Material and Method: We conducted a retrospective hormone receptor and Her2/neu study on 164 histologically confirmed invasive ductal carcinoma of the breast between the year 2002 and 2008. Immunohistochemical analysis for estrogen receptor, progestrone receptor and Her2/neu was done in all the cases. Fluorescent in situ hybridization (FISH) for Her2/neu gene amplification was performed in all 2+ cases and a few equivocal 1+ and 3+cases by immunohistochemistry. The results were then compared in a blinded fashion. Correlation between Estrogen Receptor, Progesterone Receptor and Her2/neu amplification and grade of tumour were calculated.
Result: The prevalence of estrogen receptor, progestrone receptor and Her2/neu overexpression were 64.6%, 57.3% and 35.3% respectively. The expression of estrogen receptor and progestrone receptor were significantly correlated (p<0.001). There was also a significant negative correlation between expression of hormone receptor and Her2/neu amplification. The histological grade of the tumour was also significantly correlated to the expression of both estrogen receptor and progestrone receptor. However, the relationship between Her2/neu amplification and grade of tumour was not significant and many of the grade III tumours were Her2/neu negative. In addition Her2/neu gene amplification by FISH was observed in 84.6% of breast cancers that were 3+ and in 18.75% of the cases that were 2+ by immunohistochemistry.
Conclusion: The revalence of estrogen receptor, progestrone receptor expression and Her2/neu amplification in breast cancers in Saudi Arabian population is similar to that reported internationally. There is a negative correlation between hormone receptors expression and Her2/neu amplification. However, not all high-grade breast cancers showed Her2/neu positive status.
The expression of ER is seen in 70-80% and PR in 60- 70% of cases of invasive ductal carcinoma [2]. These cases are associated with better outcome and good response to hormone therapy both in adjuvant and metastatic settings. Her2/neu, also called ErbB2, is a member of human epidermal growth factor family and encodes a transmembrane tyrosine kinase receptor. It is a protooncogene which is located on chromosome 17, and is amplified or the protein Her2/neu overexpressed in 15-25% cases of breast cancer [3]. This marker has been extensively studied and it has been shown that Her2/neu is a significant prognostic marker in both node positive and in node negative breast cancer patients [4]. The positivity of Her2/neu in breast cancer is associated with high histological grade and tumour aggressiveness. It is thus assessed for both the prognosis and prediction of response to trastuzumab treatment, chemotherapy and endocrine therapy.
Immunohistochemistry (IHC) has become the method of choice for ER and PR assessment in the last two decades. The most common cutoff to define ER positivity is 10% but there is convincing data that patients with even 1% ER positive tumour cells benefit from hormone therapy [5]. Regarding the Her2/neu status, there is no agreement as to the best method of assessing amplification and over-expression as no standardised validated method exists for Her2/neu testing. The Food and Drug Administration (FDA) has approved the Hercep Test for IHC and PathVysion for fluorescent in situ hybridization. These are the most commonly used methods for Her2/neu assessment nowadays as well as in our present study.
The aim of this study was to demonstrate the pattern of expression of hormone receptor and Her2/neu in breast carcinoma, the correlation between histological grade of the lesion, and ER and PR hormonal status and Her2/neu expression. Cases equivocal for Her2/neu expression by IHC were resolved by FISH analysis.
ER/PR analysis was carried out in all 164 cases according to the manufacturer’s instruction as a routine procedure on 4- micrometer sections of formalin fixed, paraffin embedded tumour after microwave antigen retrieval (0.01M Citrate buffer, pH 6.0), using commercially available mouse monoclonal antibodies to ER and PR (clone 6F11 and Clone 16 respectively, Ventana, AZ, USA). The staining for ER and PR was classified as positive if more than 10% of the tumour cells exhibited nuclear staining. Her-2/ neu immunohistochemical analysis was performed using the Hercep Test kit (clone CB11, Dako, CA, USA) according to the manufacturer’s instruction and the results were interpreted using the original FDA and new ASCO/CAP guideline recommendations [6]. Scoring was done on a 0-3 scale. Positive (3+) was defined as strong complete membranous staining in more than 30% of the tumour cell population. Borderline (2+) was defined as moderate membranous staining in more than 10% of tumour cells. 1+ was defined as either weak or barely perceptible membranous staining in more than 10% of the tumour cells. 0 was completely negative staining or membranous staining in less than 10% of the tumour cells. Scores of 0 and 1+ were considered as negative for Her2/neu expression 3+ as immunopositive while 2+ was weakly or borderline positive. Positive and negative controls were included in each batch.
