Material and Method: 163 mastectomy materials which had tumor diagnosis between 1995 and 2016 in our clinic and had findings of fibrocystic changes in non-tumor areas, and 151 excisional breast biopsy materials which had diagnosis of fibrocystic change but had not malignant mass were included into the study. The tissues containing fibrocystic changes in both groups were stained with HER-2 by immunohistochemical methods.

Results: It was found that HER-2 staining was more frequent in FCC areas with in mastectomy cases due to tumor (χ² = 37,079; p =,000).

Conclusions: We found out that HER 2 staining was significantly more positive in fibrocystic change cases with in mastectomy cases due to tumor than non-malignant cases and that the risk of malignancy was higher in HER-2 positive cases. We believe that HER-2 staining for breast biopsies will be helpful for determining malignancy potential in benign lesions of breast.

The HER-2/neu protooncogene known as C-erbB-2 is localized in the short arm of chromosome 17 (17q21). This gene encodes transmembrane HER-2 glycoprotein with a molecular weight of one hundred and eighty five kilodaltons (kDa) and comprising of 1255 amino acids. Normal breast epithelial cells contain 2 copies of the HER-2 gene and express low levels of HER-2 receptor on the cell surface. However, in some individuals, the number of gene copies in the cell is increased due to oncogenic transformation and gene amplification re-sults in increased membrane expression of HER-2 protein. For this reason, the basic mechanism for HER-2 protein overexpression is assumed to be gene ampli-fication. Increased membrane expression of HER-2 protein activates HER-2 tyrosine kinase by increasing dimerization. The transmission of this activation signal to the cell nucleus causes cell replication and mitosis in the cell undergoing oncogenic transformation. Thus, HER-2 gene amplification is considered to contribute to the developmental process of breast carcinoma, i.e. carcinogenesis ^7-9.

Over the years, it has been one of the major issues to determine whether these lesions are a risk factor for the subsequent development of breast cancer ¹⁰. In this study, we aimed to answer the question “Can we pre-dict the malignancy potential of patients with fibrocys-tic changes using the HER-2 gene?”

For immunohistochemical staining, sections with a thickness of 4μm were taken from parafin blocks with the help of Leica RM 2255 microtome to be mounted onto poly-L-lysin coated slides. After de-paraffinization and rehydration, the sections were soa-ked in 3% H2O2 for 15 minutes to block endogenous peroxidase activity and then protein blockage was performed. HER-2 was incubated for 1 hour with its primary antibody, and then it was incubated with secondary antibody binding to the primary antibody, and streptavidin- biotin- peroxidase enzyme complex for 15 minutes. It was washed with a buffer solution at each stage. The evaluation was made by a single pathologist under light microscopy. According to the recommendations of the American Society of Clinical Oncology / College of American Pathologists (ASCO / CAP), it was scored as follows:

Score 0: No membrane staining
Score 1: Whatever the rate in invasive carcinoma cells is, there is poor staining in non-membranous staining which is not surrounding the entire membrane and are difficult to identify, or poor staining which surrounds the entire membrane in less than 10% of cells. Immunohistochemistry refers to negative, in situ hybridization is not recommended.

Score 2: The membranous staining which completely surrounds the cytoplasmic membrane in at least 10% of invasive carcinoma cells but has moderate intensity, or the membranous staining which is less than 30% but has strong intensity is observed. It indicates a suspicious positive immunohistochemically, it should be confirmed by in situ hybridization.

Score 3: Strong uniform immunostaining which surrounds the entire cytoplasmic membrane in at least 30% of invasive carcinoma cells is seen. It is strong positive immunohistochemically, in situ hybridization is not recommended ¹¹.

Statistical Analysis
All analyses were performed with the Statistical Package for Social Sciences (SPSS) for Windows 17.0 program. For relationship between FCC and risk of malignancy in this study we were performed with Pear-son's χ2 test.

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Table 1: Results of Chi-Square Test for Differences in staining between group 1 and group 2.

