HER-2 Protein Overexpression in Metastatic Breast Carcinoma Found at Autopsy

HER-2 Protein Overexpression in Metastatic Breast Carcinoma Found at Autopsy Shigeya Kyoda 1, Satoki Kinoshita 2, Hiroshi Takeyama 1, Ken Uchida 2 and Toshiaki Morikawa 2 1 Department of Surgery, Jikei University Daisan Hospital, Komae and 2 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Received November 5, 2007; accepted September 3, 2008; published online October 4, 2008 Jpn J Clin Oncol 2008;38(11)743 747 doi:10.1093/jjco/hyn103 Objective: The overexpression of HER-2 protein has generally been considered to be consistent in primary and metastatic tumor tissues. We evaluated HER-2 protein overexpression levels in 31 autopsied cases. Methods: Hematoxylin eosin staining and immunohistological staining Hercep Test II TM were performed on the primary tumors and the lung, liver, brain and bone metastatic tumors. Results: Nine (29%) of the 31 primary tumors were HER-2 score 3þ and HER-2 score 3þ cases were significantly more frequent in carcinomas of nuclear Grade 3 than in those of Grade 1 or 2. In these 31 patients, the HER-2 status in the primary tumors was consistent with the metastatic foci of the lung, liver, brain and bone in 96% (25 of 26), 91% (21 of 23), 100% (12 of 12) and 85% (11 of 13), respectively. With regard to the nine patients with HER-2 score 3þ primary tumors, the HER-2 status in the primary tumors was consistent with the metastatic foci of the lung, liver, brain and bone in 87% (seven of eight), 78% (seven of nine), 100% (only one) and 33% (one of three), respectively. In 11 (92%) of the 12 patients with brain metastasis, the HER-2 was not overexpressed. Conclusions: Even in the far-advanced stages of autopsy, HER-2 status of the primary tumors appeared to be maintained especially in the foci of the lung, liver and brain metastases. As there was a high degree of agreement in HER-2 status between the primary tumors and the metastatic foci to the lung, liver and brain, it is considered to be reasonable to treat patients with such metastatic foci based on the HER-2 status of the primary tumors. Key words: breast cancer nuclear grade HER-2 protein overexpression autopsied patients INTRODUCTION Since Bloom and Richardson (1) reported the degree of histological malignancy in breast cancer, it has been widely accepted as a prognostic factor and used as one of the determinants for the selection of postoperative adjuvant therapies. At the 9th St Gallen Expert Consensus Meeting in 2005, the overexpression of HER-2/neu oncogene product (HER-2 protein) was added to the breast cancer risk categories (2). The overexpression of HER-2 protein has generally been considered to be consistent in primary and metastatic tumor tissues (3,4). However, only a few studies have HER-2 protein overexpression levels between primary and metastatic breast cancer tissues been compared (5,6) and reports on autopsied patients that would allow searching of systemic For reprints and all correspondence: Shigeya Kyoda, Department of Surgery, Jikei University Daisan Hospital, 4-11-1 Izumi-honchou, Komae, Tokyo 201-8601, Japan. E-mail: vez06074@nifty.ne.jp metastatic foci are hardly available (7). In this study, we evaluated the relationship between the HER-2 score and nuclear grade of primary breast cancers and the overexpression levels of HER-2 protein between primary and metastatic breast cancer tissues. PATIENTS AND METHODS Of 89 autopsies performed on cases with breast cancer at our hospital between January 1980 and December 2001, 31 for whom death from breast cancer was histopathologically confirmed and the paraffin blocks of both primary tumors and metastatic tumors were obtained at the time of autopsy were included in this study. All were females aged 35 71 (median 55 years) at the time of first presentation to our hospital. When surgical therapy was performed to the primary breast cancer, clinical stages of the disease were I in # The Author (2008). Published by Oxford University Press. All rights reserved.

