HER2/neu Amplification in Breast Cancer Stratification by Tumor Type and Grade

Anatomic Pathology / HER2/NEU AMPLIFICATION IN BREAST CANCER HER2/neu Amplification in Breast Cancer Stratification by Tumor Type and Grade Elise R. Hoff, MD, Raymond R. Tubbs, DO, Jonathan L. Myles, MD, and Gary W. Procop, MD Key Words: Breast cancer; HER2/neu; c-erbb-2 Abstract The presence of HER2/neu gene amplification is prognostically and therapeutically significant for patients with breast cancer. We sought to determine whether a relationship exists between HER2/neu gene amplification and the histologic type and grade of tumor. The histologic features and corresponding HER2/neu amplification results of 401 cases of invasive breast carcinoma were reviewed. Lobular carcinomas were less likely than ductal carcinomas to have HER2/neu amplification. Amplification was less frequent in Scarff-Bloom-Richardson grade 1 ductal carcinomas than in grades 2 and 3. Metastatic carcinomas frequently displayed HER2/neu amplification (6/20 [30%]). Our results support a correlation between HER2/neu amplification and the histologic type and grade of breast cancer. We suggest reexamination of tumors diagnosed as Scarff-Bloom- Richardson grade 1 invasive ductal carcinomas or lobular carcinomas if the lesion displays HER2/neu amplification to assure the exclusion of a higher grade of lesion or of missed ductal components. The annual incidence of breast carcinoma in the United States in 2000 was estimated at 182,000 cases, and the annual number of deaths was estimated at 41,000 persons. 1 Although mortality rates declined during the mid-1990s, breast cancer remains a leading cause of cancer death among women, second only to lung cancer. Morphologic variables, such as tumor size, grade, and metastases to regional lymph nodes, are important prognostic indicators for patients with breast cancer. In addition, amplification of the HER2/neu proto-oncogene, which has been reported to occur in 10% to 34% of invasive breast carcinomas, also has been shown to be of both prognostic and therapeutic significance. 2,3 This molecular variable may be used to guide therapy and to stratify patients into clinically relevant risk groups. Detection of the amplification of the HER2/neu oncogene or the expression of the protein product it encodes is now performed widely in the United States. HER2/neu testing is performed routinely on all invasive breast carcinomas at the Cleveland Clinic Foundation, Cleveland, OH. We sought to determine whether a relationship exists between the presence of HER2/neu amplification and the type of breast cancer (ie, invasive ductal carcinoma or invasive lobular carcinoma) and the Scarff-Bloom-Richardson (SBR) grade for invasive ductal carcinomas. Materials and Methods Clinical Samples The files of the Cleveland Clinic Foundation were searched for all cases of invasive breast carcinoma that were diagnosed between July 1999 and July 2000, on which fluorescent in situ 916 Am J Clin Pathol 2002;117:916-921 American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE hybridization was performed for the HER2/neu gene. We identified 401 cases. Of these, 388 were diagnosed as invasive ductal, invasive lobular, or metastatic breast carcinoma. The remaining 13 consisted of special-type tumors, such as tubular, medullary, inflammatory, secretory, and colloid carcinomas; HER2/neu results on these tumors were recorded, but statistical analysis was not performed because of the low number of each tumor type. Histologic assessment of tumor type and modified SBR grading were routinely performed on 5-µm-thick, H&E-stained sections of the formalin-fixed, paraffin-embedded tumors by the attending pathologist. For this study, all tumors were assigned to 1 of 3 groups: invasive ductal carcinoma, invasive lobular carcinoma, or metastatic breast cancer. Invasive ductal carcinomas were further separated into 3 subgroups based on SBR grade (SBR grade 1, SBR grade 2, or SBR grade 3). The metastatic carcinomas were analyzed together as a group, regardless of type or grade of the primary lesion, which was not always known. Cases designated as invasive lobular carcinoma included tumors that demonstrated complete lack of duct formation and had typical lobular features; pleomorphic lobular carcinomas also were included in the invasive lobular carcinoma subgroup. In Situ Hybridization Unstained sections on electrostatically charged slides were heated for 30 minutes at 60 