The mean AgNOR values in relation to HER-2/neu overexpression In a series of Iraqi women with breast cancer

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1 The mean AgNOR values in relation to HER-2/neu overexpression In a series of Iraqi women with breast cancer Dr. Esraa Abdul-Aal Department of pathology and Forensic medicine Kufa College of Medicine Kufa, P.O. Box 18 Iraq esraad@yahoo.co.uk The mean AgNOR values in relation to HER-2/neu overexpression In a series of Iraqi women with breast cancer. Esraa A. AL-Dujaily, M.Sc.path Short running title of no more than 40 characters: The mean AgNOR values in relation to HER-2/neu overexpression. Department of Pathology and Forensic Medicine, Faculty of Medicine, Kufa University, Kufa, P.O. Box 18, Iraq. Address correspondence and reprints request to: Dr. Esraa Abdul-Aal, Department of Pathology and Forensic Medicine, Faculty of Medicine, Kufa University, Kufa, P.O. Box 18, Iraq. Mobile: ( ) esraad@yahoo.co.uk Abstract Background: This study aimed to assess the usefulness of argyrophilic nucleolar organizer regions (AgNORs) in distinguishing HER-2/neu positive from HER-2/neu negative malignant breast lesions. Methods: The present investigation was performed over a period starting from September 2007 to August Formalin fixed, paraffin-embedded blocks from 90 patients with breast cancer were included in this study. A group of 25 patients with benign breast lesions (fibroadenoma) was included as a comparative group. Labeled Streptavidin-Biotin Complex (LSAB) method was employed for immunohistochemical detection of HER-2/neu. Standardized AgNORs analysis was performed on the above mentioned samples. Results: The mean AgNOR values ±standard error of the ninety studied breast cancer cases were as follow: for invasive ductal carcinoma 6.4±0.2,invasive lobular carcinoma 2.8±0.4, IDC+DCIS 7.5±0.4,and for IDC+ Paget s 8.1±1.1. There was significant difference of HER-2/neu overexpression between ductal and lobular carcinoma and also Paget s disease and the study improves significant HER-2/neu overexpression in higher histological grades,paget's disease.there was no significant difference between HER-2/neu overexpression and stage of tumor.the differences in mean AgNORs counts between benign and malignant cases was significant p<0.05,also there was significant difference between mean AgNOR values of HER-2/neu positive and negative ILC cases and IDC with DCIS cases respectively p value<0.05,while there was no significant difference between mean AgNOR values of HER-2/neu positive and negative IDC cases p>0.05.also there was significant difference between mean AgNOR values of HER-2/neu positive and negative GIII cases p<0.05 ;while well and moderately differentiated cases showed no significant difference between mean AgNOR values of HER-2/neu positive and negative cases p>0.05. Regarding tumor stage there 128