Of the 163 tumor tissues of Group 1, 93 were diagnosed with invasive carcinoma NST, 57 were diagnosed with invasive lobular carcinoma, 4 were diagnosed with intraductal papillary carcinoma, 4 were diagnosed with medullary carcinoma, 3 were diagnosed with mucinous carcinoma and 2 were diagnosed with metastatic carcinoma. Bidirectional chi-square test was used to reveal the significant difference between tumor types with FCC and staining scores. It was found that there was no significant difference between tumor types with FCC and staining scores (χ² = 11,612; p =0.708). Results related to the analyses are given in Table 2.

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Table 2: Chi-Square Test Results for the Difference between Tumor Types and Staining Degrees IC-NST: Invasine Carcinoma NST, ILC: Invasive Lobuler Carcinoma, IDPC: Intraductal Papillary Carcinoma, MUC: Mucinous Carcinoma, MEC: Medullary Carcinoma, MTC: Metaplastic Carcinoma.

In areas of fibrocystic changes positive staining was detected in the cyst epithelium of 4 cases and in ductal adenosis areas in 3 cases.

There are different opinions on whether fibrocystic changes are related to malignancy ²¹. Fibrocystic changes are found in the lower-category benign breast disease (LC-BBD) group according to the classification of the Cancer Committee of the College of American Pathologists, and in this group of diseases (Adenosis, ductal ectasia, fibroadenoma without complex features, fibrosis, mastitis, mild hyperplasia without atypia, ordinary cysts, simple apocrine metaplasia, squamous metaplasia) increased risk of malignancy has not been reported ^12,13. Likewise, Moinfar ²² reported in his book Essentials of Diagnostic Breast Pathology that fibrocystic disease is not associated with increased risk of cancer. On the other hand, in a study conducted on 11307 female patients by Wang et al. ^15, LC_BBD was diagnosed in 1376 cases and cyst was detected in 674 of them. Invasive breast carcinoma developed in 264 of 674 patients with cyst diagnosis and risk of developing malignancy for cysts was determined as 1.79, while this risk was reported to be 1.42 for other LC-BBD. An elevated risk of breast cancer associated with a diagnosis of a cyst has been indicated by studies of case series ^23-25. In a cytogenetic analysis of 69 FCC cases and 10 normal breast tissues by Lundin et al. ^26, clonal chromosome aberration was detected in 6 FCC cases, but all normal tissues were found to have normal karyotypes. The frequency of chromosomal abnormalities found in BBD, although lower than in breast carcinoma, correlates with the corresponding risk of developing invasive carcinoma.

The molecular identification of high risk breast lesions could improve the effect of current preventive strategies and the application of selected therapeutic interventions ^27,28. According to the field cancerization hypothesis, as widely reported in head and neck cancer ^27,29,30, it is likely that benign breast tumors, precursors for invasive cancer and the normal appearing peritumoral tissue, may harbor molecular changes heralding early stages of cancer development ^26,31. Moreover, the benign parenchyma of cancer-containing breasts and the contralateral normal epithelium in patients who experienced cancer in one breast, can share the same pattern of chromosomal abnormalities with invasive carcinoma ³².

HER-2 gene amplification is considered to contribute to the development of breast carcinoma, i.e. carcinogenesis ^13,14. In a HER-2 study performed by Rohan ³³ et al to determine the risk of cancer, HER-2 staining was performed by immunohistochemical methods on 74 female patients who were diagnosed with BBD and who subsequently developed malignancy and 309 female patients who did not develop malignancy, and HER-2 staining was not associated with malignancy development. In a study by Gusterson et al. ³⁴ on 150 BBDs, none of the 22 FCC cases was reported for HER2 staining.

In this study that we aim to determine the risk of malignancy development in patients with fibrocystic changes using the HER-2 gene, it was found that there was a significant difference in the degree of staining in areas of FCC with breast tumor cases. HER-2 was positive in 7 of the FCC cases with malignancy, but no HER-2 positivity was found in any of the FCC without breast carcinoma. There was a significant difference between FCC with breast tumor cases and without tumor in terms of staining scores and it was found that HER-2 staining was more frequent in FCC areas with tumors, (χ² = 37,079; p <0,05).

