orIginal ARTICLES
BCL-2 EXPRESSION IN BREAST CARCINOMAS IN POSTMENOPAUSAL WOMEN Alis Dema1, Simona Dragan2, Elena Lazar1, Danina Munteanu3, Sorina Taban1, Codruta Lazureanu1, Traila Nicola4 REZUMAT Introducere: Proteina anti-apoptotica bcl-2 este investigată extensiv în relaţie cu prognosticul şi cu predicţia raspunsului la terapie al carcinoamelor mamare. Lucrarea de faţă analizează, pe un lot de carcinoame mamare diagnosticate la femei în postmenopauză, expresia proteinei bcl-2 în corelaţie cu alţi factori clinici şi imunohistochimici (IHC) de prognostic. Material şi metodă: Au fost analizate 40 de carcinoame mamare diagnosticate la femei în postmenopauză. Studiul IHC s-a realizat cu anticorpii monoclonali anti-bcl2, anti-ER, anti-PR, anti-Ki-67, tehnica LSAB2, vizualizare cu DAB. Rezultate: Expresia protooncogenei bcl-2 a fost identificată în 55% din carcinoamele mamare invazive. ER si PR au fost pozitivi în 55% şi respectiv 57,5% din cazuri. 30% din carcinoame au prezentat un indice Ki-67≤10%, în timp ce restul de 70% au demonstrat un indice Ki-67 > 10%. Bcl-2 a fost exprimat în 100% din tumorile T1 şi în 64% din tumorile T1+T2. 63,2% din tumorile GI+GII au prezentat reacţie pozitivă pentru bcl-2. 77% şi 83% din tumorile ER şi respectiv PR–pozitive au fost bcl-2-pozitive. 92% din tumorile cu activitate proliferativă joasă au prezentat reacţie pozitivă pentru bcl-2. Tumorile N0 au fost în proporţie de 67% bcl-2 pozitive, în timp ce în grupul tumorilor N+, 46% au fost bcl-2 pozitive. Concluzii: La femeile postmenopauzale cu cancer mamar, proteina bcl-2 a fost semnificativ exprimată în tumorile de dimensiuni reduse, bine/mediu diferenţiate, cu receptori hormonali prezenţi şi cu activitate proliferativă redusă. Sunt necesare studii mai largi care să stabilească utilitatea includerii markerului în panelul de anticorpi ce stabileşte profilul molecular al carcinoamelor mamare. Cuvinte cheie: sân, carcinom, menopauză, bcl-2, imunohistochimie
ABSTRACT Introduction: The bcl-2 antiapoptosis protein is extensively investigated in correlation with prognosis and prediction of therapy response of breast carcinomas. The current study analyzes the expression of bcl-2 protein linked with other clinical and immunohistochemistry (IHC) prognostic factors in a group of postmenopausal women diagnosed with breast carcinomas. Material and methods: 40 breast carcinomas diagnosed on postmenopausal women were analyzed. IHC study was accomplished by monoclonal antibodies anti-bcl2, anti–ER, anti-PR, anti-Ki-67, LSAB2 technic, DAB visualization. Results: The bcl-2 protooncogene expression was certified in 55% of invasive breast carcinomas. ER and PR were positive in 55% respectively 57.5% of cases. Thirty percent of carcinomas presented a Ki-67 index ≤10%, while the rest had a Ki-67 index >10%. Bcl-2 was expressed in 100% T1 tumors and in 64% T1+T2 tumors. In 63.2% of GI+GII tumors, the reaction for bcl-2 was positive. In 77% and 83% of ER respectively PR positive tumors bcl-2 was positive. Furthermore, 92% of low proliferative activity tumors presented positive reaction for bcl-2. In 67% of N0 tumors and in 46% of N+ tumors, bcl-2 reaction was positive. Conclusions: bcl-2 protein in breast carcinomas of postmenopausal women was significantly expressed by small sized, well/ intermediate differentiated, hormone-dependent and low proliferative activity tumors. Larger studies are necessary for demonstration of the utility of this marker in the antibodies panel used for characterization of the breast carcinomas molecular profile. Key words: breast, carcinoma, menopause, bcl-2, immunohistochemistry
INTRODUCTION
Department of Pathology, 2 Department of Medical Polyclinics, Department of Physiopathology, 4 Department of Oncologic Surgery, Victor Babes University of Medicine and Pharmacy, Timisoara 1 3
Assoc. Prof. Alis Dema, Department of Pathology, Victor Babes University of Medicine and Pharmacy, 2 Eftimie Murgu Sq., 300041 Timisoara, Tel. +40748331268 E- mail:
[email protected] Received for publication: Jul. 11, 2008. Revised: Oct. 16, 2008.