2 was significant difference between mean AgNOR values of HER-2/neu positive and negative cases of stage II and III cases. Conclusion: Based upon the findings of this study, it can be concluded that the use of standardized AgNORs analysis may be regarded as an objective tool in distinction between those who are positive for HER-2/neu from those negative for HER-2/neu and thus its possible use in the future in developing countries like Iraq as a simple and cheap test that seem to have a significant impact on predicting aggressive behavior of breast cancer and hence in selecting patients that could benefit from Herceptest and its targeted therapy. Background Breast cancer is the commonest malignant tumor and the leading cause of cancer death in women, with more than one million new cases occurring worldwide annually (1). In Iraq, where the population was exposed to high levels of depleted uranium following the first and second Gulf Wars, breast cancer is the most common tumor in females (2). Over the last ten years, there has been a three-fold increase in the incidence of breast cancer, with most of this increase being attributed to a particularly aggressive type of the cancer (3). It is well known that proto-oncogenes and tumor suppressor genes are two types of genes that play a key role in the regulation of cell growth and differentiation. Hence, any alteration in one or more of these genes appears to play an important role in the pathogenesis of most human malignancies (4).. Her-2/neu proto-oncogene amplification and or over expression is one of the most important alterations encountered in breast cancer. This proto-oncogene (also called c-erbb2) is located on chromosome 17q11 (4,5), which encodes for p185 a transmembrane glycoprotein with tyrosine kinase Activity that belongs to the family of epidermal growth factor Receptors (5,6).HER2/neu protooncogene is amplified and or over expressed in approximately 20-25% of invasive primary breast cancers (6-8). An association have been found to exist between amplification and or over expression of HER-2/neu and advanced stage, early relapse, and reduced overall survival (9,10). Nucleolar organizer regions (NORs) represents loops of DNA which have the genes for ribosomal RNA. The argyrophilic method for NOR staining (Ag-NOR technique) identifies NOR associated proteins. This simple technique can be used to show the presence of proteins associated with nucleolar organizer regions in interphase chromosomes in conventional histological sections, and these can be counted under light microscope ( 10).The AgNORs parameter was found to be useful for assessing the prognosis of breast cancer (11).Several studies have shown that there is appositive correlation between transcriptional activity of ribosomal RNA represented by Nucleolar organizer region (NORs) and different tumor markers like Ki67,p53,and others (12,13). Indeed, there is much of evidence to document the essential role AgNORs as a prognostic marker for the aggressiveness of breast cancer (9,10,12). Therefore we plan to study the possibility of finding a relation between Nucleolar organizer regions assessed by a cheap and available silver technique and HER-2/neu genetic marker assessed by the expensive Herceptest in a series of Iraqi women with breast cancer and hence whether such relation could be used as an objective tool for the early management of breast cancer patients. Patients and Methods: Ninety specimens, collected from breast cancer patients over a period from September 2007 to August 2008, were included in this study. All the cases were collected from 129

3 major hospitals and private laboratories in Kufa district area (located in the middle of Iraq). The age range of patients was 21 to 70 years, with a mean age of 48.8 years. A group of 25 patients with benign fibroadenoma breast lesions was included as a comparative group Labeled Streptavidin-Biotin (LSAB) method was employed for immunohistochemical detection of HER-2/neu. All biopsies were classed, according to the modified WHO classification, into three grades: malignant grade I, malignant Grade II and malignant Grade III. Standardized AgNORs analysis was performed on the above mentioned samples. The results were statistically evaluated by a Chi-squared test and correlation-regression using SSPS software. Results: The mean AgNOR values of the 25 benign fibroadenoma breast lesions was (1.9±0.14); for the 90 malignant breast cases was (9.2±0.33) ;for the 61 invasive ductal carcinoma (IDC) it was (6.4±0.2); for the 8 invasive lobular carcinoma cases was (2.8±0.4) ;for the 18 IDC with insitu component of comedo type ;for the 3 cases of IDC with paget's disease was(8.1±1.1).regarding the tumor grade.the mean AgNOR values of the 2 grade I cases was 4.2±5; for the 17 grade II cases was 5.8±0.4; for the 71 grade III cases was (5.2±0.9 ).Regarding the staging of breast cancer cases there was 5 cases of stage I with mean AgNOR values of (5.2±0.9); for the 23 stage II cases was (6.4±0.4 ) ;for the 41 stage III cases was (6.6±0.3); for the 3 stage IV cases was (6.6±0.4) (table.1). HER-2/neu over-expression was detected in 70.6% of grade II and 69.0% of grade III tumor samples while none of grade I show HER-2/neu positivity (Table.2). There was no significant difference in HER-2/neu over-expression among these different grades of breast cancer (p>0.05). HER-2/neu over-expression was highly correlated with grade of tumor, indicating that HER-2/neu positive breast cancers are biologically aggressive (R = 0.96).There was no significant difference between Her-2/neu overexpression and tumor stage (p>0.05)(table.2). All the benign fibroadenoma breast lesions were negative for HER-2/neu overexpression and the mean AgNOR count of the 25 HER-2/neu negative cases was (1.9±0.14) with significant difference when compared with that of malignant cases (p value <0.05). The mean AgNOR count of the 7 HER-2/neu negative invasive lobular carcinoma cases was (2.4±0.52) and of the only 1 HER-2/neu positive case the mean AgNOR count was (5.5±0 ) with significant difference (p value <0.05). The mean AgNOR count of the 17 HER-2/neu positive invasive ductal carcinoma with comedo insitu carcinoma cases was (7.8±0.4) ;while the mean of the only 1 HER-2/neu negative case of IDC+DCIS was (4.2±0 ) with significant difference (p value <0.05). (Table.3) showing significant difference of mean AgNOR values (7.42±0.2, 5.1±0.4 respectively) between HER-2/neu positive and negative grade III cases(p value <0.05). (Table.3)showing significant differences of mean AgNOR values (6.9±0.3, 4.32±0.82 respectively )of HER-2/neu positive and negative stage II cases (p value <0.05) ; and significant difference of mean AgNOR values(7.2±0.4, 5.24±0.6 respectively ) of HER-2/neu positive and negative stage III cases (p value <0.05)(table.3). Discussion: In our study we found that the mean AgNOR count of benign fibroadenoma breast lesions was (1.9±0.14); Kumar et al (14), found that mean AgNOR count of benign breast lesions was(1.88± 0.19) and this is near to the AgNOR count of benign breast lesions in our study. We found that the mean AgNOR count of pure IDC cases was (6.4±0.2),for ILC was (2.8±0.4), for IDC with DCIS of comedo type was (7.5±0.4),while for IDC with overlying paget's disease was (8.1±1.1).Furthermore we found 130

