Romanian Journal of Morphology and Embryology 2008, 49(4):517–523

OORRIIGGIINNAALL PPAAPPEERR

E-cadherin expression in invasive breast cancer

C. SUCIU1), ANCA MARIA CÎMPEAN1), ANCA MARIA MUREŞAN2), D. IZVERNARIU1), M. RAICA1)

1)Department of Histology and Cytology 2)Department of Pathology

“Victor Babeş” University of Medicine and Pharmacy, Timisoara

Abstract There were investigated 22 cases with invasive breast carcinoma from the archive material, and the immunohistochemical expression of E-cadherin was correlated with clinical stage, pathological type, grade of the tumor, and lymph nodes status. The expression of E-cadherin was assessed by the pattern of the final product of reaction and number of positive cells. The reaction was appreciated as normal if over 70% of tumor cells showed membrane of membrane and cytoplasmic pattern, and aberrant, if less than 70% of tumor cells were positive. There were found 45.5% cases with normal expression and 54.5% with aberrant expression. Strong positive reaction was found mainly in tumor cells invading the adipose tissue, but the reaction was weak or negative in tumor cells within the vessels. Both normal and aberrant expression of E-cadherin correlates with grade and with clinical stage. In our study was found that the increase of the tumor grade is associated with decrease in the expression of E-cadherin. Tumors over 2 cm in their larger diameter showed a decreased expression of E-cadherin. Seventeen from 22 cases with lymph node metastasis had positive reaction in the primary, but there were not found significant differences between the normal and aberrant expression. Keywords: breast cancer, invasive, E-cadherin, immunohistochemistry, prognosis.

Introduction

Mammary carcinoma is nowadays the most frequent malignant tumor in females. In spite of many progresses realized in the field of diagnosis, prognosis, and therapy, morbidity and specific mortality continue to increase. This is why many investigations are conducted in order to evaluate new diagnostic markers and new therapeutic strategies. In this order, the introduction of the molecular classification of mammary carcinoma is not a surprise, and it is largely based on the immunohistochemical expression of hormone receptors, monoclonal cytokeratins, HER-2/neu protein, and c-erbB2 expression. It was already proved that mammary carcinoma consists of many molecular subtypes, which differ in terms of prognosis and therapy. This classification is not necessary enough, and in last years, researches focused on the involvement of adhesion molecules in mammary carcinogenesis, local invasion and spread.

Breast cancer consists of glandular epithelial cells from the terminal ductal-lobular unit. It is the second cause of death in female patients, and the most important factor that determines this position is cancer cells metastasis [1]. Tumor progression and metastasis have different successive stages, such as detachment of tumour cells from primary tumour, attachment of the basal membrane of the vascular endothelium with its destruction and the releasing of the tumour cells into the bloodstream or into the lymphatic system, and the colonization of the target organ. During all these events,

the junction structures between the malignant cells, particularly adherens junctions and E-cadherin, have an important role [2]. Based on these data, we investigated the expression pattern of E-cadherin in invasive breast carcinomas, and also the potential correlations between E-cadherin expression and tumour grade, tumour dimensions and the presence of tumour metastases.

Epithelial cells attach each other by a series of junctions, such zonula occludens, zonula adherens, desmosomes, and gap junctions. They are responsible for the morphological and functional integrity of the epithelial tissue, but they have a significant importance in malignant transformation of these tissues as well [3]. E-cadherin is a transmembrane calcium-dependant adhesion protein, with molecular weight of 120 kDa, and is part of the type I classical cadherin family, localized in adherens junctions. It has an extracellular domain formed by five repetitive units, which assure the homotypic cell–cell adhesion, a transmembrane domain and an intracellular domain, which is attached to the actin cytoskeleton by catenins (cytoplasmic proteins). Although the basal function of E-cadherin is mechanical adhesion between epithelial cells, it has an important role in establishing the cell polarity, the glandular differentiation, and cellular layering; it is also an important regulator of morphogenesis, thus maintaining the normal epithelial architecture [3, 4]. E-cadherin is considered as a tumour suppressor protein, because it was shown that expression and/or functional losses of this protein are associated with tumorigenesis and tumour progression [5].