C, then deparaffinized in 2 changes of xylene (5 minutes each), followed by 2 changes of absolute ethanol (1 minute each). The sections then underwent cell conditioning in a 95 C water bath, immersed in a target-retrieval solution (DAKO, Carpinteria, CA) for 40 minutes. After cooling at room temperature for 20 minutes, they were rinsed in distilled water and digested with Proteinase K (150 µl diluted 1:5,000 in 50 mmol of tris(hydroxymethyl)aminomethane hydrochloride, ph 7.6, DAKO). Enzymatic action was stopped with distilled water rinses. After dehydration in graded alcohol, digoxigeninlabeled HER2/neu probe (10 µl, Ventana, Tucson, AZ) was applied to the sections, which then were heated at 90 C for 6 minutes to allow for denaturing of DNA. Hybridization of probe and target tissue DNA took place overnight in a 37 C incubator. Stringency washes of 0.5 standard saline citrate for 5 minutes at 72 C followed. The slides then were washed in 1 phosphate-buffered saline containing 0.1% polysorbate-20 for 5 minutes, after which fluorescein-labeled antidigoxigenin antibody was applied. The slides then were counterstained with 20 µl of 4',6-diamidino-2-phenylindole in antifade solution. Signals were visualized on an Axioskop (Zeiss, Oberkochen, Germany). Gene copies were counted in 2 preselected fields, 20 nuclei in each field, for a total of 40 nuclei. Amplification was recorded as absent (1-4 gene copies) or amplified (>4 copies). Statistical Methods The following parameters were determined for each of the histologic subgroups: frequency of HER2/neu gene amplification, mean number of gene copies, and range of gene copy number for cases with HER2/neu amplification. To further stratify the data, the number of cases in each category was separated into cases with 5 to 10 signals per nucleus and those with more than 10 signals per nucleus. A comparison of the frequency of HER2/neu amplification with the type and grade of carcinoma was performed, using the chi-square test with Yates correction. Results A total of 388 cases of invasive ductal carcinoma, invasive lobular carcinoma, and metastatic breast cancer were identified. Of these, 300 were invasive ductal carcinomas (SBR grade 1, 73; SBR grade 2, 106; SBR grade 3, 121), 68 were invasive lobular carcinomas, and 20 were metastatic tumors (3 to axillary lymph nodes, 8 to the chest wall, 4 to bone, 2 to lung, and 3 to the liver). Only 1 (<1%) of 73 SBR grade 1 invasive ductal carcinomas demonstrated HER2/neu amplification compared with 17.0% (18/106) of the SBR grade 2 and 23.1% (28/121) of the SBR grade 3 invasive ductal carcinomas Image 1, Image 2, Image 3, and Image 4. Only 2 (3%) of 68 invasive lobular carcinomas demonstrated HER2/neu amplification. The metastatic carcinomas, although limited in number in this review (n = 20), demonstrated the highest frequency of amplification: 30% (6/20). None of the 13 special-type tumors, which consisted of tubular, medullary, inflammatory, secretory, and colloid carcinomas, demonstrated HER2/neu gene amplification. Comparison of these frequencies using the chi-square test with Yates correction revealed statistically significant differences in the frequencies of amplification between invasive lobular (2/68) and invasive ductal carcinomas (47/300) (P <.005). Significant differences also were found between the frequency of amplification in SBR grade 1 invasive ductal carcinomas compared with SBR grade 2 and grade 3 invasive ductal carcinomas (P <.005 and P <.001, respectively). There was no significant difference in HER2/neu amplification between SBR grade 2 and SBR grade 3 invasive ductal carcinomas. Of 18 SBR grade 2 invasive ductal carcinomas that demonstrated HER2/neu amplification, 6 (33%) had 5 to 10 copies per nucleus, while 12 (67%) had more than 10 copies per nucleus. Of 28 SBR grade 3 invasive ductal carcinomas that demonstrated HER2/neu amplification, 13 (46%) had 5 to 10 copies per nucleus, while 15 (54%) had more than 10 copies per nucleus. Of 6 metastatic carcinomas, 4 (67%) had more than 10 copies per nucleus, American Society for Clinical Pathology Am J Clin Pathol 2002;117:916-921 917

