Considerable advances in the therapy of breast cancer

HER-2/neu Status in Breast Cancer Metastases to the Central Nervous System Kelly C. Lear-Kaul, MD; Hye-Ryoung Yoon, MD; Bette K. Kleinschmidt-DeMasters, MD; Loris McGavran, PhD; Meenakshi Singh, MD Context. Breast cancer is lethal when it metastasizes; one frequent site for spread is the central nervous system (CNS). Approximately 15% to 3% of breast cancers overexpress the protein HER-2/neu at the primary site, but there are few data on whether metastases from these tumors overexpress HER-2/neu and might be responsive to the potentially toxic anti HER-2/neu immunotherapy (trastuzumab [Herceptin]) used in patients with disseminated disease. Objective. To assess CNS breast cancer metastases for HER-2/neu protein overexpression by immunohistochemistry and gene amplification by fluorescence in situ hybridization (FISH) and to compare the status in primary and metastatic sites in the same patient, whenever possible. Design. Central nervous system breast cancer metastases (n 53) from 33 patients and corresponding primary breast cancer specimens in a subset of these patients (n 12) were retrospectively identified in surgical pathology and autopsy databases. Fluorescence in situ hybridization analysis using PathVysion probes for HER-2/neu and chromosome enumeration probe 17 (CEP 17) and immunohistochemistry using the c-erb-b2 antibody (Dako A485) were compared. Immunohistochemical sections were evaluated by both visual and image analysis techniques. Results. Of 31 cases assessable by FISH, 26% showed gene amplification. One hundred percent concordance for HER-2/neu status was detected between the primary and CNS metastatic lesions in 1 of 1 patients analyzed by FISH; lesser concordance was noted in 12 cases compared by immunohistochemistry. In 9 patients with multiple CNS metastases, FISH showed concordance among different lesions within the same patient. Conclusions. When FISH is the detection method, CNS metastases accurately reflect the HER-2/neu status of the primary tumor. Central nervous system metastases from breast cancer received as surgical specimens can therefore be used to assess HER-2/neu status in patients in whom the primary tumor is unavailable for analysis. (Arch Pathol Lab Med. 23;127:1451 1457) Considerable advances in the therapy of breast cancer have been forthcoming during the past decade. One of the most exciting therapies that has been used is trastuzumab (Herceptin), an immunotherapy-based treatment that is approved for use in patients with metastatic disease whose breast cancers overexpress HER-2/neu protein or have HER-2/neu gene amplification. Clinical trials have revealed benefit in patients treated with trastuzumab in addition to chemotherapy, specifically demonstrating a longer time to disease progression, a longer duration of response, a longer survival, and a reduction in the risk of death. 1 Selection of appropriate patients for treatment is critical because along with the benefits come possible se- Accepted for publication June 2, 23. From the Departments of Pathology (Drs Lear-Kaul, Kleinschmidt- DeMasters, McGavran, and Singh), Pediatrics (Dr McGavran), and Neurology (Dr Kleinschmidt-DeMasters), University of Colorado Health Sciences Center, Denver; and Section of Cytogenetics, Seoul Clinical Laboratories, Seoul Medical Science Institute, Seoul, Korea (Dr Yoon). Dr Yoon is now with the LabGenomics Institute of Medical Genetics, Yongin, South Korea. Presented in part at the 8th Annual Breast Multidisciplinary Symposium, Amelia Island, Fla, February 23. Reprints: Meenakshi Singh, MD, Department of Pathology, Box B216, University of Colorado Health Sciences Center, 42 E 9th Ave, Denver, CO 8262 (e-mail: meenakshi.singh@uchsc.edu). rious side effects, including cardiac dysfunction, leukopenia, anemia, and infection. 1 Breast cancers overexpress HER-2/neu protein in 15% to 3% of cases. 1 5 In breast cancer patients with metastatic disease, testing has predominantly been conducted on the tumor at the primary site, to ascertain which patients are eligible for therapy. It is generally unknown whether there is equal expression or differences of protein expression between multiple metastases, either in the same organ or at different sites, although there have been several recent studies comparing HER-2/neu overexpression and amplification in primary versus metastatic lesions. 