Ordinary apocrine cells are an important constituent feature of FCC. Several studies have analyzed the possible relationship between apocrine change in FCC and breast carcinoma. Thus, Foote and Stewart in a study of 300 breast cancer specimens and 200 noncancerous specimens, analyzed the frequency of apocrine epithelium ³⁵. They found no significant difference in the frequency of apocrine change. Their conclusion was that apocrine metaplasia is unlikely a precursor of breast cancer, given the high prevalence of this feature. Furthermore, Wellings and Alpers ³⁶ in their subgross analysis of 186 autopsy breast tissue specimen and 107 breast cancer specimens, failed to demonstrate a continuous spectrum of apocrine metaplasia to overt carcinoma. However, in this study, no staining was observed in apocrine metaplasic areas. In areas of fibrocystic, positive staining was detected in the cyst epithelium in 4 of the samples, in ductal adenosine areas in 3 of the samples. We were convinced that apocrine metaplasia has a lower risk of malignancy than cyst formation and ductal adenosis.

In this study that we conducted on FCC with tumor cases and FCC without malignancy. The number of cases was relatively low. Performing these studies in larger groups will increase their significance. In this study, we found out that HER 2 staining was significantly more positive in FCC with malignancy than without malignancy and that the risk of malignancy was higher in HER-2 positive cases by immunohistochemistry. Early diagnosis is very important in breast carcinoma. Screening programs in our country are quite common. However, these programs pose a financial burden. We therefore believe that HER-2 staining for breast biopsies will be helpful for determining malignancy potential in FCC of the breast, and that positive staining will be critical about adjustment of patient follow-up frequency according to this potential.

1) Bartow SA, Pathak DR, Black WC, Key CR, Teaf SR. Prevalence of benign, atypical, and malignant breast lesions in populations at different risk for breast cancer. A forensic autopsy study. Cancer 1987; 60: 2751- 60.

2) Fiorica JV. Fibrocystic changes. Obstet Gynecol Clin North Am 1994; 21: 445-52.

3) Hutter RVP. Consensus meeting. Is fibrocystic disease of the breast precancerous. Arch Pathol 1986; 110: 171-3.

4) Hockenberger SJ. Fibrocystic breast disease: every woman is at risk. Plast Surg Nurs 1993; 13: 37-40.

5) Vorherr H. Fibrocystic breast disease: pathophysiology, pathomorphology, clinical picture, and management. Am J Obstet Gynecol 1986; 154: 161-79.

6) Wellings SR, Alpers CE. Apocrine cystic metaplasia: subgross pathology and prevalence in cancer-associated versus random autopsy breasts. Hum Pathol 1987; 18: 381-6.

7) Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement. Am J Clin Pathol 2000; 113: 251-8.

8) Couturier J, Vincent-Salomon A, Nicolas A et al. Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 (HER-2/neu) gene status in breast carcinoma. Mod Pathol 2000; 13: 1238-43.

9) Ridolfi RL, Jamehdor MR, Arber JM. HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach. Mod Pathol 2000; 13: 866-73.

10) Hartmann LC, Sellers TA, Frost MH et al. Benign breast disease and the risk of breast cancer. N Engl J Med 2005; 353: 229–37.

11) Wolff AC, Hammond ME, Schwartz JN et al. American Society of Clinical Oncology; College of American Pathologists. American Society of Clinical Oncology/College of American Pathologists Guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007; 25: 118-45.

12) Hutter RVP. Consensus meeting: is“fibrocystic disease” of the breast precancerous? Arch Pathol Lab Med 1986; 110: 171–83.

13) Fitzgibbons PL, Henson DE, Hutter RVP. Benign breast changes and the risk for subsequent breast cancer. Arch Pathol Lab Med 1998; 122: 1053–5.

14) Gail MH, Brinton LA, Byar DP et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually. J Natl Cancer Inst 1989; 81: 1879-86.