744 HER-2 overexpression metastatic breast cancer Figure 1. (a) Microscopic findings of the primary tumor, HER-2 score 3þ, (b) site of brain metastasis, HER-2 score 1þ (Hercep Test II, TM 100). But, HER-2 gene was amplified by fluorescence in situ hybridization. four (13%), II in eight (26%), IIIa in seven (23%), IIIb in four (13%) and IV in eight (26%). The mean number of the metastasized organs identified at autopsy per patient was 5.3 [standard deviation (SD), 2.5], and mean survival after the start of treatment was 33.9 months (SD, 24.7 months), ranging from 2 to 107 months. Thirty patients died before the clinical use of trastuzumab as the drug was available to only one. The most commonly seen histological type of breast cancer was scirrhous carcinoma (15 cases, 48%), followed by solid-tubular carcinoma (eight cases, 26%), papillotubular carcinoma (five cases, 16%) and others (three cases). Hematoxylin eosin (HE) staining and immunohistological staining using Hercep Test II TM (DAKO, Denmark) were performed on the primary tumors and on the lung, liver, brain and bone metastatic tumors in accordance with the standard testing procedure. HER-2 protein overexpression was scored from 0 to 3þ in accordance with the criteria for evaluation (Fig. 1). Then, HER-2 status was categorized into twogroups:overexpression(her-2score3þ) and nonoverexpression (HER-2 score 2þ, 1þ or 0). For those cases in which the HER-2 status of the primary and each metastatic foci were inconsistent, fluorescence in situ hybridization (FISH) test was conducted. Nuclear atypia and mitotic counts were scored from one to three based on the malignancy criteria specified in the National Surgical Adjuvant Study of Breast Cancer (NSAS-BC) protocol (8 10) and nuclear grades were based on the combined scores. The chi-square test was used for statistical analysis, with the significance level set at 5%. RESULTS CASE DISPOSITION The numbers of primary carcinomas of nuclear Grades 1, 2 and 3 were two (7%), eight (26%) and 21 (67%) (Table 1). The HER-2 score in the primary tumor was 0 in 15 (48%), 1þ in five (16%), 2þ in two (7%) and 3þ in nine (29%). The lung, liver, bone and brain metastases were found on autopsy in 26, 23, 13 and 12 patients, respectively. Metastases to multiple organs occurred in 25 patients. HER-2 STATUS AND NUCLEAR GRADE OF PRIMARY TUMOR The relationship between HER-2 status and nuclear grade is shown in Table 2. In 10 cases, the nuclear grade was 1 or 2 and HER-2 status was termed non-overexpression. Of 21 cases with nuclear Grade 3, the HER-2 status overexpression was found in nine cases (43%). Frequency of the combination of HER-2 status overexpression and nuclear Grade 3 was significantly higher (P ¼ 0.0140). Table 1. Histological features and HER-2 status in 31 primary breast cancers Histological type Papillo-tubular carcinoma 5 Solid-tubular carcinoma 8 Scirrhous carcinoma 15 Others 3 Nuclear grade Grade 1 2 Grade 2 8 Grade 3 21 HER-2 score in primary tumor Score 0 15 Score 1þ 5 Score 2þ 2 Score 3þ 9 Metastatic sites Lung 26 Liver 23 Brain 12 Bone 13