C. Suciu et al.

518

Material and Methods

Selection of patients

Twenty-two cases of primary invasive breast cancer were selected for the study. Data of patients, including age, size of the tumor, pathological grade and type, lymph node status and metastasis are shown in Table 1.

Table 1 – Clinical and pathological data of cases No. of patients (n) 22

2 (31–40) 4 (41–50) 6 (51–60) 5 (61–70) 4 (71–80)

Age groups – n (interval)

1 (81–90) Average age – years (interval) 60.6 (34–81) Average size of the tumour – cm (interval) 2.5 (1–8)

G1 2 (10.5) G2 9 (47.4) Histopathological grade – n (%) G3 8 (42.1)

Ductal invasive 19 (86.5) Mucinous 1 (4.5) Histopathological type – n (%) Papillary 2 (9)

Yes 17 (77.28)Lymph node – n (%)

No 5 (22.72)I 1 (4.5) II 9 (41) III 11 (50)

Anatomoclinical stage – n (%)

IV 1 (4.5)

Primary histopathological evaluation was made on 5 µm thick sections stained with usual Hematoxylin–Eosin. Classification was made according WHO’s criteria, and the majority (n = 19) of the cases were invasive ductal carcinoma, two were invasive papillary carcinomas and one was mucinous carcinoma. The grading of the invasive ductal carcinoma was made using the Scarff–Bloom–Richardson system, which is the modified Elston–Ellis system [4].

Immunohistochemistry

Paraffin embedded blocks were cut at 5mµ, the sections were laid on electrically charged slides, dewaxed in benzene at 580C and then rehydrated in alcohol baths with decreasing concentrations. Antigen retrieval was performed for 20 minutes in the microwave oven at 95–990C, in phosphate buffer with pH 6.0 (DakoCytomation, Denmark), and was followed by cooling of slides to room temperature in phosphate buffer. The incubation with the primary anti-E-cadherin antibody clone NCH38, dilution 1:75 was done for 30 minutes. Next, the LSAB plus working system was applied. Visualization of the positive reaction was done with 3,3’-diaminobenzidine, and nuclei were stained with Lillie’s modified Hematoxylin.

Assessment of immunohistochemical results

Immunoreaction was assessed as normal (strong) or aberrant (weak or absent), and the internal positive control was represented by the neighboring normal

glandular structures. Aberrant expression was stated when the immunohistochemical reaction was negative or less than 70% of the tumor cells show positive reaction with membrane pattern.

Normal expression was stated when ≥70% of the tumor cells showed positive reaction with membrane pattern. In addition, we have assessed the positive reaction pattern as membrane, membrane and cytoplasmic, and cytoplasmic without membrane intensification.

Statistical analysis

Paired t-test was used for the correlation between E-cadherin expression and tumour grade, anatomo-clinical stage and lymph nodes status.

Results

The average age of the patients was of 60.6 years, with values between 34 and 81 years; one patient was male. The average tumour size was 2.5 cm, with values between 1 and 8 cm.

The majority of the tumors were ductal invasive carcinomas (n = 19), of which 47.36% (n = 9) had G2 differentiation grade, 42.11% (n = 8) were poorly differentiated (G3), and only two of the cases had G1 histopathological grade. Two of the cases were papillary invasive carcinomas, and one was a mucinous carcinoma. The majority of the cases (77.2%, n = 17) had lymph node metastases.

In normal mammary tissue, the immunoreaction for E-cadherin was positive in all cases. All epithelial cells of the normal terminal duct-lobular unit were stained with moderate intensity, and the final reaction product was restricted to the plasma membrane (Figure 1).

In benign lesions, containing dilated ducts and apocrine metaplasia E-cadherin was intensely expressed in almost all epithelial cells (Figure 2a). The dominant pattern was membraneous with enhanced signal at the basal pole; some cells showed diffuse cytoplasmic pattern without membrane enhancement, and more rarely, with moderate staining only in the periphery of the cytoplasm (Figure 2b).