6 1 With the exception of a study that focused on axillary lymph nodes as metastatic sites, 1 these reports have not addressed the HER-2/neu status in metastases to specific organs. Almost no information exists on HER-2/neu protein status in brain metastases from breast carcinoma. Often patients with breast cancer develop metastatic disease months or years remote from the occurrence of the primary breast carcinoma. Usually these additional metastatic lesions are not biopsied and hence are not available to the pathologist for HER-2/neu testing. The exception is brain metastases. Metastases from breast cancer to brain and dura are frequent, occurring in up to 3% of patients. 11 Owing to the distinctive anatomy of the central nervous system (CNS), biopsies or resections of metastatic Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al 1451

lesions to brain or dura are more likely to be performed for palliation of symptoms and for pathologic confirmation of diagnosis, thus providing material for additional tests. Indeed, at times, the brain metastases may be the only tissues available to the pathologist for HER-2/neu testing. In this study, we asked the following questions: (1) do brain metastases accurately reflect the HER-2/neu status of the primary tumor, (2) are multiple brain metastases within a single patient concordant or discordant for HER-2/neu expression, and (3) which technical method is optimal for testing these metastases? MATERIALS AND METHODS Formalin-fixed, paraffin-embedded blocks from 33 patients with CNS metastases from breast cancer (n 53) were retrospectively identified in the surgical pathology databases (198 22, inclusive; 14 patients) and the autopsy databases (1983 22, inclusive; 19 patients) from the University of Colorado Health Sciences Center, Denver. If more than 1 metastatic lesion to the CNS was present in the same patient, the 3 largest lesions were selected for evaluation. Tumor material from the breast cancer at its primary site was additionally available for immunohistochemical (IHC) analysis in 12 of the 33 patients. This availability allowed comparison for HER-2/neu status between the metastases and the primary tumor in the same patient in these 12 cases. The time from surgical removal or biopsy of the primary tumor until diagnosis of the brain lesion (at surgery or autopsy) ranged from less than 2 months to 2 years. In 1 patient, the diagnosis of breast cancer was initially made after resection of metastatic CNS lesions. At the time of primary diagnosis, the patients ranged in age from 25 to 62 years, with a mean age of 45 years. In cases in which the surgical pathology reports provided information on the primary carcinoma, tumor size ranged from.6 cm to 6. cm; 18 (9%) lesions were invasive ductal carcinomas, and 2 (1%) were invasive lobular carcinomas. The modified Bloom-Richardson grade of the tumor gathered from these surgical pathology reports was as follows: 43% were poorly differentiated (grade III/III), 43% were moderately differentiated (grade II/III), and 14% were well differentiated (grade I/III) tumors. Immunohistochemistry Immunohistochemical staining for HER-2/neu protein overexpression was performed on 5- m sections using the Dako polyclonal c-erb-b2 oncoprotein antibody A485 at a 1:2 dilution (Dako Corporation, Carpinteria, Calif). A modified HercepTest method (Dako) was used. In brief, the sections were deparaffinized, hydrated, and antigen retrieval was performed in a decloaking chamber at 15 C in citrate buffer for 2 minutes. The slides were then cooled to room temperature, washed with a TRIS-buffer for 3 3 minutes, and peroxidase blocking was performed for 1 minutes. After rewashing, the primary antibody was applied for 1 hour. Following a 3-minute incubation in Dako Envision plus rabbit secondary antibody, the substrate-chromogen solution (diaminobenzidine) was added as a visualization reagent. Finally, the slides were counterstained with hematoxylin. Positive and negative controls were included in each run. Light microscopy evaluation of hematoxylin-eosin stained and immunostained sections was performed. The results of membrane staining were scored using the manufacturer s recommendation. Each sample was placed into 1 of 4 categories (,,, ). Tumors with complete absence of staining or membrane staining in less than 1% of the tumor cells were scored as. Tumors with faint or barely perceptible membrane staining in more than 1% of the cells and cells with only part of the cell membrane staining were scored as. Tumors with a moderate complete membrane staining in more than 1% of the cells were scored as. Tumors with a strong complete membrane staining in more than 