FISH analysis was performed for Her2/neu gene amplification in all 2+ cases (32 cases) by IHC, and 28 equivocal cases of 1+ and 3+ (showing some technical fault as staining of the blood vessel or benign ducts) between 2005 and 2008. The cases were sent to Kassel Klinikum in Germany (as this test is not available in our institution) for further assessment by FISH analysis to confirm or exclude gene amplification. Her2/neu gene amplification was determined in paraffin-embedded tissue sections by FISH using PathVysion (Abbott/Vysis, IL, USA) as a ratio of HER2/neu gene copies-to chromosome 17 centromere copies.
Statistical Methods
Statistical significance was set at p <0.05. Data were analyzed
by the chi-square test. Computations were performed with
the sigma STAT for Windows software (version 2.03, SPSS
Inc., IL, USA).
As regards the Her2/neu expression by IHC, 58 cases were 3+ (Figure 3), 23 cases were 2+ (Figure 4) and the remaining were either 1+ or 0, out of the total of 164 cases,. It was found that ER and PR expression was increased significantly in Her2/neu negative tumours compared to Her-2 positive tumours. However, a substantial number of Her-2 positive tumours still expressed ER or PR (Table II). Thus there was a statistically significant negative correlation between the expression of hormone receptor and Her2/ neu overexpression.
Out of 164 cases of invasive ductal carcinoma of the breast, 9 cases were grade I, 85 cases were grade II and 70 cases were grade III. The reactivity for steroid receptors was observed to decrease with increasing grade. Grade II tumours expressed ER and PR more than grade III tumours (Table III). Thus the grade of tumour was significantly correlated to the expression of both ER and PR (Pearson chi-square value = 6.085 and 11.169 respectively with p values of 0.047 and 0.025). The relationship between Her2/neu overexpression and grade of tumour was found to be not significant (p value > 0.25) and vast majority of Her2/neu negative tumours were grade III.
Sixty cases out of 164 (those that were either 2+ and a few 1+ or 3+) were confirmed by FISH analysis (Table IV). The cases assigned by IHC as suspicious for 1+ (two cases) group were Her2/neu negative by FISH analysis. Among the samples described immunohistochemically as 2+ (32 cases), amplification of Her2/neu gene was discovered in 18.25% of the cases. (n=6). The remaining 81.25% of the cases (n=26) were negative by the FISH method. In the group of specimen assessed by IHC as equivocal for 3+ (26 cases), Her2/neu gene amplification was detected in 84.6% (n=22). Lack of gene amplification by FISH was seen in 4 cases (15.3%).
Figure 1: IHC showing estrogen receptor positive tumour cells in breast cancer (x400).
Figure 2: IHC showing progesterone receptor positive tumour cells in breast cancer (x400).
Table I: Relationship between estrogen receptor and progesterone receptor
Table II: Association between Her2/neu status and estrogen and progesterone receptor expression
Table IV: Comparison of Her2/neu status determined by FISH and immunohistochemistry
We performed a retrospective study in a cohort of 164 patients to correlate hormone receptor and Her2/neu status with histological grade of the lesion and ER, PR hormonal status. Consistent with most widely adapted clinical practice, and the practice at our institution, we considered a tumour as Her2/neu positive as being either IHC score of 3+ or FISH positive (ratio of more than or equal to 2.2).
In our study, the prevalence of ER, PR expression and Her2/neu amplification were 64.6%, 57.3%, and 35.3% respectively. These fall within the ranges given in the English literature (7-10). ER and PR determination are established procedures in the routine management of patients with breast cancer, primarily as a predictive factors for response to therapeutic and adjuvant hormonal therapy [11],[12]. In the published literature [13] approximately 50% of tumours are ER +, PR +, 25% ER -, PR –, 20% ER +, PR- and 5% ER –, PR +. Our study reports a lower proportion of ER + PR- (8.5%) and a higher proportion (34.2%) of hormone receptor negative cases. Desai et al. [14] and Dutta et al. [15] also found a high incidence of steroid non-reactivity in breast cancer patients. In general, there was a statistically significant positive correlation between the expression of ER and PR in our study, as reported by others [8],[16],[17]. This also abides by the theory of ER-dependent PR synthesis [15].
The co-expression of hormone receptors with that of Her2/neu is infrequent as previously reported. In our study, Her2/neu Receptor revealed a significant inverse association with hormone receptor status. We found that ER and PR expression was increased significantly in Her2/ neu negative tumours, compared to Her2/neu positive tumours. Similar results were shown in previous studies [7],[8],[18]-[20]. The generally accepted inverse correlation between the expressions of hormone receptors and Her2/ neu amplification is explained by hormone-dependent downregulation of Her2/neu involving a complex molecular interaction as reviewed by Ciocca et al [21] and as shown by in vitro studies [18],[19]. This is one of the reasons why women who overexpress Her2/neu may be resistant to tamoxifen. However, as demonstrated in our study and by others, ER positive cases can have Her2/ neu overexpression/amplification. Both ER and Her2/ neu positive tumours have poorer disease-free and overall survival than ER positive, Her2/neu negative tumours suggesting that Her2/neu overexpression may be better predictor of response to tamoxifen therapy than ER status alone [22].