Atypical ductal hyperplasia was found in only one case, adjacent to a ductal invasive carcinoma. All epithelial cells were stained for E-cadherin, and the pattern and intensity of the immunoreaction was similar to that of the normal mammary tissue (Figure 3a). Ductal in situ carcinoma was identified in a single case, associated to an invasive lobular carcinoma. The reaction for E-cadherin was positive in almost all tumor cells, with membrane pattern, but the intensity of the reaction product was significantly lower than in normal ducts or invasive carcinoma (Figure 3b).

Positive reaction for E-cadherin was found in almost all cases, with homogeneous (Figure 4a) or heterogeneous pattern (Figure 4b). In only one case the reaction was intense positive with diffuse cytoplasmic pattern and without membrane enhancement (Figure 4, c and d). Papillary carcinoma was negative, and mucinous carcinoma showed a slight membrane positive reaction in a limited tumor area (Figure 4e).

E-cadherin expression in invasive breast cancer

519

In ductal invasive carcinoma with heterogeneous pattern, usually malignant cells in the periphery of the tumor were more intensely stained, and the intensity was even stronger for small groups invading the adipose tissue (Figure 5a). On the other hand, tumor cells found within the vascular lumen showed only a low expression of E-cadherin, with discontinuous and granular pattern (Figure 5b).

Ten from 22 cases with invasive breast carcinoma

(45.5%) showed normal immunostaining, whereas 54.5% (n = 12) showed aberrant staining. The staining pattern with both membrane and cytoplasmic staining was found in 17 cases; four cases had cytoplasmic staining only, and one case was negative. Even in cases with normal expression, the pattern was predominantly combined, both membrane and cytoplasmic (n = 8). The positive staining was restricted to the cell membrane in only two cases.

Figure 1 – Normal interlobular mammary duct

(E-cadherin, ×400)

(a) (b)Figure 2 – Cystic change and apocrine metaplasia (a, ×400), membraneous,

diffuse cytoplasmic and heterogeneous pattern (b, ×1000)

(a) (b)Figure 3 – Atypical ductal hyperplasia (a),

DCIS (b), E-cadherin, ×400

C. Suciu et al.

520

(a) (b)

(c) (d)

(e)

Figure 4 – Ductal invasive carcinoma, all tumor cells stained with membrane pattern (a). Ductal invasive carcinoma, few

positive cells (b). Cytoplasmic pattern in ductal invasive carcinoma (c). Citoplasmic pattern with moderate membrane

enhancement (d). Mucinous carcinoma (e). E-cadherin immunostaining, ×400

(a) (b)Figure 5 – Ductal carcinoma invasive in the adipose tissue. Note the intense staining of tumor cells (a). Tumor cell with weak positive reaction in the lumen of a venule (b), E-cadherin immunostaining, ×400

E-cadherin expression in invasive breast cancer

521

The model of E-cadherin expression related to clinical and pathological parameters is shown in Table 2.

Table 2 – Pattern of E-cadherin expression E-cadherin expression n (%)

Normal Aberrant

Number of cases n (%) 10 (45.5) 12 (54.5) I 0 1 II 3 6 III 7 4

Clinical stage

IV 0 1 Yes 8 9

Lymph node metastases No 2 3 1 0 2 2 5 4 Histopathological grade G 3 5 3 I 1 5 II 7 2 III 0 1

Primary tumour T

IV 2 4 I 1 5 II 5 3 Lymph nodes N III 2 1

Discussion

In normal epithelial tissues, when the cell density reaches a certain value, the growing and proliferation rate decrease, and this phenomenon is called contact-inhibition. This is one of the mechanisms through normal epithelial cells control their own evolution, and at the same time, is the element that differentiates them from tumour cells. In the latter, the contact inhibition is lost, which leads to aberrant, uncontrolled proliferation. It is well known that the losses of contact inhibition are due to abnormal E-cadherin and p27 [3]. There are a series of cadherin’s modifications associated with breast cancer, such as cadherin level reduction compared to the normal tissue, abnormal localizations (not only on the membrane), mutations, a.o.