1% of the cells were scored as. Tumors with and scores were interpreted as positive for protein overexpression, and tumors with and scores were interpreted as negative for protein overexpression. These results were correlated with image analysis of the immunostained sections performed using the ChromaVision Automated Cellular Imaging System (ACIS; ChromaVision Medical Systems, Inc, San Juan Capistrano, Calif), with scores less than 2. interpreted as negative, and 2. or greater interpreted as positive for protein overexpression. Clinically significant discordance was identified if the results were negative versus positive via visual examination and/or image analysis. Fluorescence In Situ Hybridization Adjacent 5- m sections from each paraffin-embedded block were used for fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification, and hematoxylin-eosin stained slides were used to identify target areas. Fluorescence in situ hybridization was performed as specified by the manufacturer, with some modifications, using the PathVysion system (Vysis, Inc, Downers Grove, Ill). Briefly, the sections were deparaffinized, pretreated in SSC at 75 C for 15 minutes, and digested in.25 mg/ml proteinase K solution at 45 C for 15 minutes; they were then dehydrated through a series of ethanol baths. Before hybridizing with the dual-probe mixture for HER-2/neu gene and alpha-satellite DNA for chromosome 17, probe and target were co-denatured. The locus-specific identifier HER-2/neu DNA probe is a 19-kb SpectrumOrange (Abbott-Vysis, Inc, Abbott Park, Ill) directly labeled fluorescent DNA probe specific for the HER-2/ neu gene locus (17q1-q12). The chromosome enumeration probe (CEP) 17 DNA probe is a 5.4-kb SpectrumGreen (Abbott- Vysis, Inc) directly labeled fluorescent DNA probe specific for the -satellite DNA sequence at the centromere region of chromosome 17 (17p1-q1). After posthybridization washing in M urea/. SSC, ph 7. 7.5 at 45 C for 3 minutes and then in SSC at room temperature for 2 minutes with shaking, the slides were counterstained with 4,6-diaminido-2-phenylindole dihydrochloride (DAPI) and coverslipped. Whenever possible, a minimum of 6 tumor nuclei per slide were scored according to the manufacturer s guidelines. The observers had no knowledge of the results obtained from IHC studies. The samples with small tumor areas having fewer than 6 scorable tumor nuclei were included if the probe signal intensities and background were adequate. HER-2/neu gene copy level was determined as a ratio of HER-2/neu gene copies to chromosome 17 centromere copies. HER-2/neu gene amplification was defined as a ratio of 2. or greater. Results obtained by FISH analysis were compared with those obtained by visual light microscopy and image analysis of the IHC sections. RESULTS Immunohistochemistry Results of immunohistochemistry are detailed in Table 1. Briefly, of the 33 patients with 53 CNS metastases successfully analyzed by IHC with both light microscopic and image analysis scores, the brain metastases showed HER-2/neu protein overexpression in 5% (17 of 33 patients) by light microscopic visual analysis and 42.4% (14 of 33 patients) by image analysis. When comparing visual examination and image analysis, IHC sections showed clinically significant discordance in 6 (1%) of the 53 metastatic lesions. All of the discordant cases belonged to the score category on visual examination. These cases had a range of scores between and 1.9 on image analysis (Figures 1 through 3). Fourteen (42%) of the 33 patients had more than 1 CNS metastasis. In cases in which there was more than 1 metastatic lesion, 78.6% (11/14) demonstrated concordance between HER-2/neu status of the different CNS metasta- 1452 Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al

Table 1. HER-2/neu Status of Central Nervous System Metastases of Breast Cancer* Immunohistochemistry Visual Image Case No. Analysis Analysis Patients with multiple metastases 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b 6a 6b 6c 7a 7b 8a 8b 8c 9a 9b 1a 1b 1c 11a 11b 11c 12a 12b 12c 13a 13b 14a 14b 14c Patients with single metastasis 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 32 33 3.7 3.8.. 5.5 5.5 1.9.8 2.4 2.6.7. 3.6.5.2.... 1.8.1 3.9 3. 5.3 1.7 3.1 1.9 2...1 2.1 4.3. 2.8.. 3.3 2. 1.8 4.8..4 5.3....7...5 FISH 5.4 5.1 1.7 5.8 4.7 1. 2.5 2.4 1.9 15.2 13.5 14.9 1.4 1. 1.7 1. 5.7 1.4 1.4 1. 