Jpn J Clin Oncol 2008;38(11) 745 Table 2. Relationship between HER-2 status and nuclear grade (31 cases) HER-2 status FREQUENCY OF METASTASIS BY ORGAN Nuclear grade Metastasis was found in the lung, liver, bone and brain in 26 (84%), 23 (74%), 13 (42%) and 12 cases (39%), respectively. It was also seen in the adrenal gland, heart, kidney, ovaries, peritoneum, spleen, skin (local recurrence to skin was excluded from the number) and thyroid in 12, 11, 7, 5, 4, 4, 4 and 4 patients, respectively. HER-2 STATUS IN METASTATIC TUMORS Grade 1,2 Grade 3 Non-overexpression 10 12 Overexpression 0 9 Frequency of the combination of HER-2 status and nuclear Grade 3 was significantly higher (P ¼ 0.0140). The correlations of HER-2 expression status of the primary tumors with metastatic foci of the lung, liver, brain and bone are given in Table 3. In those 31 patients, the HER-2 status in the primary tumors was consistent with the metastatic foci of the lung, liver, brain and bone in 96% (25 of 26), 91% (21 of 23), 100% (12 of 12) and 85% (11 of 13) patients, respectively. HER-2 overexpression was detected in seven of 26 (27%) lung metastatic tumors, seven of 23 (30%) liver metastatic tumors, only one of 12 (8%) brain metastatic tumors and only one of 13 (8%) bone metastatic tumors. The frequency of HER-2 overexpression in the cases with brain or bone metastasis was significantly lower than that in the cases with the lung or liver metastases (Table 3). With regard to the nine patients with HER-2 score 3þ primary tumors, the HER-2 status in the primary tumors was consistent with the metastatic foci of the lung, liver, brain and bone in 87% (seven of eight), 78% (seven of nine), 100% (only one) and 33% (one of three) patients, respectively. In eight of the nine patients, a score of HER-2 3þ was detected not only in the primary tumors but also in any metastatic foci (Table 4). And there were no pairs of primary and metastatic tumors that showed HER-2 overexpression only in the metastatic tumors. For the four patients showing discordance in the HER-2 status between the primary tumors and metastatic foci, HER-2geneamplificationinthesixmetastasisfociwas examined using FISH. Immunohistochemically, HER-2 score in these foci was 0 or 1þ in five and 2þ in one. Of these foci, HER-2 gene amplification was detected by FISH in brain metastatic foci in one patient (Fig. 1). Table 3. Comparison of HER-2 status between primary and metastatic tumors in 31 patients with breast cancers HER-2 score in primary tumor DISCUSSION HER-2 score in lung metastasis Total Number of cases (%) 3þ 0, 1þ, or2þ Lung metastasis 3þ 8 7 (87) 1 (13) 0, 1þ, or2þ 18 0 (0) 18 (100) Total 26 7 (27) 19 (73) Liver metastasis 3þ 9 7 (78) 2 (22) 0, 1þ, or2þ 14 0 (0) 14 (100) Total 23 7 (30) 16 (70) Brain metastasis 3þ 1 1 (100) 0 (0) 0, 1þ, or2þ 11 0 (0) 11 (100) Total 12 1 (8) 11 (92) Bone metastasis 3þ 3 1 (33) 2 (67) 0, 1þ, or2þ 10 0 (0) 10 (100) Total 13 1 (8) 12 (92) Since it was first reported in the Lancet (11), hormone therapy has been used for the treatment of breast cancer for over 100 years. A wide range of chemotherapies for breast cancer has now become available because numerous anti-cancer drugs, such as the taxanes, have been developed. Table 4. HER-2 score in primary and metastatic foci with HER-2 3þ primary tumors Primary Lung Liver Brain Bone 3þ none 1þ!FISH (2) 1þ!FISH (þ) 0!FISH (2) 3þ 3þ 0!FISH (2) None None 3þ 2þ!FISH (2) 3þ None None 3þ 3þ 3þ None 0!FISH (2) 3þ 3þ 3þ None 3þ FISH, fluorescence in situ hybridization. For the four patients showing discordance in the HER-2 status between the primary tumors and six metastatic foci, HER-2 gene amplification was examined by FISH. Five cases showed discrepancy in the HER-2 status, and amplification was detected in the primary tumors and metastatic foci of the brain.