The reduction of cadherin level in breast cancer is a common aspect, more obvious in the lobular type, and from this reason, E-cadherin is considered to be important in tumour spread. Therefore, some data from the literature show that in ductal invasive carcinoma the decrease of cadherin level (aberrant expression) is found in 52% of the cases, whereas in lobular invasive carcinoma is found in 82% of the cases [3]. We have found an aberrant E-cadherin expression in 54.5% of the cases (n = 22) of invasive carcinoma. Referring strictly to ductal invasive carcinoma, we found aberrant expression in 47.4% of the cases (n = 9), similar with results published by Kowalski PJ et al. [6], of approximately 45%. The two cases of papillary invasive and the single case of mucinous carcinoma have shown aberrant E-cadherin expression.

Other authors have found much lower values of aberrant expression of E-cadherin in breast cancer, of approximately 16%, claiming that its expression is normal until advanced tumour stages [7]. This result has been obtained using other evaluation intervals than used

by us. The authors consider aberrant reaction when the percent of the positive cells is lower than 10%. Therefore, they consider normal reaction also when, for example, 20% of the cells are positive. Therefore, there are conditions when the tumour cells are negative for E-cadherin in a large proportion and the tumour is considered with normal reaction. Other authors, who used the same evaluation interval, have obtained similar results with Howard ME et al. [7]. Thus, Qureshy HS et al. [1] have obtained aberrant E-cadherin expression in 12% of the cases of ductal invasive carcinomas. By studying these data, we found that if we would have used the same evaluation intervals, we would have obtained similar results, which makes the method for choosing these intervals questionable.

Generally, tumors with low level of E-cadherin expression are more invasive, less differentiated, and have a higher ability to invade lymph nodes. The expression of E-cadherin was assessed in breast cancer, and it was found that the decrease of its expression level is correlated with lymph node metastases [8–10], tumor grade [10–12], and behavior [13].

Some of these data show that breast tumors with lymph node metastases have aberrant (decreased) expression of E-cadherin as compared with the tumors without lymph node metastases. One of the hypotheses is that the expression and function of E-cadherin reduced temporarily may be correlated with migration of tumor cells in the blood vessels and in peritumoral tissue [14, 15]. The process already fulfilled is followed by the re-expression of E-cadherin in tumour cells, and this fact is enough to explain why tumor metastases have an equal or even higher level of E-cadherin expression than the primary.

Another hypothesis is that of Zschiesche W et al. [16], which consider that only the correct function of cadherin/catenin complex determines the lack of tumour metastases. Thus, Kowalski PJ et al. [6] have shown that aberrant E-cadherin expression found in 45% of the cases is a common phenomenon in the tumors with distant metastases. In our study, from the cases with lymph node metastasis (n = 17), approximately 53% (n = 9) had aberrant E-cadherin expression. There have been though 48% of the cases, in which the expression of cadherin is normal although lymph node metastases are present and therefore these results are somehow in contradiction with the literature data regarding the involvement of E-cadherin in tumour metastases. One of these explanations is that in these cases the level of cadherin may be normal, but not from the functional point of view. It is possible that altering of the cadherin/catenin complex without altering cadherin expression to be an explanation for tumor progression and metastases [16].

It is interesting to notice that in our study, 22.8% (n = 5) of the cases had no lymph node metastases and from these, more than half had aberrant E-cadherin expression. Moreover, in one of these cases, the reaction was negative, although the tumor largely infiltrated the skeletal striated muscle and the tegument, fact that classified the tumour as T4. Moreover, the

C. Suciu et al.

522

grade assessment has shown a tumour with moderate cell pleiomorphism and intense mitotic activity, with predominant solid areas, which graded the tumour as G3.