The grade of tumour in our study was significantly correlated to the expression of both ER and PR (p value of 0.04 and 0.025, respectively). The lower grade tumours showed high ER and PR positivity, similar to other studies [8],[23]. The overexpression/amplification of Her2/neu in different histological grades of invasive ductal carcinoma of the breast has been a subject of interest. In our study the relationship between Her2/neu amplification and grade of tumour was found to be not significant and vast majority of Her2/neu negative tumours were grade III. This is in contrast with previous studies [20],[24]-[26], which reported histologically high-grade tumours’ association with increased rate of Her2/neu overexpression. We confirmed the results of Her2/neu protein and overexpression of all 2+ and equivocal 1+ and 3+ cases on IHC, using FISH analysis in total of 60 cases. As only patients with Her2/neu protein overexpression and/or gene amplification benefit from Trastuzumab treatment our aim was to identify those cases, using a more accurate and reliable method – i.e. FISH. Our results indicate that the IHC 2+ group is characterized by high heterogeneity as only 18.75% of the cases showed Her2/neu gene amplification. In samples described by IHC as 3+, amplification of Her2/neu gene was discovered in 84.6% of the cases by FISH analysis. These results are in accordance with many previous reports [27]-[31] in the evaluation of Her2/neu status of breast cancer using formalin-fixed paraffin-embedded specimens which found a high degree of correlation between IHC 3+ staining and amplification detected by FISH. In conclusion, the prevalence of ER, PR and Her2/neu amplification among Saudi Arabian breast cancer patients is similar to the rest of the world. There is also a negative correlation between expression of hormone receptor and Her2/neu expression. The lower grade of tumour is related to hormone receptor expression in accordance to that reported in the literature, however not all our high-grade tumours were Her2/neu positive.
ACKNOWLEDGEMENT
I thank Dr Shaesta Naseem Zaidi, MD for her assistance
in the study and preparation of the manuscript. A special
thanks to Rouch Company who supported performing the
FISH analysis at Kassel Klinikum in Germany.
1) Horita K, Yamaguchi A, Hirose K, Ishida M, Noriki S, Imamura Y, Fukuda M: Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer. Eur J Histochem 2001, 45(1):73-84
2) Zafrani B, Aubriot MH, Mouret E, De Crémoux P, De Rycke Y, Nicolas A, Boudou E, Vincent-Salomon A, Magdelénat H, Sastre-Garau X: High sensitivity and specificity of immunohistochemistry for the detection of hormone receptors in breast carcinoma: comparison with biochemical determination in a prospective study of 793 cases. Histopathology 2000, 37: 536-545
3) Hanna W, Kahn HJ, Trudeau M: Evaluation of HER-2/neu (erbB-2) status in breast cancer: from bench to bedside. Mod Pathol 1999, 12:827-834
4) Ross JS, Fletcher JA: The HER-2/neu oncogene in breast cancer: Prognostic Factor, Predictive Factor, and Target for Therapy. Oncologist 1998, 3:237-252
5) Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010, 28:2784-2795
6) Brunelli M, Manfrin E, Martignoni G, Bersani S, Remo A, Reghellin D, Chilosi M, Bonetti F: HER-2/neu assessment in breast cancer using the original FDA and new ASCO/CAP guideline recommendations: impact on selecting patients for herceptin therapy. Am J Clin Pathol 2008, 129:907-911
7) Huang HJ, Neven P, Drijkoningen M, Paridaens R, Wildiers H, Van Limbergen E, Berteloot P, Amant F, Vergote I, Christiaens MR: Hormone receptors do not predict the HER2/neu status in all age groups of women with an operable breast cancer. Ann Oncol 2005, 16:1755-1761
8) Ratnatunga N, Liyanapathirana LV: Hormone receptor expression and Her/2neu amplification in breast carcinoma in a cohort of Sri Lankans. Ceylon Med J 2007, 52:133-136
9) Perez EA, Pusztai L, Van de Vijver M: Improving patient care through molecular diagnostics. Semin Oncol 2004, 31:14-20