Hoff et al / HER2/NEU AMPLIFICATION IN BREAST CANCER Image 1 Typical low-grade (Scarff-Bloom-Richardson grade 1) invasive ductal carcinoma demonstrating prominent tubule formation, low nuclear grade, and few to absent mitoses (H&E, 400). Image 2 Lack of amplification of the HER2/neu gene was seen typically in low-grade invasive ductal carcinomas (2 gene copies per nucleus) ( 1,000). Image 3 Typical high-grade (Scarff-Bloom-Richardson grade 3) invasive ductal carcinoma demonstrating lack of tubule formation, high nuclear grade, and prominent mitoses (H&E, 400). Image 4 Fluorescence in situ hybridization image of a high-grade (Scarff-Bloom-Richardson grade 3) carcinoma showing presence of amplification of the HER2/neu gene. The large green signals represent the blurring of signals that occurs when large numbers of copies are present in proximity to each other ( 400). while the remaining 2 (33%) had only 5 to 10 copies per nucleus. The single SBR grade 1 invasive ductal carcinoma that demonstrated HER2/neu amplification had 19.4 copies per nucleus. The 2 lobular carcinomas that had amplification of the HER2/neu gene both had 5 to 10 copies per nucleus. The mean and range of HER2/neu copy numbers for cases with gene amplification are given in Table 1. Because only 2 of 68 invasive lobular carcinomas demonstrated HER2/neu amplification, the lobular carcinomas were reexamined to exclude the possibility of a misclassification. One of these cases was a pleomorphic variant of lobular carcinoma, with a nuclear grade of 3/3. The other case was reexamined by 3 pathologists with expertise in breast pathology and determined to represent not an 918 Am J Clin Pathol 2002;117:916-921 American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE Table 1 HER2/neu Amplification Stratified by Tumor Type and Grade No. of Cases No. of Cases With Mean (Range) of Gene No. (%) of Cases With 5-10 Copies >10 Copies Copy Nos. in Cases Tumor Type No. of Cases With Amplification per Nucleus per Nucleus With Amplification Ductal (SBR grade) 1 73 1 (1) 0 1 2 106 18 (17.0) 6 12 14 (6 to 20.0) 3 121 28 (23.1) 13 15 11.6 (5.1 to >20.0) Lobular 68 2 (3) * 2 0 6.4 (6.2 to 6.7) Metastatic 20 6 (30) 2 4 12.7 (5.2 to 19.0) Total 388 55 (14.2) 23 32 SBR, Scarff-Bloom-Richardson. * Reexamination of the histopathologic features of the 2 cases of lobular carcinoma that demonstrated HER2/neu amplification revealed one to be a pleomorphic variant of lobular (nuclear grade 3/3), while the other had been misclassified; it was determined to be an invasive ductal carcinoma, SBR grade 2. Therefore, 1/67 (1%) of invasive lobular carcinomas demonstrated HER2/neu amplification. A recalculation because of the reclassified case was not performed, since significant differences already had been established. invasive lobular carcinoma but rather an invasive ductal carcinoma (SBR grade 2); rare ductal structures were identified. Based on this reassessment, we believe only 1 (<1%) of the now 67 cases of invasive lobular carcinoma, a pleomorphic variant of lobular carcinoma, actually had amplification of the HER2/neu oncogene. In a similar manner, the 1 SBR grade 1 invasive ductal carcinoma that demonstrated HER2/neu amplification was reassessed to ensure the exclusion of a higher-grade lesion. In this case, however, all 3 pathologists agreed with the original diagnosis. Discussion HER2/neu is a proto-oncogene located on the long arm of chromosome 17. 4 Many adult tissues, including breast, endometrium, prostate, and ovary, normally express low levels of the protein encoded for by this gene. Amplified levels of this gene and its protein product have been found in between 10% and 35% of invasive breast carcinomas. 2 HER2/neu amplification in breast cancer has been associated with a number of adverse outcomes, including decreased overall and disease-free survival, especially for patients with disease metastatic to lymph nodes, 5-7 Because of its numerous adverse associations, HER2/neu amplification status has become an increasingly important and reliable predictor of patient outcome, and testing for this variable is now widely performed. Determination of this variable also has become an important aid in the determination of which patients will be candidates for the new anti-her2/neu drug, trastuzumab (Herceptin), which has been reported to be of benefit to patients with breast cancers that overexpress HER2/neu. 8-10 At our institution, all cases of invasive breast carcinoma are tested for HER2/neu amplification. In this retrospective review, we sought to ascertain whether significant differences exist between the type of breast cancer, the grade of invasive ductal carcinomas, and the HER2/neu amplification status. We found that overall, invasive ductal carcinomas were significantly more likely to show HER2/neu