, max: 17 cm). The correlation between tumor size and benign/malignant subgroup was statistically significant (P < 0.001).

All the benign cases showed mild to moderate stromal cellularity. Of the nine malignant cases, eight cases showed severe cellularity and one case showed mild/moderate cellularity. The difference was statistically significant between the benign and the malignant groups in terms of stromal cellularity (P < 0.001).

In all the benign cases, the mitotic rate was less than 10/10 HPF. In the malignant group, the mitotic rate was found to be more than 10/10 HPF in eight cases and less than 10/10 HPF in one case. Although the threshold value of mitosis was >10/10 HPF for the malignant subgroup, one case showing 8/10 HPF mitosis was put into the malignant category as the case met all other criteria for malignancy mentioned previously. [4],[5],[6]

In the malignant PT cases, the mitotic rate was statistically significantly higher than that in the benign subgroup (P < 0.001). In the benign group, the p53 positivity was <20% in 15 cases and 21-42% in two cases. In the malignant group, the p53 positivity was >41% in five cases, 21-40% in three cases and <20% in one case [Figure 1]. p53 expression was statistically significantly higher in the malignant tumors than in the benign ones (P < 0.001). Stromal p53 expression rates in the malignant PTs were shown to be statistically significantly higher than that in the benign ones (P < 0.001). Moreover, the stromal p53 expression was statistically significantly correlated with stromal cellularity and mitotic rate (P < 0.001, P = 0.002).
Figure 1: Nuclear p53 staining in tumor cells (Diaminobenzidine (DAB), X200)

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All the benign tumors showed Ki-67 positivity less than 10% of the stromal cells. But, in the stroma of the malignant tumors, Ki-67 was <10% in four cases and ≥10% in five cases [Figure 2]. Stromal Ki-67 expression in malignant tumors was statistically significantly higher than that in the benign tumors (P < 0.001). The epithelial Ki-67 expression ratios did not show a significant difference between the benign and malignant groups.
Figure 2: Nuclear high Ki-67 positivity (DAB, X200)

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While ER and PR expressions in the epithelial component were observed in both groups, there was no expression in the stromal component. Epithelial ER and PR expression did not show a statistically significant difference between the two groups.

We did not note any staining in the stromal component with HER2. Of the 26 cases, 25 cases (96%) showed epithelial staining with HER2. The case without epithelial HER2 staining was malignant (4%).

The epithelial component showed different staining patterns with HER2. Three types of staining patterns were observed, which were epithelial basal granular staining (benign: 2, malignant: 1) (11.5%), epithelial basolateral membranous staining (benign: 14, malignant: 6) (76.9%) and both of these (benign: 1, malignant: 1) (7.6%) [Figure 3] and [Figure 4]. The clinical features and IHC results are summarized in [Table 1] and [Table 2].
Figure 3: Basal granular HER2 staining in the epithelial component of the tumor (DAB, X400)

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Figure 4: Basolateral membranous HER2 staining in the epithelial component of the tumor (DAB, X400)

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Table 1: Pati ents ages, tumor subtypes, sizes, stromal cellularity rates, stromal p53 and Ki-67 expressions

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Table 2: Epithelial ER and PR expression rates and c-erbB-2 expression patterns

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   Discussion Top


PT is a rare fibroepithelial neoplasm of the breast. The stroma is the neoplastic component of the tumor that determines the clinical behavior. [2] Recently, loss of interaction between the epithelium and the stroma is believed to be an important factor in the development of PT. As a result, increase of mitotic activity in the stroma and stromal overgrowth progress into malignancy. [3] PT most often occurs between the ages of 45 and 49 years. Malignant PT is seen more commonly in older ages than benign tumor. [10] In our study, patients with malignant and benign tumors were, on an average, 17 years apart (P = 0.002). This is not likely to explain the reason for this difference because our serial contains a limited number of cases. Researches on larger series can shed light on this issue.

Patients usually present with a mass in the breast. [10] Even though pain, rapid growth of the lesion, sudden increase in the tumor size, skin changes and nipple retraction are thought to be the features favoring malignancy, they can also be seen in benign lesions. [10]

The average tumor size reported in the literature is 4-5 cm. Although there are exceptions, mostly, malignant PTs are larger in size. [2],[11] In our study, we detected statistically significant differences in sizes between the benign and the malignant tumors (P < 0.001).