Referring to the size of the tumor, data from the literature are controversial. The assessment made by Heimann R et al. [17], had a score with a cut-off value of 25%, thus all the tumors with over 25% positive cells are considered with intense reaction, and the ones with low than 25% positive cells with weak reaction. They have noticed that as the size of the tumour increases, the same does the percent of the tumors with weak expression of E-cadherin. Thus, for tumors less than 2 cm in size, the percent of the cases with weak expression of E-cadherin is 14%, this percent increase to 25% for tumors more than 2 cm in size.

Data obtained by these authors [17], and by others [1] suggest that the percent of the tumors with aberrant expression of E-cadherin increases with the size of the tumors, and that weak expression is more frequent in large tumors. Other authors have shown that the percent of the tumors with aberrant expression of E-cadherin decreases with the increase of the tumour size [8].

From the tumors assessed by us, those with less than 2 cm in size (T1 stage) have aberrant expression in 83.3% of the cases, those with size over 2 cm (T2 + T3 + T4) having this percent of approximately 56.25%. An interesting aspect we want to emphasize on is the fact that in T2 stage tumors (sizes between 2 and 5 cm) the percent of the cases with aberrant reaction is 22.2%.

Some data from the literature show a strong correlation with tumor grade, thus the expression of E-cadherin decreases with the increase of the tumour grade [18–21].

Heimann R et al. [17] have shown that the number of the tumors with aberrant expression of E-cadherin increases from 11% for G1 to 49% in G3 tumors. Other authors have shown that there is no correlation with the tumour grade [7].

We have noticed that the percent of the tumors with aberrant expression of E-cadherin decreases with the increase of the tumour grade. Thus, 100% of G1 cases had aberrant expression, which decreases to 44.5% for G2 and to 37.5% for G3 tumors, respectively. On the other hand, the number of cases included in this study is low, and therefore, we cannot draw definitive conclusions regarding the relationship between E-cadherin expression and grade.

Conclusions

Invasive breast carcinomas are characterized by the decrease of E-cadherin expression, being found in almost a half of the cases of ductal invasive carcinomas. The percent of the cases with aberrant E-cadherin expression is higher in well-differentiated tumors (G1), percent that decreases with the increase of the differentiation grade, being the lowest in undifferentiated tumors (G3). Tumors larger than 2 cm are characterized by the decrease of aberrant E-cadherin expression.

References [1] QURESHI H. S., LINDEN M. D., DIVINE G., RAJU U. B.,

E-cadherin status in breast cancer correlates with histologic type but does not correlate with established prognostic parameters, Am J Clin Pathol, 2006, 125(3):377–385.

[2] HIROHASHI S., Inactivation of the E-cadherin-mediated cell adhesion system in human cancers, Am J Pathol, 1998, 153(2):333–339.

[3] JIANG W. G., MANSEL R. E., E-cadherin complex and its abnormalities in human breast cancer, Surg Oncol, 2000, 9(4):151–171.

[4] NOLLET F., KOOLS P., VAN ROY F., Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members, J Mol Biol, 2000, 299(3):551–572.

[5] TAKEICHI M., Cadherins in cancer: implications for invasion and metastasis, Curr Opin Cell Biol, 1993, 5(5):806–811.

[6] KOWALSKI P. J., RUBIN M. A., KLEER C. G., E-cadherin expression in primary carcinomas of the breast and its distant metastases, Breast Cancer Res, 2003, 5(6):R217–R222.

[7] HOWARD M. E., LAU K. S., LYLES H. R., BIRDSONG G. G., UMBREIT J. N., KOCHHAR R., Expression of e-cadherin in high-risk breast cancer, J Cancer Res Clin Oncol, 2005, 131(1):14–18.

[8] OKA H., SHIOZAKI H., KOBAYASHI K., INOUE M., TAHARA H., KOBAYASHI T., TAKATSUKA Y., MATSUYOSHI N., HIRANO S., TAKEICHI M., MORI T., Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis, Cancer Res, 1993, 53(7):1696–1701.