In the past two decades, numerous oncology studies concerned investigations of prognostic valuable factors in malignant tumors with different sites. Classical prognostic factors of breast cancer, represented by tumor size, axillary lymph nodes status, histologic subtype and grade, do not offer enough predictive information for disease evolution and treatment response of all tumors.1,2 Therefore, researches in the field have focused on a larger range of molecular markers with prognostic and prediction _____________________________ Alis Dema et al
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value, that are identified by immunohistochemical (IHC) or biochemical methods, but only few were validated through clinical trials. The breast carcinoma therapeutic planning routinely uses estrogen receptors (ER), progesterone receptors (PR) and HER2/neu oncoprotein determination, but recent data point out the prognostic and predictive importance of some markers involved in apoptosis regulation: p53 protein, bcl-2 family proteins.3-7 The bcl-2 gene, an anti-apoptosis gene situated on 18th chromosome, encodes a protein involved in programmed cellular death (apoptosis) in various physiologic and pathologic conditions.8 The bcl-2 proto-oncogene expression was described in different epithelial tumors (colo-rectal, prostate, pulmonary, etc.), including breast carcinomas.9-13 The value of bcl-2 identification in breast carcinomas is currently under scrutiny, until now, the majority of researches have signalized a positive relationship between bcl-2 expression and favorable prognostic factors.14-16 There are certain molecular and therapeutic peculiarities of breast tumors depending on the menopausal status. On one hand, the majority of tumors diagnosed in menopausal women present hormonal receptors, and on the other hand, there are significant differences of adjuvant therapy connected with this menopausal status: tamoxifen vs. aromatase inhibitors.17,18 The present study analyzes the IHC bcl‑2 protein expression and the relationship with other prognostic factors, on a group of postmenopausal women diagnosed with breast cancer.
MATERIAL AND METHOD From the Oncology, Surgery and Radiotherapy Services of City Emergency Hospital Timisoara we have selected a group of 40 postmenopausal patients with breast cancer stages I to IV stages. Thirty-six patients underwent histopathological evaluation of axillary lymph node status. Primary tumor tissue samples after surgical resection or fine needle biopsy were fixed in buffered formalin 24-48 hours, then embedded in paraffin. The standard histopathological examination on HE stain enables the following specifications: the tumor type, the differentiation grade, lymphovascular invasion. The tumors were diagnosed corresponding to WHO classification of breast tumors, the tumor grade being appreciated base upon the Scarff - Bloom Richardson classification (SBR) modified by Elston _____________________________ TMJ 2008, Vol. 58, No. 3 - 4
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and Ellis (grade I – well differentiated tumor; grade II –intermediate differentiated tumor; grade III – poorly differentiated tumor).19 In situ ductal carcinoma lesions were classified in: “comedo” and “noncomedo”. The tumor pathological stage was specified accordingly to the 6th Edition criteria of AJCC Cancer staging manual.20 The IHC study done on paraffin-embedded tissue, concerned the ER and PR status, the Ki-67 index of cellular proliferation and the bcl-2 anti-apoptosis protein expression. Serial sections, 4 µm thick, were made from paraffin-embedded tumoral representative areas, and then stretched against silanized sides to prevent detachment during antigen unmasking procedures. From the cases with more paraffin blocks, we selected the tissue section without extended necrotic areas, but with normal mammary gland or benign lesions associated to the carcinoma (for IHC positive control). Tissue sections were deparaffined in xylene, rehydrated in gradual concentrations of ethanol in distillated water, with blocking of endogenous peroxidase with hydrogen peroxide 3%, for 5 min. For unmasking the antigens, we used the heat procedure by boiling 20 min in Target retrieval solution (Dako) pH6 and pH9 in microwaves. Subsequently, we applied immunostain with prediluted DAKO antibodies: anti-Bcl-2 (clone 124), anti-ER (clone 1D5), anti-PR (PgR636), anti-Ki-67 (clone MIB1). The working system used was LSAB2 (labelled streptavidin biotin) – HRP and diaminobenzidine (DAB) was the chromogen used for reactions visualization. The final reaction product was brown with nuclear site for ER, PR, Ki-67 and cytoplasm and/or membrane for bcl-2. Finally, the sections were counterstained with Mayer hematoxylin, dehydrated, clarified and mounted with Eukitt. Immunostained sections were examined with a Zeiss microscope and images were captured using a Canon digital camera. IHC reactions results quantification Only the nuclear stain of the invasive tumor component was considered for ER, PR and Ki-67 regardless the intensity of stain. Randomly chosen 10 high power fields (x400) were evaluated and the final percentage of positive nuclei was the mean value of calculated percentage for each field. - For ER/PR the threshold was 10% (≥10%) positive nuclei in order to consider ER/PR positive cases.