2.2.9 1.7 * Cases with discrepancies between immunohistochemistry (image analysis) and fluorescence in situ hybridization (FISH) results are highlighted in italic type. Cases showed a subpopulation of cells with HER-2/neu gene amplification. Case showed polysomy of CEP 17. ses on both light microscopic visual analysis and image analysis. In cases in which primary breast tumor material was available (12 [36%] of 33 patients), we found HER-2/neu protein overexpression in 25% (3 of 12 patients) of the primary breast tumors compared with 5% (6 of 12 patients) of the CNS metastases (see Table 2). By visual interpretation, the IHC sections showed clinically significant discordance between the levels of HER-2/neu protein overexpression in the breast tumor versus the CNS metastases in 7 (58.3%) of 12 paired cases. In 3 cases, the breast tumor had protein overexpression but the CNS metastases were negative, whereas in 4 cases the CNS metastases were positive and the breast tumor was negative for protein overexpression. By image analysis, there was discordance in the HER-2/neu status between the primary and metastatic lesions in 5 (41.7%) of 12 patients. Fluorescence In Situ Hybridization Results of FISH analyses are detailed in Table 1. Of the 31 cases successfully analyzed by FISH, 25.8% (8 of 31 patients) demonstrated HER-2/neu gene amplification in the CNS metastases. In the 9 patients in whom more than 1 metastatic lesion was identified, all 9 patients (1%) demonstrated concordance between the HER-2/neu status of the different CNS metastases. In cases in which primary breast tumor material was available (1 [32%] of 31), HER- 2/neu gene amplification was identified in 4% (4 of 1 patients) of the primary breast tumors and their corresponding CNS metastases; none showed discordance in the presence or absence of HER-2/neu gene amplification in the breast tumor versus the CNS metastases (see Table 3). When comparing the image analysis scores of immunohistochemical results for protein overexpression with the FISH data for gene amplification, 86% of lesions showed concordance when comparing individual metastatic lesions (43 lesions total). Five metastatic lesions showed no gene amplification, although IHC indicated protein overexpression, whereas 1 metastatic lesion demonstrated gene amplification by FISH when the IHC was negative (see Table 1). One of the metastatic lesions with positive IHC that was negative by FISH did show amplification of a minor subpopulation of tumor cells within the metastasis; in this unusual metastatic lesion, the number of cells with amplification was 33 of 124 total cells counted. An additional case, which was positive for protein overexpression by IHC yet negative for gene amplification by FISH, showed polysomy of CEP 17 signals (4 of 63 nuclei counted) but no overall HER-2/neu gene amplification. COMMENT In this study, we demonstrated that the HER-2/neu gene is amplified and its protein is overexpressed in breast cancer metastases to the CNS, but the percentages vary considerably. When FISH was used as the testing method, we detected gene amplification in 26% of all CNS metastases in our study. Fluorescence in situ hybridization appears to be the test method that demonstrates optimal correlation for HER-2/neu status between the primary tumor and metastases in the same patient. Using FISH, there was a 1% concordance for the presence or absence of amplification between the primary and metastatic tumors. There was also 1% concordance by FISH for HER-2/neu status Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al 1453

Figure 1. A through C, Case showing concordance between image analysis results and fluorescence in situ hybridization (FISH) analysis of a primary breast carcinoma, in which both studies are negative. A, Invasive ductal carcinoma of the breast (hematoxylin-eosin, original magnification 4). B, Immunohistochemical section in which image analysis was negative for HER-2/neu protein overexpression (score.4; original magnification 4). C, HER2/neu FISH without gene amplification (HER-2/neu copy number of 1.; original magnification 1). D through F, Case showing discordance between image analysis results and FISH analysis in the primary breast carcinoma. D, Invasive ductal carcinoma of breast (hematoxylin-eosin, original magnification 4). E, Immunohistochemical section in which image analysis was negative for HER-2/neu protein overexpression (score ; original magnification 4). F, HER-2/neu FISH with HER-2/neu gene amplification (HER-2/neu copy number of 2.; original magnification 1). between multiple brain metastases within the same patient. Hence, FISH, but not IHC testing of the metastases, appears to accurately