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Expression of vascular endothelial growth factor, CD10, and factor 8 in phyllodes tumor: A 5-year study of 38 cases


1 Department of Pathology, Maulana Azad Medical College, New Delhi, India
2 Department of Surgery, Maulana Azad Medical College, New Delhi, India

Date of Submission07-May-2018
Date of Decision30-Jun-2018
Date of Acceptance04-Dec-2018
Date of Web Publication28-Jan-2020

Correspondence Address:
Meeta Singh,
Department of Pathology, Maulana Azad Medical College, New Delhi
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_300_18

 > Abstract 


Introduction: Phyllodes tumor is a group of biphasic fibroepithelial tumors of the breast, graded as benign, borderline, and malignant. The grading of breast phyllodes remains a challenging task for the pathologists as the prognosis, and further treatment depends on it. In this study, an effort has been made to grade phyllodes tumor on the basis of immunohistochemistry.
Aims and Objectives: Vascular endothelial growth factor, CD10, and factor 8 have been used as immunohistochemical markers for grading.
Results and Conclusion: We have found a significant correlation between the expression of these markers and grading of phyllodes tumor. Positive correlation was also found amongst expression of all three markers. To conclude, increased expression of these markers with increasing grade can aid in diagnosis and guide treatment.

Keywords: Fibroepithelial, immunohistochemistry, phyllodes



How to cite this URL:
Chaudhary D, Singh M, Kushwaha P, Agarwal R, Singh V, Mandal S, Khurana N, Mishra A. Expression of vascular endothelial growth factor, CD10, and factor 8 in phyllodes tumor: A 5-year study of 38 cases. J Can Res Ther [Epub ahead of print] [cited 2020 Aug 15]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=277103




 > Introduction Top


Phyllodes tumor is a rare fibroepithelial neoplasm with risk of recurrence and metastasis. It accounts for 0.3%–1.5% of female breast tumors, although there is an increased incidence in the Asian population.[1],[2] Phyllodes tumors are classified as benign, borderline, or malignant based on various histological criteria, of which mitosis is the most important parameter.[3] However, it is challenging at times to classify phyllodes on the basis of morphology alone. The expression of various immunohistochemical markers such as CD10, vascular endothelial growth factor (VEGF), CD 34, beta-catenin, factor 8, CD117, actin, and Bcl2 has been studied for subcategorization of phyllodes in the past; however, any single marker has not been found to be conclusively useful. Therefore, the aim of the study is to evaluate the utility of VEGF, CD10, and factor 8 in the grading of phyllodes tumor.


 > Materials and Methods Top


The study comprised of 38 patients diagnosed with phyllodes tumor at the Department of Pathology, Maulana Azad Medical College, from January 2013 to December 2017. Relevant clinical data in terms of age, tumor size, and laterality were noted. Phyllodes tumors were classified into benign, borderline, and malignant as per the World Health Organization (WHO) 2012 classification using histological parameters into consideration: stromal cellularity, atypia, overgrowth, mitosis, and margins.[4]

Immunohistochemistry (IHC) was performed using antibodies against CD10, VEGF, and factor 8 with Avidin-Biotin method and diaminobenzidine as chromogen. The scoring of the individual marker was done as follows.

Scoring for vascular endothelial growth factor

Granular cytoplasmic staining was evaluated in stromal cells in ×400, with the vascular endothelium serving as a positive internal control.[5]

  • Negative: No stromal staining
  • Low (+1): Staining was weaker than the vascular endothelium in less than half of the cells
  • Intermediate(+2): Staining was the same as or higher than the vascular endothelium in less than half of the cells
  • High (+3): Staining was the same as or higher than the vascular endothelium in more than half of the cells.


Scoring for cell development marker 10

Stromal cell staining was assessed, using cytoplasmic staining of the breast, myoepithelium serving as an internal control in ×400.[6]

Grading:

  • Negative: No stromal staining
  • Grade 1: <10% stromal cells were positive
  • Grade 2:10%–30% stromal cells were positive
  • Grade 3: >30% stromal cells were positive.