- For Ki-67 the percentage refers to Ki-67 positive nuclei of the invasive tumor component, using the following semiquantitative score of tumor proliferative activity: 0 – Ki-67 absent; low proliferative activity – positive reaction in ≤ 10% of tumor cells; high proliferative activity – positive reaction in > 10 % Ki67 positive nuclei. - bcl-2 expression represented by cytoplasm
Table 2. Clinical and pathological characteristics of the
Table 2. Clinical and pathological characteristics of the studied cases. Clinical /
Number
%
34
85
4
10
Mammary biopsy
2
5
pathological feature Surgical
Sectorectomy/mastectomy
approach
with DALN*
and/or membrane immunostain was evaluated as follows: negative reaction – absence of tumor cells stain or weak heterogenous positive stain in less than 25% of tumor cells, with an intensity inferior to the lymphocytes’ stain; positive reaction – more than 25% of tumor cells stained, with the same or higher intensity comparative to the lymphocytes’ stain.
Tumor
Ductal
34
85
histologic type
Others
6
15
Tumor grade
GI
0
0
GII
31
77.5
RESULTS
TNM
Forty patients with breast carcinoma aged between 51 and 80 years old (mean age: 62 years) were included in the study. Only patients with installed menopause at the time of breast carcinoma diagnosis were selected. The majority of breast carcinomas of postmenopausal women were diagnosed between 55‑64 years old (37.5%) and 65-74 years old (35%) as resulting from Table 1. From the 40 primary breast tumors, 34 (85%) were ductal invasive carcinomas and 6 (15%) nonductal or mixt carcinomas (infiltrative lobular, medullary, papillary, invasive ductal and lobular).
Sectorectomy/mastectomy without DALN*
GIII
9
22.5
T1
6
15
T2
21
52.5
T3
0
0
T4
12
30
Tx
1
2.5
N0
12
30
N1
24
60
Nx
4
10
*DALN = Dissection of axillary lymph nodes
IHC studied markers expression and their interrelations Bcl-2 The bcl-2 protein was expressed in 22 of 40 tumors (55%). The bcl-2 expression from carcinomas invasive had heterogenous distribution Table 1. Age-related incidence of breast carcinoma (n = 40). Table 1. Age-related incidence of breast carcinoma (n =component 40) and variable stain intensity within the same tumor section, even in the same tumor area. (Fig. 1) The 45-54 55-64 65-74 75-84 Age group staining pattern was cytoplasmic and membranous, sometimes with perinuclear enhancement. (Fig. 2) 7 15 14 4 Patients No. (%)
(17.5%)
(37.5%)
(35%)
(10%)
Due to the primary tumor size, the 40 tumors were designated: pT1- 6 tumors (15%); pT2 – 21 tumors (52.5%); pT3 – 0, pT4 -12 tumors (30%); in one case (2.5%) data on primary tumor dimensions was lacking (pTx). Pathological evaluation of axillary lymph nodes status (in 36 cases out of 40), indicated the presence of lymph node metastases in 24 (60%) of 40 cases. Accordingly to the histologic grade, tumors were classified as follows: GI - 0, GII - 31 (77.5%) and GIII – 9 (22.5%). Table 2 reveals the main clinical and pathological features of IHC studied cases.