amplification than were invasive lobular carcinomas (P <.005). In addition, higher grade invasive ductal carcinomas (SBR grades 2 and 3) were more likely to demonstrate HER2/neu amplification (17.0% and 23.1%, respectively) than lower grade (SBR grade 1) ductal carcinomas (1%). These differences were statistically significant (P <.005 and P <.001, respectively). The fact that most of the invasive lobular carcinomas (66/67 [99%]) and low-grade (SBR grade 1) invasive ductal carcinomas (72/73 [99%]) in our study lacked HER2/neu amplification suggests that amplification is highly unlikely in these types of carcinomas. The presence of HER2/neu amplification in pleomorphic lobular carcinoma was less surprising, given its high nuclear grade. In this study, we identified a tumor that demonstrated HER2/neu amplification; the tumor originally was suspected to be a lobular carcinoma, but on further study was found to be an invasive ductal carcinoma, SBR grade 2. Therefore, the presence of HER2/neu amplification in an invasive lobular carcinoma or an SBR grade 1 invasive ductal carcinoma should prompt reevaluation of the tumor to exclude the possibility of misclassification. This low frequency of amplification among the lobular carcinomas in our study correlates with results found by Porter et al, 11 who examined c-erbb-2 expression in cases containing in situ and invasive lobular carcinomas. c-erbb-2 expression was found in none of their 15 cases containing invasive lobular carcinoma, and it was present in only 1 of the 57 cases with in situ lobular carcinoma. This case was described as having only weak staining, which may not have represented true amplification. 11 Similarly, Rosenthal et al 12 also found that invasive lobular carcinomas were much less likely than invasive ductal carcinomas to demonstrate American Society for Clinical Pathology Am J Clin Pathol 2002;117:916-921 919

Hoff et al / HER2/NEU AMPLIFICATION IN BREAST CANCER HER2/neu amplification (13% compared with 48%). The lobular carcinomas they tested, however, demonstrated a much higher frequency of amplification than the lobular carcinomas in the present review: 13% compared with 1% (1/68). Rosenthal et al 12 also found HER2/neu amplification to be as significant an adverse prognostic factor among the lobular carcinomas as it was among the ductal carcinomas. Other investigators also have reported similar differences in rates of HER2/neu amplification between ductal and lobular carcinomas. 13-16 In contrast, however, Rosen et al, 17 in a study of HER2/neu expression and tumor phenotype, reported HER2/neu amplification in ductal and lobular carcinomas, and they found almost equal rates of amplification in these groups: 49% in ductal carcinomas and 43% in lobular carcinomas. These authors did not find a relationship between the grade of tumor differentiation and HER2/neu expression. The reason for the higher percentage of amplified lobular carcinomas in the study by Rosen et al 17 compared with ours may be related to the different methods used (immunohistochemical analysis vs fluorescent in situ hybridization) and to the criteria they used to define positivity: 25% or more of carcinoma cells showing membrane immunoreactivity. Some authors may consider this cutoff value as too low to qualify for overexpression of HER2/neu. 18 Therefore, the higher percentage of amplified lobular carcinomas found by Rosen et al 17 may have been due to overinterpretation of the immunohistochemical staining, resulting in the inclusion of possible false-positive results. Other studies have compared HER2/neu expression with the tumor grade of invasive ductal carcinomas. 14,15,17,19-22 In a 1991 study by Rilke et al, 21 consisting of 1,210 patients, amplification was found in 3.9% of grade 1 carcinomas, 20.4% of grade 2 carcinomas, and 38.9% of grade 3 carcinomas. Similarly, Tsuda et al 19 found c-erbb-2 amplification in 33% of grade 3 invasive ductal carcinomas, 10% of grade 2 carcinomas, and 0% of grade 1 carcinomas. The values from these studies are similar to those found in the present study. These data support the existence of a correlation between HER2/neu amplification and both tumor type and histologic grade of the invasive ductal carcinoma. Only 1 of our grade 1 ductal carcinomas and 1 of the lobular carcinomas, a pleomorphic variant, demonstrated HER2/neu amplification; none of the nonpleomorphic variants of lobular carcinoma demonstrated HER2/neu amplification. Interestingly, one