746 HER-2 overexpression metastatic breast cancer HER-2/neu oncogene was first identified in 1984 (12) and HER-2 protein overexpression was found to be correlated with poor breast cancer prognosis in 1987 (13). Since then, HER-2/neu oncogene has received much attention. Clinical use of trastuzumab was started in the USA in September 1998 (National Health Insurance listing of trastuzumab in Japan started in June 2001). Thus, treatment for HER-2 positive breast cancer has dramatically advanced in the past 10 years. Since the publication of a report by Bloom and Richardson (1), the relationship between a higher histological grade that is almost equivalent to nuclear grade of breast cancer and a poorer prognosis has been widely recognized. The proportion of breast cancer with nuclear Grade 3 is said to be 25 67%, in general (14). As the patients of the current study were those subjected to autopsy, as high as 68% of those examined showed nuclear Grade 3. None of the cases with nuclear Grade 1 or 2hadHER-2score3þ, whereas 43% of those with nuclear Grade 3 had HER-2 score 3þ. Of 31 primary tumors, all nine with HER-2 score 3þ showed nuclear Grade 3. These findings suggest that patients with a potentially poor prognosis may be identified based on the combination of nuclear grade and HER-2 score. Nuclear grade was adopted as one of the breast cancer risk categories at the 9th St Gallen Expert Consensus Meeting in 2005 (2), and HER-2/neu was also newly added. This may be attributed to the worldwide recognition of the importance of an HER-2 score as a prognostic factor. As trastuzumab specifically binds to HER-2 protein, a receptor with tyrosine kinase activity that penetrates the cellular membrane, its therapeutic efficacy is largely affected by the overexpression level of HER-2 protein, which is generally seen in 10 30% of primary breast cancers (13,15). The frequency of HER-2 score 3þ in primary tumors in the current study was comparable to 29%. The frequency of HER-2 score 3þ in metastatic tumors was found in 27% in lung metastases and 30% in liver metastases. The HER-2 protein overexpression level is said to be consistent between the primary and metastatic tumors (3,4). In the current study, a high degree of agreement was seen between the HER-2 status of the primary foci and that of each of the metastatic foci of the lung, liver, brain and bone (85 100%). Among them, the frequency of HER-2 protein overexpression was highly consistent between the primary and metastatic tumors, as seen in seven of eight cases with lung metastases (88%), seven of nine with liver metastases (78%) and one brain metastasis (100%). However, the frequency of HER-2 overexpression was as low as 33% in bone metastases. In view of these findings, for those cases with the lung, liver or brain metastasis and HER-2 overexpression, it appears appropriate to make a judgment on the use of trastuzumab based on the HER-2 status of the primary foci in treating these metastatic foci. Although brain metastasis of breast cancer is generally seen in 18 30% of autopsied cases (16,17), the frequency in living patients is relatively low (14 20%) (18,19). However, an increase in brain metastases in patients with HER-2 positive recurrent breast cancer has recently been noted due to prolonged survival associated with the wide use of trastuzumab. Reported incidences of brain metastases are as high as 25 35% (20) and HER-2 positive breast cancer is likely to metastasize to the brain at high frequency (21). In the current study on 31 autopsied cases, 11 of 21 (52%) patients with HER-2 non-overexpression of primary tumors had brain metastases, whereas only one of nine cases (11%) with HER-2 overexpression did. Among the 12 cases with brain metastasis, the primary foci of 11 were HER-2 negative, which suggested that those cells that are characterized by non-overexpression of HER-2 are more likely to cause such metastasis. In conclusion, even in the far-advanced stages of autopsy, HER-2 status of the primary tumors appeared to be maintained, especially in the foci of the lung, liver and brain metastases. Because there was a high degree of agreement in HER-2 status between the primary tumors and the metastatic foci of the lung, liver and brain, it is considered to be reasonable to treat patients with such metastatic foci based on the HER-2 status of the primary tumors. Acknowledgements The authors express their gratitude to Ms Michiko Kasai, Ms Naoko Ikeda and the staff of the Department of Pathology, The Jikei University School of Medicine who participated in the preparation of the specimens. Conflict of interest statement None declared. References 1. Bloom HJG, Richardson WW. 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