Microvessel density (factor 8)

The areas of highest vascularization were chosen at low power (×10) and microvessel counting followed at high power (×400).[7]

After counting of 10 high-power field, an average count was obtained. Only vessels with a clearly defined lumen were counted.

Statistics

The Chi-square test was used to determine differences in CD10, VEGF, and factor 8 expression between benign, borderline and malignant phyllodes tumors. One-way analysis of variance was used to determine the level of significance of markers (VEGF, CD10, and factor 8) between the groups (benign, borderline, and malignant). Chi-square test was used to calculate the P value. Statistical significance was established at P < 0.05.

The test used for calculating the correlation coefficient was Pearson's coefficient correlation test. Data were collected, coded, and analyzed by SPSS for Windows Version 9.0 (Chicago, SPSS Inc) under Microsoft Windows XP.


 > Observations and Results Top


This study included 38 cases of phyllodes tumor classified as 21 cases of benign phyllodes (55.2%), 4 cases of borderline phyllodes tumors (10.5%), and 13 cases of malignant phyllodes tumors (34.2%) [Figure 1].
Figure 1:(a) Mammography – malignant phyllodes on the left side. (b) Gross – fungating growth of malignant phyllodes

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In 21 cases of benign phyllodes tumors, the patients' age ranged from 14 to 55 years, and tumor sizes ranged from 2.5 cm to 15 cm in maximal diameters with mean of 3.75 cm. H & E of benign phyllodes with immunohistochemistry with VEGF, CD 10 and factor 8 is shown in [Figure 2]. In four cases of borderline phyllodes tumors, the patients' ages ranged from 17 to 50 years, and tumor sizes ranged from 10 cm to 20 cm with mean of 15 cm. H & E sections of borderline phyllodes with grade 2 expression of CD 10 and MVD = 60 of factor 8 is shown in [Figure 3]. In the remaining 13 cases of malignant phyllodes tumors, the patients' ages ranged from 21 to 66 years, and tumor sizes ranged from 7 cm to 29 cm with mean of 18 cm. H & E section of malignant phyllode and expression of VEGF, CD 10 and factor 8 is depicted in [Figure 4]. These results showed highly significant correlation between patients' ages (P = 0.04) and tumor sizes (P = 0.001) with the tumor grade (Chi-square test).
Figure 2: (a) Trucut biopsy of benign phyllodes. (b) Grade 2 staining of vascular endothelial growth factor in benign phyllodes. (c) Grade 2 staining of cell development marker 10 in benign phyllodes. (d) Factor 8 (microvessel density = 45) in benign phyllodes

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Figure 3: (a) H and E of borderline phyllodes. (b) Grade 2 staining of cell development marker 10 in borderline phyllodes. (c) Factor 8 (microvessel density = 60) in borderline phyllodes

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Figure 4: (a) H and E of malignant phyllodes. (b) Grade 2 staining of vascular endothelial growth factor in malignant phyllodes. (c) Grade 3 staining of cell development marker 10 in malignant ohyllodes. (d) Factor 8 (microvessel density = 96) in malignant phyllodes

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The staining intensity and the expression of VEGF rose with worsening of tumor grade. Highly significant correlation was found between VEGF expression and tumor grade (P =0.003) (Chi-square test). The results are summarized in [Table 1].
Table 1: Vascular endothelial growth factor expression

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There was a significant correlation between CD10 expression and tumor grade with a P = 0.010. Results are summarized in [Table 2]. The expression of factor 8 significantly correlated with tumor grade with a P = 0.017 (Chi-square test). Results are summarized in [Table 3].
Table 2: CD10 expressio

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Table 3: Factor 8 microvessel density