of the carcinomas that was diagnosed originally as an infiltrating lobular carcinoma but displayed HER2/neu amplification was found to be an invasive ductal carcinoma on review. Therefore, we suggest that if HER2/neu amplification is present in SBR grade 1 invasive ductal carcinomas or in invasive lobular carcinomas, reexamination of the morphologic features of the neoplasm should be performed to confirm the tumor type and grade as a matter of quality assurance. From the Departments of Anatomic and Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH. Address reprint requests to Dr Procop: Dept of Pathology, MailStop L40, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. References 1. American Cancer Society. Facts and Figures, 2000 [pamphlet]. Baltimore, MD: Williams & Wilkins; 2000. 2. Ross JS, Fletcher JA. Her2/neu (c-erbb-2) gene and protein in breast cancer. Am J Clin Pathol. 1999;112(suppl 1):S53-S67. 3. Bloom HJG, Richardson WW. Histological grading and prognosis in breast cancer: a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957;11:359-377. 4. Schecter AL, Hung MC, Vaidyanathan L, et al. The neu gene: an erbb-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science. 1985;229:976-978. 5. Tandon AK, Clark GM, Chamness GC, et al. Her-2/neu oncogene protein and prognosis in breast cancer. J Clin Oncol. 1989;7:1120-1128. 6. Paik S, Hazen R, Fisher ER, et al. Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: prognostic significance of erbb-2 protein overexpression in primary breast cancer. J Clin Oncol. 1990;8:103-112. 7. Wright C, Angus B, Nicholson S, et al. Expression of c-erbb- 2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res. 1989;49:2087-2090. 8. Vogel C, Cobleigh MA, Tripathy D, et al. First-line, singleagent Herceptin (trastuzumab) in metastatic breast cancer: a preliminary report. Eur J Cancer. 2001;37(suppl 1):S25-S29. 9. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-her2 monoclonal antibody in women who have HER2- overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol. 1999;17:2639-2648. 10. Slamon D, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783-792. 11. Porter PL, Garcia R, Moe R, et al. C-erbB-2 oncogene protein in in situ and invasive lobular breast neoplasia. Cancer. 1991;68:331-334. 12. Rosenthal SI, Depowski PL, Sheenan CE, et al. Her2/neu oncogene amplification detected by fluorescence in-situ hybridization (FISH) in lobular breast cancer [abstract]. Mod Pathol. 1999;12:29A. 13. Van de Vijver MJ, Peterse HL, Mooi WJ, 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;319:1239-1245. 14. Marx D, Schauer A, Reiche C, et al. C-erbB-2 expression in correlation to other biological parameters of breast cancer. J Cancer Res Clin Oncol. 1990;116:15-20. 920 Am J Clin Pathol 2002;117:916-921 American Society for Clinical Pathology

Anatomic Pathology / ORIGINAL ARTICLE 15. Parkes HC, Lillycrop K, Howell A, et al. C-erbB2 mrna expression in human breast tumours: comparison with c-erbb2 DNA amplification and correlation with prognosis. Br J Cancer. 1990;61:39-45. 16. Allred DC, Clark GM, Tandon AK, et al. Her-2/neu in nodenegative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol. 1992;10:599-605. 17. Rosen PP, Lesser ML, Arroyo CD, et al. Immunohistochemical detection of Her2/neu in patients with axillary lymph node negative breast carcinoma: a study of epidemiologic risk factors, histologic features, and prognosis. Cancer. 1995;75:1320-1326. 18. Wang S, Saaboorian MH, Frenkel E, et al. Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridization assays. J Clin Pathol. 2000;53:374-381. 19. Tsuda H, Hirohashi S, Shimosato Y, et al. Correlation between histologic grade of malignancy and copy number of c-erbb-2 gene in breast carcinoma: a retrospective analysis of 176 cases. Cancer. 1990;65:1794-1800. 20. Suzuki M, Okuyama T, Yoshikawa K, et al. Overexpression of p53, c-erbb-2 and epidermal growth factor receptor in human breast carcinomas. Pathol Int. 1996;46:46-53. 21. Rilke F, Colnaghi MI, Cascinelli N, et al. Prognostic significance of Her2/neu expression in breast cancer and its relationship to other prognostic factors. Int J Cancer. 1991;49:44-49. 22. Schimmelpenning H, Eriksson ET, Falkmer UG, et al. Prognostic significance of immunohistochemical c-erbb-2 proto-oncogene expression and nuclear DNA content in human breast cancer. Eur J Surg Oncol. 1992;18:530-537. American Society for Clinical Pathology Am J Clin Pathol 2002;117:916-921 921