4 increase in the mean AgNOR values with progression of the tumor from well to poorly differentiated ;(4.2±5) for HER-2/neu positive GI cases(fig 1(a)),( 5.8±0.4) for positive GII and (6.7±0.2) for positive GIII cases(fig1(c)). Suramanian S etal (16) found that AgNOR count was significantly related to the histological grade. Regarding the tumor stage there was slight increase of mean AgNOR values (5.2±0.9, 6.4±0.4, 6.6±0.3 and 6.6±0.4 )for stage I, II, III and IV respectively. This is can be explained by the low number of stage I and IV cases, 5 and 3 respectively which can not provide a confidant results. HER-2/neu Immunohistochemical study: This study showed that HER-2/neu over-expression was completely absent in benign breast lesions. but the finding is that HER-2/neu over-expression appears to be a biomarker for malignant breast tissue and does not play any role in the benign (fibroadenoma) breast lesions. This observation confirms the results of many previous studies that reached the same conclusion (17, 18). Indeed, our study reported HER-2/neu over-expression in (67.8%) out of 90 breast cancer cases. This result is significantly higher than those reported by many western researches, as reported by Marc J. et al.,(1988) (19). Furthermore, most of ductal carcinoma cases were purely invasive ductal carcinoma of non otherwise specified (NOS)(74.4%) that showed significant HER- 2/neu over-expression (65.6%). The current study also demonstrated HER-2/neu expression in (12.5%) of lobular carcinoma cases, which was of pleomorphic type Anita et al.,(2003) (20) found a similar result. (Figure.1(e)). From the above results, a significant difference in HER-2/neu over-expression was observed between invasive ductal and lobular carcinoma (P<0.05). Our finding that HER-2/neu expression, as detected by this assay, is seen more frequently in DCIS than in invasive carcinoma, implicates HER-2/neu signaling is playing a critical role in the early stages of breast tumorigenesis. Regarding HER-2/neu overexpression in invasive ductal carcinoma with overlying Paget's disease, HER-2/neu over-expression was seen in all cases of both the invasive ductal carcinoma and Paget's disease components, when both components were seen jointly Mark J. et al., (1988) (19). A high proportion of HER- 2/neu overexpression was seen in moderately and poorly differentiated cases of breast cancer, while a lower proportion of HER-2/neu overexpression was reported in cases of well differentiated breast cancer. HER-2/neu overexpression appears to be well correlated with tumor grade (R = 0.96). AgNOR silver staining results in relation to HER-2/neu Immunohistochemical overexpression: In our study we did a comparison between the mean AgNOR values of the HER-2/neu positive and HER-2/neu negative cases of different pathological parameters to find a correlation between the increased and decreased mean AgNOR count in relation to the HER-2/neu positivity and negativity of the studied cases included in our study. Our study revealed that there was significant difference between the mean AgNOR values of HER-2/neu positive malignant cases in comparison to HER-2/neu negative cases (p value <0.05) ; also there was significant difference between mean AgNOR values of positive ILC and IDC+DCIS cases respectively in comparison to negative cases(p value <0.05) ;furthermore our study demonstrated significant difference of mean AgNOR values of positive grade III cases in comparison to negative cases ; regarding tumor stage there was significant difference of mean AgNOR values of positive stage III cases in comparison to negative cases,while stage IV showed no significant difference between mean AgNOR values of positive and negative cases, this is can be explained by the low number of the stage IV cases only 3 cases which could not provide us with confident results. 131