10) Bast RC Jr, Ravdin P, Hayes DF, Bates S, Fritsche H Jr, Jessup JM, Kemeny N, Locker GY, Mennel RG, Somerfield MR: 2000 update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001, 19:1865-1878
11) Bast RC Jr, Ravdin P, Hayes DF, Bates S, Fritsche H Jr, Jessup JM, Kemeny N, Locker GY, Mennel RG, Somerfield MR: American Society of Clinical Oncology Tumor Markers Expert Panel. 2000 update of recommendations for the use of tumor markers in breast and colorectal cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001, 19:1865-1878
12) Donegan WL: Tumor-related prognostic factors for breast cancer. CA Cancer J Clin 1997, 47:28-51
13) Barnes DM, Millis RR: Oestrogen receptors: the history, the relevance and the methods of evaluation. In: Kirkham N, Lemoine NR (eds) Progress in pathology, Vol 2. Edinburgh, Churchill Livingstone, 1995, 89-114
14) Desai SB, Moonim MT, Gill AK, Punia RS, Naresh KN, Chinoy RF: Hormone receptor status of breast cancer in India: a study of 798 tumours. Breast 2000, 9:267-270
15) Dutta V, Chopra GS, Sahin K, Nema SK: Hormone Receptors, Her-2/Neu and chromosomal aberrations in breast cancer. MJAFI 2008, 64:11-15
16) Horwitz KB, McGuire WL: Estrogen control of progesterone receptor induction in human breast cancer: role of nuclear estrogen receptor. Adv Exp Med Biol 1979, 117:95-110
17) Grann VR, Troxel AB, Zojwalla NJ, Jacobson JS, Hershman D, Neugut AI: Hormone receptor status and survival in a populationbased cohort of patients with breast carcinoma. Cancer 2005, 103:2241-2251
18) Ciocca DR, Gago FE, Fanelli MA, Calderwood SK: Coexpression of steroid receptors (estrogen receptor alpha and/or progesterone receptors) and Her-2/neu: Clinical implications. J Steroid Biochem Mol Biol 2006, 102:32-40
19) Konecny G, Pauletti G, Pegram M, Untch M, Dandekar S, Aguilar Z, Wilson C, Rong HM, Bauerfeind I, Felber M, Wang HJ, Beryt M, Seshadri R, Hepp H, Slamon DJ: Quantitative association between HER-2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer. J Natl Cancer Inst 2003, 95:142-53
20) Ivkovic-Kapicl T, Knezevic-Usaj S, Djilas-Ivanovic D, Panjkovic M: Correlation of HER-2/neu protein overexpression with other prognostic and predictive factors in invasive ductal breast cancer. In Vivo 2007, 21:673-678
21) Ciocca DR, Fujimura FK, Tandon AK, Clark GM, Mark C, Lee- Chen GJ, Pounds GW, Vendely P, Owens MA, Pandian MR, et al: Correlation of HER-2/neu amplification with expression and with other prognostic factors in 1103 breast cancers. J Natl Cancer Inst 1992, 84:1279-1282
22) Ferrero-Poüs M, Hacène K, Bouchet C, Le Doussal V, Tubiana- Hulin M, Spyratos F: Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis. Clin Cancer Res 2000, 6:4745-4754
23) Thoresen S, Thorsen T, Tangen M, Hartveit F: Oestrogen and progesterone receptor content and the distribution of histological grade in breast cancer. Breast Cancer Res Treat 1982, 2:251-255
24) Hoff ER, Tubbs RR, Myles JL, Procop GW: HER2/neu amplification in breast cancer: stratification by tumor type and grade. Am J Clin Pathol 2002, 117:916-921
25) Ariga R, Zarif A, Korasick J, Reddy V, Siziopikou K, Gattuso P: Correlation of her-2/neu gene amplification with other prognostic and predictive factors in female breast carcinoma. Breast J 2005, 11:278-280
26) Huang HJ, Neven P, Drijkoningen M, Paridaens R, Wildiers H, Van Limbergen E, Berteloot P, Amant F, Vergote I, Christiaens MR: Association between tumour characteristics and HER-2/neu by immunohistochemistry in 1362 women with primary operable breast cancer. J Clin Pathol 2005, 58:611-616
27) Gramlich TL, Cohen C, Fritsch C, DeRose PB, Gansler T: Evaluation of c-erbB-2 amplification in breast carcinoma by differential polymerase chain reaction. Am J Clin Pathol 1994, 101:493-499
28) Pauletti G, Godolphin W, Press MF, Slamon DJ: Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene 1996, 13:63-72
29) Persons DL, Borelli KA, Hsu PH: Quantitation of HER-2/neu and c-myc gene amplification in breast carcinoma using fluorescence in situ hybridization. Mod Pathol 1997, 10:720-727
){kind=link}
){kind=link}
){kind=link}
){kind=link}
){kind=link}
){kind=link}
){kind=link}
){kind=link}