reflect the HER-2/neu status of the patient s primary tumor. This capability has practical implications because it allows the pathologist to test brain metastases from a given patient for HER-2/neu status by FISH techniques when the primary tumor tissues are not available for analysis. Conversely, patients with breast cancer who develop metastases to the brain do not have to undergo resection of their brain lesions solely to determine HER-2/neu status, 12 since it appears from our study that the presence or absence of amplification does not change over time between the primary and the metastatic tumor(s), given that the time between the primary and metastatic tumors in our study was as long as 2 years. The duration of formalin fixation for our autopsy and surgical tissues did not appear to negatively influence our IHC or FISH results, despite information in the insert for the Dako HercepTest, which states that formalin fixation time for test tissues should be 18 to 24 hours. Indeed, many of our autopsy tissues (which were often fixed 2 7 days before processing) were positive either by IHC or by FISH. 1454 Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al

Figure 2. A through D, Case in which the immunohistochemical (IHC) image analysis scores of the primary breast carcinoma were discordant from that of the metastasis in the spinal cord dura. A, Breast carcinoma (hematoxylin-eosin, original magnification 4). B, Paired IHC-stained section that was negative for HER-2/neu protein overexpression by image analysis (score of.4; original magnification 4). C, Spinal cord dural metastasis from the same case (hematoxylin-eosin, original magnification 4). D, Paired IHC-stained section of the dural metastasis with HER- 2/neu protein overexpression by image analysis (score of 2.; original magnification 4). Figure 3. A and B, Fluorescence in situ hybridization (FISH) analysis of a case in which the primary breast tumor and the metastasis both showed gene amplification. The green signals represent the CEP 17 probe, and the red signals represent the amplified HER-2/neu signals on chromosome 17 (original magnification 1). A, Breast carcinoma with gene amplification and a HER-2/neu copy number of 4.1. B, Spinal cord dural metastasis of same case as shown in A, with gene amplification and a HER-2/neu copy number of 5.7. Currently, HER-2/neu status testing methodology and algorhythms are not uniform among different laboratories for breast cancer tissue specimens. The HercepTest immunohistochemical method is a relatively easy and inexpensive method to measure HER-2/neu protein overexpression, and it is used widely. It does have a reported higher rate of false-positive and false-negative results compared with FISH analysis. Some of these differences may be attributed to the more subjective interpretation of recommended guidelines for assessing the positive cell membrane staining of HER-2/neu immunohistochemical stains. Image analysis methods for evaluating immunohistochemical stains for HER-2/neu are supposed to provide a more objective method for interpreting stain results, although data on this are somewhat limited. Alternatively, FISH measures gene amplification with a reported higher degree of reproducibility, especially if performed in a central facility, as in many ongoing clinical Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al 1455

Table 2. HER-2/neu Protein Overexpression by Immunohistochemistry [Visual Analysis Score (Image Analysis Score)] in Paired Primary and Metastatic Tumors* Case No. Primary Metastasis a b c 5 (.) (2.4) (2.6) 6 (.) () (.7) (.) 8 (1.9) (.2) (.) (.) 12 (.) (1.7) () (3.1) 13 (.6) (1.9) (2.) 17 (4.) (2.8)...... 21 (.4) (2.) 26 (4.) (5.3) 27 (.) (.) 29 () (.) 3 (2.2) (.7) 31 (.) (.)...... * Discrepant cases are highlighted in italic type. Table 3. HER-2/neu Copy Number by Fluorescence In Situ Hybridization in Paired Primary and Metastatic Tumors Case No. 5 12 13 17 21 26 27 29 3 31 Primary 2.3 4.1 1. 2.6 Metastasis a b c 2.5 1.4 5.7 2.2 2.4 trials. It is, however, more costly, labor-intensive, and needs equipment (fluorescence capable microscopes) that is not widely available in smaller clinical laboratories. In previously published series, the concordance between IHC and FISH for HER-2/neu status in breast cancers at the primary site has ranged from 75% to 96%. 5,13 15 In addition, reportedly 5% to 3% of breast cancers from the primary site exhibit true HER-2/neu protein overexpression without gene amplification, as assessed by combined FISH, IHC, and Western blot analyses. 