5 Conclusion AgNOR method is a reliable and inexpensive method that can be used as a complementary test that seem to have a significant impact on predicting aggressive behavior of breast cancer and hence in selecting patients that could benefit from the expensive Herceptest and hence its targeted therapy in developing countries like Iraq. Hopefully,in the near future HER-2/neu testing will become a routine examination for all breast cancer patients in our country. References: 1. Parkin DM, Bray F, Ferlay J et al. Estimating the world cancer burden. Globocan Int. J. Cancer 2001;94 : Results of Iraqi cancer registry ,Iraqi Cancer Board,Iraqi Cancer Registry,Ministry of Health,Baghdad-Iraq. 3. Al-Azzawi SN. Depleted Uranium radioactive Contamination In Iraq: An Overview. Global Research. August 31, 2006,1:page 4 4. Joe W, Gray and Danial Pinkel.Molecular cytogenetics in human cancer society and American Joint Committee on cancer workshop on molecular markers in the classification and staging of cancer,atlanta, Georgia.Patho.J 1990:9: Hung MC, Lau YK. Basic science of HER-2/neu : Areview. Sem. Oncol 1999: 12: Coussens L, Yang-Feng TL, Lioa YC, Chen E, et al.. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 1985: 230: Schecter AL, Stern DF, Vaidyanathan L, Decker SJ, Drebin JA, Greene MI, Weinberg AR. The neu oncogene: An erbb-related gene encoding a 185,000-Mr tumor antigen. Nature 1984: 312: Slamon DJ, Goldolphin W, Jones LA, et al..studies of the HER-2/proto-oncogene in human breast cancer and ovarian cancer. Science 1989:44: Anwar N,Mokhtar N, Mourad M,et al., c-erbb2 and its relation to lymph node positivity :Short term follow up in Egyptian cancer patients.cmb Simha M, Menon M, Doctor V. prognostic value of argyrophylic nucleolar organizer regions (AgNORs) in breast lesions. Indian J Cancer. 33(2):76-85; Crocker J, Hall PA, Macartney JC, Stansfield AG. A comparative study of nucleolar organizer regions (AgNOR), Ki67 staining and DNA flow cytometry in non-hodgkin's. J Pathol; 154:37, Rosai J. Ackermans surgical pathology. 8th ed. Philadelphia Mosby; Davide Treré, Claudio Ceccarelli, Lorenzo Montanaro,et al. University of Bologna, Italy. Nucleolar Size and Activity Are Related to prb and p53 Status in Human Breast Cancer DOI: /jhc.4A Kumar A, Kushwaha AK, kumar M, Gupta S. argyrophylic nuclealar organizer region: their value and correlation with clinical prognostic factors in breast carcinoma. J Surg oncol, 65(3):201-4, Abdel-Ghani A. Selim, Ghada El-Ayat, Clive A. Wells 1 Expression of c-erbb2, p53, Bcl-2, Bax, c-myc and Ki-67 in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast. Pubmed;2002,60: Supramanian S, Shariff S, Andradi C. AgNORs and their relationship to cell size, histological grade, lymph node involvement, metastases and survival pattern in carcinoma of the breast : a study from south India. J Surg Oncol ; 62(2):139-43;