Figure 1. bcl-2 positive reaction in a breast invasive ductal carcinoma. Anti-bcl-2, LSAB2 system, DAB visualization, hematoxylin counterstain. _____________________________ Alis Dema et al
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stained for Ki-67), while 28 tumors (70%) were characterized by high proliferative activity (>10% Ki67 positive nuclei). Table 3 is presenting the immunohistochemically determined molecular characteristics of the analyzed tumor group.
Table 3. IHC molecular markers of the analyzed tumors (n = 4 Table 3. IHC molecular markers of the analyzed tumors (n = 40). IHC
Specimens
molecular
No.
%
characteristic ER
Figure 2. Cytoplasm and membrane stain for bcl-2. Anti-bcl2, LSAB2 system, DAB visualization, hematoxylin counterstain.
The bcl-2 expression was present in the majority of ductal and myoepithelial cells of nonmalignant peritumoral component, except few cystic dilated ductal structures and apocrine metaplastic ducts, which were non-reactive. In ductal carcinoma in situ “non-comedo” type, foci associated to invasive tumor presented bcl-2 overexpression of variable intensity, while ductal carcinoma in situ “comedo” type was without reaction. The tumor stroma lymphocytes with positive reaction for bcl-2 represented the intern positive control of IHC reaction in cases without bcl-2 reactivity in tumor cells. ER, PR ER and PR were identified in the tumor cells nuclei of invasive and intraductal components, together with ductal epithelial cells of nonmalignant component associated to some of analyzed tumors. The majority of cases presented a heterogenous distribution of ER and PR within the same section, and variable stain intensity: from weak to moderate or strong/intense. Some cases had a positive cytoplasmic reaction, in the absence of nuclear stain, which was not counted. ERs were positive in 22 cases (55%) and PRs in 23 cases (57.5%). Ki-67 The positive reaction for Ki-67 antigen, exclusively intranuclear, presented an important stain intensity variation, heterogenous expression as topographic distribution and diverse stain patterns: fine granular, nucleolus isolated stain, nucleolus and nuclear membrane, intense and complete stain and perichromosome stain. Twelve (30%) of the 40 analyzed tumors demonstrated low proliferative activity (≤10% nuclei _____________________________ TMJ 2008, Vol. 58, No. 3 - 4
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PR Bcl-2 Ki-67
+
22
55
-
18
45
+
23
57.5
-
17
42.5
+
22
55
-
18
45
- low proliferative activity
12
30
- high proliferative activity
28
70
Figure 3. bcl-2 expression in an intermediate differentiated invasive ductal carcinoma. Anti-bcl-2, LSAB2 system, DAB visualization, hematoxylin counterstain.
Figure 4. Intense expression of ER in an invasive ductal carcinoma (same case as previous Fig. 3). Anti-ER, LSAB2 system, DAB visualization, hematoxylin counterstain.
Figure 5. Intense expression of PR in an invasive ductal carcinoma (same case as Fig. 3). Anti-PR, LSAB2 system, DAB visualization, hematoxylin counterstain.
respectively PR-positive tumors demonstrated bcl-2 protein overexpression. (Figs. 3 - 5) An important percentage of low proliferative activity tumors (92%) showed positive reaction for bcl-2 (Fig. 6), compared to high proliferative activity tumors, which were positive for bcl-2 up to 39%. Analyzing the relationship between bcl-2 expression and tumor differentiation grade, we observed that 64.5% of well and moderate differentiated tumors were positive for bcl-2, in contrast with poorly differentiated tumors, with only 22% reactive for bcl-2. The tumors without lymph nodes metastases (N0) were positive for bcl-2 in 67% cases, while tumors with lymph nodes metastases (N+) were bcl-2 positive in 46% cases.
The results of the analysis concerning the relationship between bcl-2 expression and other prognostic factors reveal that bcl-2 was significantly expressed by small sized tumors (100% T1 and 60% T1+T2) comparative with larger tumors (T3+T4) up to 42% positive reaction for bcl-2. (Table 4) Table 4. bcl-2 expression associated with other prognostic factors (n=
Table 4. bcl-2 expression associated with other prognostic factors (n= 40 cases)
40 cases).
Prognostic factor
bcl-2 expression -
+
T1
0
6 (100%)
Figure 6. Proliferation index Ki-67