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Positive correlation was found between the expression of all the three markers with a correlation coefficient of +0.37 between CD10 and VEGF and correlation coefficient of +0.26 between VEGF and factor 8 (Pearson's coefficient correlation test). Results are summarized in [Table 4].
Table 4: Correlation between vascular endothelial growth factor, CD10, and factor 8

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 > Discussion Top


Phyllodes tumor is a rare fibroepithelial lesion, the peak of which occur in women aged 45–49 years. They are biphasic breast tumors, having a tendency to recur and metastasize. Several criteria are used to classify phyllodes; the most recent one is the 2012 WHO classification. However, there always remains interobserver variability among pathologists as the grading lacks standard interpretation.[8] Studies have been done correlating the significance of IHC markers with the grade of phyllodes.[8] In the present study, the IHC expression of VEGF, CD10, and factor 8 was correlated with the grade of phyllodes.

VEGF (also known as vascular permeability factor) has a role in angiogenesis and endothelial survival. VEGF A subtype is the most extensively studied in context to breast cancer.[9] As phyllodes progress, increase stromal overgrowth results in areas of relative hypoxia which triggers hypoxia-inducible factor and VEGF expression to promote angiogenesis. Several studies demonstrated increase in VEGF expression with increasing degree of malignancy.[5] Tse et al. also correlated VEGF staining with increased mitotic rate and infiltrative margins in malignant phyllodes.[5] In our study, VEGF staining correlated with the grade of phyllodes with P = 0.003 with an expression of VEGF in 62% of benign, 50% borderline, and 100% of malignant phyllodes.

Another biomarker, CD10, is a metalloproteinase which degrades many bioactive amines and thus provide tumor capacity of invasion and metastasis.[10] Few studies are also available on the expression of CD10 in myoepithelial cells in the breast which acts as an aid to diagnose few difficult lesions.[11] This study shows that CD10 expression strongly correlates with phyllodes tumor grade, thus differentiating between benign and malignant phyllodes. CD10 expression was seen in 29% benign, 75% borderline, and 77% malignant phyllodes. A study conducted by Tse et al. concluded positive staining of CD10 in 5.9% benign phyllodes, 31.4% borderline, and 50% malignant cases.[10] In another small study by Mechtersheimer et al., three benign phyllodes tumors studied showed weak CD10 staining, while one malignant case included showed intense staining.[12]

The assessment of angiogenesis can be a useful and predictive factor in solid human cancers. Factor 8 recognizes small-caliber blood vessels required for angiogenesis in carcinoma. Many studies have shown positive correlation between microvessel density (MVD) with the prognosis in bladder carcinomas.[13] Few studies have been done on breast cancers.[7] Our study showed increased MVD with increasing grade of phyllodes with significant P value. Dacic et al. found no significant difference in MVD in low- and high-grade phyllodes tumors.[7] In a study by El Gehani et al., a significant correlation was found between MVD (calculated by CD 31 and factor 8) and tumor grade in bladder carcinomas, thus stating that angiogenesis increases in parallel with tumor stage and grade.[14] These variations can be attributed to different IHC markers used for MVD, interobserver variations in counting vessels, and various statistical analysis used. The results of various studies on the expression of these immunohistochemical markers on phyllodes tumors are summarized in [Table 5].
Table 5: Comparison of P value in different studies on vascular endothelial growth factor, CD10 and factor 8

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To conclude, in our study the expression of VEGF, CD 10 and factor 8 correlated with grade of phyllodes, though study was limited by number of cases with few borderline phyllodes. More studies on larger number of cases are required to support this fact. This observation of higher expression of these biomarkers in malignant and borderline cases can have both diagnostic and prognostic significance. Furthermore, it can further provide insights into pathogenesis and aid in planning future treatment modalities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.