15) Wang J, Costantino JP, Tan-Chiu E, Wickerham DL, Paik S, Wolmark N. Lower category benign breast disease and the risk of invasive breast cancer. J Natl Cancer Inst 2004; 96: 616-20.

16) Carter CL, Corle DK, Micozzi MS, Schatzkin A, Taylor PR. A prospective study of the development of breast cancer in 16692 women with benign breast disease. Am J Epidemiol 1988; 128: 467-77.

17) London SJ, Connolly JL, Schnitt SJ, Colditz GA. A prospective study of benign breast disease and the risk of breast cancer. JAMA 1992; 267: 941-4.

18) Schnitt SJ. Benign breast disease and breast cancer risk: potential role for antiestrogens. Clin Cancer Res 2001; 7: 4411-22.

19) Melton LJ 3rd. The threat to medical records research. N Engl J Med 1997; 337: 1466-70.

20) Page DL, Dupont WD, Rogers LW, Rados MS. Atypical hyperplastic lesions of the female breast: a long-term follow-up study. Cancer 1985; 55: 2698-08.

21) Youngson BJ, Mulligan AM. Fibrocystic Change and Columnar Cell Lesions. Breast Pathology. In: Goldblum; editor. Philadelphia; Elsevier; 2011; pp 149-58.

22) Moinfar F. Fibrocystic Change. Essentials of Diagnostic Breast Pathology In: Moinfar F; editor. Berlin; Springer; 2007; pp 16-17.

23) Jones BM, Bradbeer JW. The presentation and progress of macroscopic breast cysts. Br J Surg 1980; 67: 669–71.

24) Ciatto S, Biggeri A, Del Turco MR, Bartoli D, Iossa A. Risk of breast cancer subsequent to proven gross cystic disease. Eur J Cancer 1990; 26: 555–7.

25) Bundred NJ, West RR, Dowd JO, Mansel RE, Huges LE. Is there an increased risk of breast cancer in women who have had a breast cyst aspirated? Br J Cancer 1991; 64: 953–5.

26) Lundin C, Mertens F. Cytogenetics of benign breast lesions. Breast Cancer Res Treat 1998; 51: 1–15.

27) Peto R, Boreham J, Clarke M, Davies C, Beral V. UK and USA breast cancer deaths down 25% in year 2000 at ages 20–69 years. Lancet 2000; 355:1822.

28) Etzioni R, Urban N, Ramsey S, McIntosh M, Schwartz S, Reid B. The case for early detection. Nat Rev Cancer 2003; 3: 243 –52.

29) Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A genetic explanation of Slaughter’s concept of field cancerization: Evidence and clinical implications. Cancer Res 2003; 63: 1727 –30.

30) Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, Kummer JA, Leemans CR, Braakhuis BJ. Genetically altered fields as origin of locally recurrent head and neck cancer: A retrospective study. Clin Cancer Res 2004; 10: 3607–13.

31) Viola M, van Houten M, Leemans CR et al. Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: A prospective study. Clin Cancer Res 2004; 10: 3614–20.

32) Botti C, Pescatore B, Mottolese M et al. Incidence of chromosomes 1 and 17 aneusomy in breast cancer and adjacent tissue: An interphase cytogenetic study. J Am Coll Surg 2000; 190: 1–10.

33) Rohan TE, Hardwick W, Miller AB, Kandel RA. Immunohistochemical detection of c-erbb-2 and p53 in benign breast disease and breast cancer risk. Nat Cancer Inst 1998; 90: 1262-9.

34) Gusterson BA, Machin LG, Gullick WJ et al. CerbB-2 expression in benign and malignant breast disease. Br J Cancer 1988; 58: 453-7.

35) Foote FW, Stewart FW. Comparative studies of cancerous versus non-cancerous breasts. Basic morphologic characteristics. Ann Surg 1945; 121: 6-53.

36) Wellings SR, Alpers CE. Apocrine cystic metaplasia: subgross pathology and prevalence in cancer-associated versus random autopsy breasts. Hum Pathol 1987; 18: 381-6.