6 17. Gusterson BA, Machin LG, Gullick WJ et al. cerbb2 expression in benign and malignant breast disease.br J Cancer 1988,58: Selim A-GA, El-Ayat G, Wells GA. Expression of c-erbb2, p53, Bcl-2, Bax, c-myc and ki-67 in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast. Virchows Archive 2002;441: Marc J. Van DE Vijver, Johannes L. Peterse,Wolter J.Moor,et al..neu protein overexpression in breast cancer :Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer.n Engl J Med 1988,319: Anita L Bane, Susanna Tjan, Robert K Parkes, Irene Andrulis and Frances P O'Malley.Invasive lobular carcinoma: to grade or not to grade. Published online 17 December Modern Pathology 2005, 18: Table 1. Showing mean AgNOR values± S.D. of the different studied groups. Parameters No. Mean AgNOR values± S.D. Benign fibroadenoma ±0.14 Malignant cases ±0.33 Histological type IDC ±0.2 ILC 8 2.8±0.4 IDC+DCIS ±0.4 IDC+ Paget s 3 8.1±

7 Grade I 2 4.2±5 II ±0.4 III ±0.2 Tumor stage Stage I 5 5.2±0.9 Stage II ±0.4 Stage III ±0.3 Stage IV 3 6.6±0.4 Table 2. HER-2/neu Immunohistochemical overexpression: Parameters Total c-erbb-2 protein overexpression No. of patients ( Negative) (positive ) P R value test Benign fibroadenoma 25 25(100%) 0(-) <0.05 Malignant cases 90 29(32.2%) 61(67.8%) Histological type IDC* 82 22(26.8%) 60(73.2%) <0.05 ILC 8 7(87.5%) 1(12.5%) *including IDC+DCIS 18 1(5.6%) 17(94.4%) IDC+ paget s 3-3(100%) 134

8 Grade I 2 2 (100%) 0 (-) > II 17 5 (29.4%) 12 (70.6%) III (31.0%) 49(69.0%) Tumor stage Stage I 5 3(60%) 2(40%) > Stage II 23 5(21.7%) 18(78.3%) Stage III 41 13(31.7%) 28(68.3%) Stage IV 3 2(66.7%) 1(33.3%) Table 3. AgNOR silver staining results in relation to HER-2/neu Immunohistochemical overexpression: Parameters Mean AgNOR values± standard Error of HER-2/neu (+ve) and(-ve) cases Negative(X±E) positive(x±e) P value Benign fibroadenoma 25(1.9±0.14) 0(-) <0.05 Malignant cases 29(5.0±0.36) 61(7.1±0.20) Histological type IDC 21(5.93±0.3 ) 40(6.6±0.22) >0.05 ILC 7(2.4±0.52) 1(5.5±0) <0.05 IDC+DCIS 1(4.2±0) 17(7.8±0.4) <0.05 IDC+ paget s - 3(6.1±2.2) - Grade I 2 (4.22±5.00) 0 (-) - II 5 (5.03±0.9) 12 (6.1±0.3) >0.05 III 22 (5.1±0.4 ) 49(7.42±0.2) <

9 Tumor stage Stage I 3(4.3±1.3 ) 2(6.6±0.4) >0.05 Stage II 5(4.32±0.82) 18(6.9±0.3 ) <0.05 Stage III 13(5.24±0.6) 28(7.2±0.4) <0.05 Stage IV 2(6.24±0.2) 1(7.3±0) >0.05 Fig.(a) Showing HER-2/neu negative of score +1, IDC-GI.(X40)(b) showing regular,uniform black dots of AgNORs. Silver stain.(x1000) of IDC -G1. (c) Showing strong HER-2/neu positive IDC GIII of score +3.(d)The NORs here are the more numerous,black and tend to be more irregular.silver stain (X1000). (e)ilc pleomorphic type showing moderate complete membrane staining of score 2+ "positive reaction"(x40). (f)the NORs looks more numerous,black and tend to be more irregular(x1000). 136