[15]



 
 > References Top

1.
Rowell MD, Perry RR, Hsiu JG, Barranco SC. Phyllodes tumors. Am J Surg 1993;165:376-9.  Back to cited text no. 1
    
2.
Tse GM, Niu Y, Shi HJ. Phyllodes tumor of the breast: An update. Breast Cancer 2010;17:29-34.  Back to cited text no. 2
    
3.
Jacobs TW, Chen YY, Guinee DG Jr., Holden JA, Cha I, Bauermeister DE, et al. Fibroepithelial lesions with cellular stroma on breast core needle biopsy: Are there predictors of outcome on surgical excision? Am J Clin Pathol 2005;124:342-54.  Back to cited text no. 3
    
4.
Lakhani SR, Ellis IO, Schnit SJ, Tan PH, Van de Vijver MJ, editors. World Health Organization Classification of Tumors of the Breast. Vol. 4. Lyon, France: IARC; 2012.  Back to cited text no. 4
    
5.
Tse GM, Lui PC, Lee CS, Kung FY, Scolyer RA, Law BK, et al. Stromal expression of vascular endothelial growth factor correlates with tumor grade and microvessel density in mammary phyllodes tumors: A multicenter study of 185 cases. Hum Pathol 2004;35:1053-7.  Back to cited text no. 5
    
6.
Ibrahim WS. Comparison of stromal CD10 expression in benign, borderline, and malignant phyllodes tumors among Egyptian female patients. Indian J Pathol Microbiol 2011;54:741-4.  Back to cited text no. 6
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7.
Dacic S, Kounelis S, Kouri E, Jones MW. Immunohistochemical profile of cystosarcoma phyllodes of the breast: A study of 23 cases. Breast J 2002;8:376-81.  Back to cited text no. 7
    
8.
Ortega E, Aranda FI, Chuliá MT, Niveiro M, Payá A, Seguí J. Phyllodes tumor of the breast with actin inclusions in stromal cells: Diagnosis by fine-needle aspiration cytology. Diagn Cytopathol 2001;25:115-7.  Back to cited text no. 8
    
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Tse GM, Lui PC, Scolyer RA, Putti TC, Kung FY, Law BK, et al. Tumour angiogenesis and p53 protein expression in mammary phyllodes tumors. Mod Pathol 2003;16:1007-13.  Back to cited text no. 9
    
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Tse GM, Tsang AK, Putti TC, Scolyer RA, Lui PC, Law BK, et al. Stromal CD10 expression in mammary fibroadenomas and phyllodes tumours. J Clin Pathol 2005;58:185-9.  Back to cited text no. 10
    
11.
Moritani S, Kushima R, Sugihara H, Bamba M, Kobayashi TK, Hattori T, et al. Availability of CD10 immunohistochemistry as a marker of breast myoepithelial cells on paraffin sections. Mod Pathol 2002;15:397-405.  Back to cited text no. 11
    
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Mechtersheimer G, Krüger KH, Born IA, Möller P. Antigenic profile of mammary fibroadenoma and cystosarcoma phyllodes. A study using antibodies to estrogen- and progesterone receptors and to a panel of cell surface molecules. Pathol Res Pract 1990;186:427-38.  Back to cited text no. 12
    
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Beecken WD, Engl T, Jonas D, Blaheta RA. Expression of angiogenesis inhibitors in human bladder cancer may explain rapid metastatic progression after radical cystectomy. Int J Mol Med 2009;23:261-6.  Back to cited text no. 13
    
14.
El Gehani K, Al-Kikhia L, Mansuri N, Syrjänen K, Al-Fituri O, Elzagheid A, et al. Angiogenesis in urinary bladder carcinoma as defined by microvessel density (MVD) after immunohistochemical staining for factor VIII and CD31. Libyan J Med 2011;6:10.  Back to cited text no. 14
    
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Ho SK, Thike AA, Cheok PY, Tse GM, Tan PH. Phyllodes tumours of the breast: The role of CD34, vascular endothelial growth factor and β-catenin in histological grading and clinical outcome. Histopathology 2013;63:393-406.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

 
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