[9] HUNT N. C., DOUGLAS-JONES A. G., JASANI B., MORGAN J. M., PIGNATELLI M., Loss of E-cadherin expression associated with lymph node metastases in small breast carcinomas, Virchows Arch, 1997, 430(4):285–289.

[10] SIITONEN S. M., KONONEN J. T., HELIN H. J., RANTALA I. S., HOLLI K. A., ISOLA J. J., Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer, Am J Clin Pathol, 1996, 105(4):394–402.

[11] GURIEC N., MARCELLIN L., GAIRARD B., CALDÉROLI H., WILK A., RENAUD R., BERGERAT J. P., OBERLING F., E-cadherin mRNA expression in breast carcinomas correlates with overall and disease-free survival, Invasion Metastasis, 1996, 16(1):19–26.

[12] CHARPIN C., GARCIA S., BONNIER P., MARTINI F., ANDRAC L., CHOUX R., LAVAUT M. N., ALLASIA C., Reduced E-cadherin immunohistochemical expression in node-negative breast carcinomas correlates with 10-year survival, Am J Clin Pathol, 1998, 109(4):431–438.

[13] LIPPONEN P., SAARELAINEN E., JI H., AALTOMAA S., SYRJÄNEN K., Expression of E-cadherin (E–CD) as related to other prognostic factors and survival in breast cancer, J Pathol, 1994, 174(2):101–109.

[14] COLPAERT C. G., VERMEULEN P. B., BENOY I., SOUBRY A., VAN ROY F., VAN BEEST P., GOOVAERTS G., DIRIX L. Y., VAN DAM P., FOX S. B., HARRIS A. L., VAN MAARCK E. A., Inflammatory breast cancer shows angiogenesis with high endothelial proliferation rate and strong E-cadherin expression, Br J Cancer, 2003, 88(5):718–725.

[15] KLEER C. G., VAN GOLEN K. L., BRAUN T., MERAJVER S. D., Persistent E-cadherin expression in inflammatory breast cancer, Mod Pathol, 2001, 14(5):458–463.

[16] ZSCHIESCHE W., SCHÖNBORN I., BEHRENS J., HERRENKNECHT K., HARTVEIT F., LILLENG P., BIRCHMEIER W., Expression of E-cadherin and catenins in invasive mammary carcinomas, Anticancer Res, 1997, 17(1B):561–567.

[17] HEIMANN R., LAN F., MCBRIDE R., HELLMAN S., Separating favorable from unfavorable prognostic markers in breast cancer: the role of E-cadherin, Cancer Res, 2000, 60(2):298–304.

[18] PARKER C., RAMPAUL R. S., PINDER S. E., BELL J. A., WENCYK P. M., BLAMEY R. W., NICHOLSON R. I., ROBERTSON J. F., E-cadherin as a prognostic indicator in primary breast cancer, Br J Cancer, 2001, 85(12):1958–1963.

E-cadherin expression in invasive breast cancer

523[19] CHARPIN C., GARCIA S., BOUVIER C., DEVICTOR B.,

ANDRAC L., CHOUX R., LAVAUT M., E-cadherin quantitative immunocytochemical assays in breast carcinomas, J Pathol, 1997, 181(3):294–300.

[20] ELSTON C. W., ELLIS I. O., Pathologic prognostic factors in breast cancer. I. The value of histologic grade in breast cancer: experience from a large study with long-term follow-up, Histopathology, 1991, 19(5):403–410.

[21] GREENLEE R. T., HILL-HARMON M. B., MURRAY T., THUN M., Cancer statistics, 2001, CA Cancer J Clin, 2001, 51(1):15–36.

Corresponding author Cristian Suciu, Assistant, MD, PhD candidate, Department of Histology and Cytology, “Victor Babeş” University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300 041 Timişoara, Romania; Phone +40256–204 476, Fax +40256–490 626, E-mail: cristian_suciu@yahoo.com Received: January 25th, 2008

Accepted: October 10th, 2008