Microscopically, the tumor consists of a benign epithelial component and a cellular spindle cell stromal component (fibroblastic and myofibroblastic). [10] Subepithelial stromal areas are found to be more cellular than others. [11] The tumor is characterized by leaf-like appearances extending toward the cystic spaces. Epithelial cells line the ducts, the slit-like spaces and the leaf-like structures.

Today, PTs are classified as benign, borderline and malignant on the basis of histological criteria, including degree of cellularity, stromal cytological atypia, mitotic activity, stromal overgrowth and the tumor margins (infiltrative/pushing). [4],[5],[6]

Benign PTs show mild to moderate stromal cellularity and stromal cellular atypia, low mitotic rate (3-4 mitosis/10 HMF) and pushing tumor margins. But, in malignant PTs, severe stromal cellularity and stromal cellular atypia, increased mitotic rate (>10 mitosis/10 HPF), stromal overgrowth and infiltrative tumor margins with malignant heterologous elements are observed. [9] The cases that do not meet all these malignant criteria are considered to be borderline.

The grading of PTs is not objective as the interpretation criteria are subjective. Therefore, the incidence of borderline PTs are reported to be between 12% and 26% in large series. [12]

In our study, we analyzed 26 PT cases (benign: 17, malignant: 9). We could achieve follow-up data of all benign cases and in five of the malignant cases. Of the 17 benign cases, two cases showed new identical lesions in the contralateral breast. The remaining 15 cases did not develop any new or recurrent lesion. One of the malignant cases developed lung metastasis at the 48 th post-operative Month and local recurrences at the 55 th , 58 th and 62 nd months. The other malignant case showed local recurrences at the 8 th and 12 th months. In both these cases, local recurrences and metastatic focus were treated by surgical excision. The other three malignant cases did not show any metastatic lesions and local recurrences.

p53 is a tumor suppressor gene located on chromosome 17p13.1. It codes a 53 kDa nuclear phosphoprotein. This protein, which is found in low levels in normal cells, plays a role in normal cell cycle regulation and apoptosis. [13] In PT, expression of p53 is shown to be associated with tumor grade, nuclear atypia, stromal overgrowth, mitotic rate, stromal nuclear pleomorphism and infiltrative tumor margins. [9],[13],[14] Most of the studies revealed high p53 expression rates associated with increasing tumor grade. [15],[16],[17] However, the results of the studies on p53 protein are quite variable. Some of the studies reported p53 expression only in malignant cases, while some other publications showed p53 expression even in benign PTs. [15],[18]

In our study, stromal p53 expression rates in malignant PTs were shown to be statistically significantly higher than those in the benign ones (P < 0.001). Moreover, stromal p53 expression was statistically significantly correlated with stromal cellularity and mitotic rate (P < 0.001, P = 0.002).

Ki-67 is a non-histone nuclear protein that determines cellular proliferation immunohistochemically and exists in cells throughout the cell cycle. [12] Studies have shown the relation between Ki-67 expression and the increasing tumor grade in PTs. [15] In the literature, the Ki-67 proliferation index is 5-25% in benign and 15-100% in malignant PTs. [12] In our series, Ki-67 positivity was between 1% and 5% in the benign group and 2% and 60% in the malignant group. Ki-67 expression was significantly higher in the malignant group (P = 0.001). In addition, stromal Ki-67 expression was statistically significantly correlated with the degree of stromal cellularity and mitotic activity (P < 0.001, P < 0.001). Ki-67 expression in the epithelial component did not show a significant difference between the groups.

Detection of hormone receptors in breast carcinoma is a prognostic indicator for the response to endocrine therapy and in determining the course of the disease. [19] However, the importance of hormone receptor status is not clear in PTs.

In the studies evaluating the expression of ER and PR in PTs, it was shown that the receptor expression is limited in the epithelium There was no significant expression in the stroma. [19],[20] In a study evaluating the expression of ERα and ERβ, only ERβ expression was found in the stroma. [21] We did not observe ERα expression in the stromal component of the tumors in our study.

Tse et al. examined 143 PT cases in their study. [19] They reported that the PR expression in epithelial cells is more intense than the ER expression, like breast carcinoma. [19] Also, they found a correlation between ER/PR expression and tumor grade. This study revealed that ER expression was higher in benign tumors than in borderline and malignant tumors; PR expression was higher in benign and borderline tumors than in malignant PTs. [19]

According to these findings, it is reported that the degree of malignancy and hormone receptor status are in inverse proportion in PTs.