16 Similar to primary tumors, our study demonstrates that CNS metastases also can show discordance between amplification of the gene and overexpression of the HER-2/ neu protein. Immunohistochemistry showed a higher percentage of protein overexpression in the CNS metastases in our study, with 42% of cases demonstrating protein overexpression by IHC using image analysis techniques, versus 26% that showed gene amplification by FISH. Although the explanation for most of our metastases showing protein overexpression without gene amplification is unknown at this point, we noted a plausible explanation for the discordance in 2 of these patients. Interestingly, in 1 of these cases only a subpopulation of cells within the metastatic lesion demonstrated HER-2/neu gene amplification. In this example, the amplified subset constituted a distinct minority of the cells in the tumor (33 of 124 cells). This heterogeneity for amplification resulted in a score within the negative range for HER-2/neu amplification, using standardized guidelines; the IHC result on this patient was positive. A second patient demonstrated an increased number of CEP 17 signals per cell, that is, polysomy of CEP 17. This provided a HER-2/neu-to-CEP 17 ratio within the normal range and therefore did not demonstrate amplification based on the copy number score, although the IHC result was positive. A concurrent use of CEP 17 probe as an internal control in FISH for HER-2/neu gene amplification can exclude aneuploidy of chromosome 17 as a source of increased HER-2/neu gene copy number. Two of our cases with discrepancies between IHC image analysis and FISH results of the CNS metastases had image analysis scores in the lower positive range (2. and 2.1) and visual analysis results of. Heterogeneity for HER-2/neu status may not be all that surprising. Distant metastases from any type of cancer represent clonal outgrowths with genetic modifications that may not be representative of the entire primary tumor. Cytogenetic analyses have demonstrated profound genetic heterogeneity in breast cancer, 17 with a different genetic composition seen in up to 31% of metastases when compared with the paired primary tumors. 18 In addition, these metastatic lesions were often identified years after resection of the primary cancer, often with intervening treatment of the patient, including chemotherapy, radiation therapy, hormonal manipulation, and bone marrow transplantation. One of the more intriguing aspects of our study was the concordance for HER-2/neu status we found between multiple metastases within the same patient. Given the known postclonal modification that occurs in metastatic lesions, we might have found more variation for HER-2/neu status between multiple metastases or upregulation of HER-2/neu status in the metastases compared to the primary tumor. This did not appear to be the case. It is currently unknown whether tumors that display only one of either HER-2/neu protein overexpression or gene amplification will be equally or less responsive to trastuzumab immunotherapy than tumors in which both tests are positive. Indeed, although use of the drug is currently reserved only for patients with metastatic breast cancers that either overexpress the protein or that display gene amplification, on the average only 1 in 3 of these patients will demonstrate clinical response to the drug. The explanation for why some of these positive patients are nonresponders has not been forthcoming, but heterogeneity in HER-2/neu amplification that we have observed in a subset of metastases is a finding that may warrant further investigation. In addition, further research into mechanisms responsible for HER-2/neu protein overexpression in the absence of gene amplification may also be relevant. Financial support for this research was provided by the Department of Pathology, University of Colorado Health Sciences Center, Denver. The authors thank Carole Witka for manuscript preparation, Norma Aumen for immunohistochemical staining, Lisa Litzenberger and Billie Carstens for photographic expertise, and Lynne Meltesen for assistance with the fluorescence in situ hybridization studies. We also thank Paul Garen, MD, for his helpful comments. References 1. Slamon DJ, 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. 21;344:783 792. 2. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation 1456 Arch Pathol Lab Med Vol 127, November 23 HER-2/neu Status in Breast Cancer CNS Metastases Lear-Kaul et al

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