Ulex Europeus Agglutinin (UEA-I) Lectin Binding in Breast Carcinoma and its Relationship to Prognostic Factors
From the Departments of Surgery (Drs. Erhan, Akyildiz, Içöz, Yilmaz) and Pathology (Drs. Alkanat, Erhan), Ege University Medical School, Izmir.
Y Erhan, MB. Alkanat, M Akyildiz, G Içöz, R Yilmaz, Y Erhan, Ulex Europeus Agglutinin (UEA-I) Lectin Binding in Breast Carcinoma and its Relationship to Prognostic Factors. 1996; 16(5): 509-511
Abstract
In this study, lectin binding was compared with pathological prognostic factors and clinical follow-up details. Formalin-fixed, paraffin-embedded tissues from 43 cases of breast carcinoma were studied for binding with Ulex Europeus Agglutinin (UEA-I) lectin. Staining results were compared with tumor size, histologic and nuclear grade, lymph node status (number, capsular and pericapsular invasion), blood and lymphatic vessel invasion, ER and PR status, clinical stage and the patients' short-term follow-up details. Analysis of staining with UEA-I showed a significant relationship with blood vessel invasion (P < 0.01) and lymphatic vessel invasion (P < 0.05). Furthermore, PR showed a significant inverse correlation with lectin binding (P < 0.05). Staining with UEA-I related significantly with axillary lymph node metastases (P < 0.05). UEA-I was positive in four (66.6%) out of six cases with distant organ metastasis. This study confirms that, in breast cancer, lectin binding to the cancer cells can be a reliable indicator for axillary metastases, and the need for additional therapeutic interventions.
Ann Saudi Med 1996;16(5):509-511.
Lectins are sugar-binding proteins and glycoproteins of nonimmune origin. Their major applications include agglutination, precipitation in gels, solid phase, blotting, photobleaching, histochemistry, electron microscopy and flow cytometry. Their high affinity and specificity for certain carbohydrate residues has led to their use in histochemistry as discriminative sugar labels. Their role is analogous to the antigen-recognizing role of monoclonal antibodies, and the technical details of histochemical methods with both type of reagents are similar.1-3
Lectins are isolated from a wide range of plant and animal sources and are now readily available commercially in highly purified form. Alterations in cell surface carbohydrate expression during neoplastic development have been frequently observed in men and animals.2,4
Histochemical study shows that malignant tissues exhibit altered binding patterns and that the pattern often varies between different tumors, possibly correlating with tumor prognosis.4
There is disagreement as to the significance of altered lectin binding. In some studies previously performed in our institute, Con-A lectin-binding properties of mammary lesions were examined.5,6 In this study, we considered the cell-staining proportion of formalin-fixed, paraffin-embedded primary breast cancer tissue with Ulex Europeus Agglutinin-I (UEA-I) lectin and their relationships with known pathological prognostic factors and clinical follow-up details. UEA-I, isolated from gorse seeds, has binding specificity to L-fucose.4
Methods
Material and Method
Forty-three consecutive patients with primary operable breast cancer were selected for study. Lesions consisted of 31 invading ductal carcinomas (IDC), two infiltrating lobular carcinomas (ILC), one colloid carcinoma, one atypical medullary carcinoma (ATM), one squamous carcinoma, as well as seven mixed mammary carcinomas (Table 1).
All patients were treated with modified radical mastectomy or simple mastectomy with regional lymph node sampling. The patients were admitted between 1991 and 1992 and had a minimum follow-up time of three years. Radiotherapy and/or chemotherapy regimens were also included in the therapy when needed.
Table 1. Tumor type and UEA-I immunostaining.
|
Tumor type |
UEA-I + |
UEA-I − |
Total |
|
IDC |
22 |
9 |
31 |
|
ILC |
1 |
1 |
2 |
|
Mixed |
6 |
1 |
7 |
|
ATM |
1 |
0 |
1 |
|
Squamous |
0 |
1 |
1 |
|
Colloid |
0 |
1 |
1 |
|
Total |
30 |
13 |
43 |
Frozen sectioned tumor tissue obtained from mastectomy specimens was incubated both with fluorescein isothiocyanate conjugated ß-estradiol and progesterone (Products of SIGMA Co. USA) for two hours. These slides were examined under fluorescent microscope to cite the existence of estrogen and progesterone receptors (ER and PR) of the tumor tissues.
Slides carrying four-μm sections from formalin-fixed, paraffin-embedded tumoral tissues were used for UEA-I immunostaining. UEA-I, antilectin antibody and labeled streptavidin biotin immunostaining kits used were the products of DAKOPATTS A/S. Sections were deparaffinized, rehydrated and pretreated with 1% CaCl2 + 1% trypsin (pH 7.6) solution for 10 minutes. Endogenous peroxydases were blocked with 1% hydrogen peroxide solution. Lectin, antilectin antibody, link antibody, streptavidin-biotin solution and aminoethylcarbazole (AEC) chromogen were applied, with a tris-buffer rinse between all steps. All slides were counterstained with Mayer's hematoxylin and coverslipped with an alcohol-free aqueous mounting medium.
Histologic differentiation was assessed according to the Elston modification of Bloom-Richardson's7 system. Nuclear grade was determined according to Lash and colleagues.8 We searched for blood and lymphatic vessel invasion in peritumoral areas with HE slides.
Results of immunostaining were compared with pathological prognostic factors, ER and PR status, stage and short-term follow-up results. The statistical significance of these correlations were tested by the chi-squared test.