In our study, malignant and benign tumors did not differ significantly in terms of hormone receptor expression [Table 2]. In the literature, there are a few studies indicating the hormone receptor status; that is why we could not comment on this issue.

ErbB is the tyrosine kinase receptor family containing ErbB1 (HER1/EGFR), ErbB2 (HER2/neu), ErbB3 (HER3) and ErbB4 (HER4). [3] HER2 plays a role in cell growth, differentiation and survey by means of a complex signaling network. [22] HER2 amplification was observed in a large number of tumor types such as breast, ovarium, lung and gastric carcinomas. [3] HER2 amplification in breast carcinoma is associated with a decrease in the disease-free survival and overall survival. [22] Therefore, Trastuzumab, a monoclonal antibody targeting HER2, is effective in its treatment.

Several studies based on the idea of HER2 amplification, which plays a role in pathogenesis of PTs, were performed. As they noted that there is no expression of HER2 in the stroma of the tumor, the use of targeted therapies with monoclonal antibodies was lower. [7],[16]

HER2 expression was studied in 23 cases by Shpitz et al.[7] In this study, membranous immunostaining of the ductal epithelial cells was considered to be positive. HER2 immunostaining was found to be positive in more than 61% of the cases in the epithelial component, but they reported that there was no significant correlation between this expression and the clinical or pathological features of the tumor. [7]

In our study, HER2 expression was not seen in the stromal component of the tumors. HER2 expression in the epithelial component showing different staining patterns were noted. Basolateral membranous staining pattern was observed in 20 of 26 cases (14 benign, six malignant) (76.9%). Basal granular staining pattern was observed in three cases (two benign, one malignant) (11.5%). Two cases showed a combination of both patterns (one benign, one malignant) (7.6%). In the literature, there is no other study considering different staining patterns of HER2; this was the feature attracting our attention.

As a conclusion, the expression rates of Ki-67 and p53 can be used with other histologic parameters in grading and differentiating benign/malignant cases. Most of the PTs (either benign or malignant) express HER2 immunohistocemically. HER2 expression in the epithelial component of PTs can be seen in different patterns, and this attracting staining feature may be a new research topic for new treatment strategies.

 
   References Top

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3.Karým RZ, Scolyer RA, Tse GM, Tan PH, Putti TC, Lee CS. Pathogenic mechanisms in the initiation and progression of mammary phyllodes tumours. Pathology 2009;41:105-17.   Back to cited text no. 3
    
4.Moffat CJ, Pinder SE, Dixon AR, Elston CW, Blamey RW, Ellis IO. Phyllodes tumours of the breast: A clinicopathological review of thirty two cases. Histopathology 1995;27:205-18.  Back to cited text no. 4
    
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7.Shiptz B, Bomstein Y, Sternberg A, Klein A, Týomkýn V, Kaufman A, et al. Immunoreactivity of p53, Ki-67 and c-erbB-2 in phyllodes tumors of the breast in correlation with clinical and morphologic features. J Surg Oncol 2002;79:86-92.   Back to cited text no. 7
    
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13.Tan PH, Jayabaskar T, Yip G, Tan Y, Hilmy M, Selvarajan S, et al. p53 and c-kit (CD117) protein expression as prognostic indicators in breast phyllodes tumors: A tissue microarray study. Mod Pathol 2005;18:1527-34.   Back to cited text no. 13
    
14.Tse GM, Putti TC, Kung FY, Scolyer RA, Law BK, Lau TS, et al. Increased p53 protein expression in malignant mammary phyllodes tumors. Mod Pathol 2002;15:734-40.   Back to cited text no. 14
    
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21.Sapino A, Bosco M, Cassoni P, Castellano I, Arisio R, Cserni G, et al. Estrogen receptor-beta is expressed in stromal cells of fibroadenoma and phyllodes tumors of the breast. Mod Pathol 2006;19:599-606.  Back to cited text no. 21
    
22.Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology 2001;61 Suppl 2:1-13.  Back to cited text no. 22
    

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Correspondence Address:
Ulku Kucuk
Department of Pathology, Tepecik Research and Training Hospital, Izmir
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.118690

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