Results
Patient ages ranged between 28 and 73 years. According to TNM classification, five were in Stage I, 21 were in Stage II, 11 were in Stage III and four were in Stage IV.
A clear vision of diffuse intracytoplasmic staining in tumor cells was obtained by using labeled streptavidinbiotin immunoperoxydase technique (Figure 1).
No significant relationships were found between UEA-I immunostaining and histologic and nuclear grade, and stage of the disease. However, the relationship between UEA-I immunostaining and blood (P < 0.01) and lymphatic (P < 0.05) vessel invasion was significant (Tables 2 and 3). There was also an inverse relationship between UEA-I immunostaining and PR status (Table 4).
There was a significant relationship (P < 0.05) between UEA-I immunostaining and axillary lymph node metastases according to histopathological evaluation (Table 5). Four out of six (66.6%) cases with distant organ metastases gave a positive reaction with UEA-I (Table 6).
Table 2. Blood vessel invasion and UEA-I immunostaining (P<0.01).
|
|
Blood vessel invasion |
|
|
UEA-I status |
+ |
− |
|
+ |
12 |
18 |
|
− |
0 |
13 |
Table 3. Lymphatic vessel invasion and UEA-I immunostaining (P<0.05).
|
|
Lymphatic vessel invasion |
|
|
UEA-I status |
+ |
− |
|
+ |
16 |
14 |
|
- |
2 |
11 |
Table 4. Progesterone receptor status and UEA-I immunostaining (P<0.05).
|
|
PR status |
|
|
UEA-I status |
+ |
− |
|
+ |
18 |
12 |
|
− |
12 |
1 |
Table 5. Axillary lymph node metastases and UEA-I immunostaining (P<0.05).
|
|
Axillary lymph node metastases |
|
|
UEA-I status |
+ |
− |
|
+ |
22 |
8 |
|
− |
5 |
8 |
Discussion
Changes in carbohydrate expression on cell surfaces during neoplastic process were shown, both in human and in experimental animals' tissues. This evidence was used in the discrimination of benign and malignant tumors, as well as being a prognostic factor. Lectins are proteins or glycoproteins which can bind carbohydrate residues in fixed tissue sections. This property enables us to analyze and isolate various glycoconjugates.1,4
Table 6. Other prognostic parameters' association with UEA-I status.
|
|
UEA-I + |
UEA-I − |
Total |
|
Clinical stage |
|
|
|
|
I-II |
17 |
9 |
26 |
|
III-IV |
12 |
3 |
15 |
|
Histologic grade |
|
|
|
|
I |
5 |
2 |
7 |
|
II |
7 |
3 |
10 |
|
III |
12 |
3 |
15 |
|
Nuclear grade |
|
|
|
|
I |
9 |
3 |
12 |
|
II |
12 |
4 |
16 |
|
III |
5 |
2 |
7 |
|
Lymph node metastasis |
|
|
|
|
None |
8 |
8 |
16 |
|
1-3 nodes |
8 |
2 |
10 |
|
>3 nodes |
14 |
3 |
17 |
|
Distant metastasis |
|
|
|
|
− |
4 |
2 |
6 |
|
+ |
26 |
11 |
37 |
In recent years, lectin-binding properties of epithelial cells of normal, hyperplastic, and neoplastic mammary tissues were observed and various types of lectins were used as probes. Besides this, different results were obtained in various studies, concerning a relationship between the lectin-binding intensity of tumors and the prognosis. In the present study, we compared UEA-I immunostaining of mammary carcinomas with tumor size, nuclear and histologic grade, lymph node status, blood and lymphatic vessel invasion, ER and PR status and short-term follow-up results of the patients.9-11
In a previous study, Walker9 couldn't find a relationship between UEA-I binding and the degree of differentiation of the tumor. However, in the same study, the relationship between immunostaining and lymph node status was shown in frozen sections. Fenlon et al.4 reported a significant correlation between UEA-I binding and disease-free interval and survival in 100 cases of breast carcinoma. They reported that the high intensity of UEA-I immunostaining means early local recurrence, and shorter survival. In the present study, we observed significant relationships between lectin immunostaining, blood and lymphatic vessel invasion and axillary lymph node metastases. We observed positive UEA-I immunostaining in 66.6% of patients with distant metastatic disease. Also, there was an inverse relationship between UEA-I immunostaining and PR status.
UEA-I labels fucose residues and is a marker of O blood group. It showed different binding patterns to the epithelial cells of malignant and benign mammary lesions. The mechanism of this variation is extremely complex and mirrors the presence or absence of some glycoconjugates. It may also reflect the minor changes in the composition of oligosaccharide chains.4
As a result, UEA-I immunostaining particularly highlights the probability of axillary lymph node metastases and seems to be of use as an indicator for additional therapeutic interventions besides surgery in mammary carcinoma.
Acknowledgment
The authors wish to thank to Necmettin Özdemır, MD, from the Pathology Department of Ege University, who kindly performed statistical analysis of the results.
References
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3. Walker RA. The use of lectins in histopathology. Pathol Res Pract 1989;185:826-35.
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8. Lash RH, Bauer TW, Hermann RE, Esselstyn CB. Partial mastectomy: pathologic findings and prognosis. Human Pathol 1986;17:813-22.
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11. Brooks SA, Leathern AJC. Prediction of lymph node involvement in breast cancer by detection of altered glycosylation in the primary tumour. Lancet 1991;338:71-4.
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