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HER2: biology, detection, and clinical implications.

Human epidermal growth factor receptor 2 (ERBB2, formerly HER2/neu, c-erbB2), 1 of a family of 4 membrane tyrosine kinases (TKs), was found to be amplified in a human breast cancer cell line 25 years ago, (1) and this amplification was shown to be important in the pathogenesis and progression of human breast cancer 2 years later. (2) Since that time, HER2 amplification and resultant HER2 protein overexpression have been linked to important tumor cell proliferation and survival pathways, several drugs have been developed to target the pathway, and the detection of HER2 has become a routine prognostic and predictive factor in breast cancer.

The aim of this review is to highlight important aspects of the biology of HER2, the current standards for its detection, and the clinical importance of its assessment.

BIOLOGICAL SIGNIFICANCE OF HER2

The estrogen receptor (ER) and the HER2 (c-erbB2, HER2/neu) signaling pathways are the dominant drivers of cell proliferation and survival in most (85%) breast cancers. Targeting these pathways provides the most effective therapy in appropriately selected patients. Endocrine therapy to target ER and trastuzumab to target HER2 provide striking disease-free and overall survival benefits in the adjuvant setting when micrometastatic disease is present (50% reduction in risk of recurrence). (3-6) Remissions sometimes lasting years, although temporary, are seen in patients with metastatic disease treated with ER- and HER2-targeted therapy, the latter usually combined with chemotherapy. (7)

The HER2 pathway has been described in systems biology terms as a complex biological network composed of 3 layers: an input layer of membrane receptors and their ligands to trigger the signal coming from outside the cell; a core system processing layer of protein kinases transmitting the signal to the nucleus; and an output layer of transcription factors regulating genes that affect various cellular functions (8) (Figure 1). In turn, the genes and gene products regulating the activity of the pathway have been and are being defined. The input layer is composed of 4 membrane receptors/TKs (HER1-4) and their many ligands (at least 11) (Table 1). (8) In breast cancer, HER2 is the dominant TK receptor, being amplified in 20% of cases. (2) On ligand binding to their extracellular domains, HER proteins undergo dimerization and transphosphorylation of their intracellular domains. HER2 does not have a ligand and relies on heterodimerization with another family member or homodimerization with itself when expressed at very high levels to be activated. These phosphorylated tyrosine residues dock with numerous intracellular signaling molecules leading to activation of downstream second messenger pathways and crosstalk with other membrane signaling pathways. (8-12) Transcription factors activated by the pathway regulate many genes involved in cell proliferation, survival, differentiation, angiogenesis, and invasion and metastasis. HER2 has the strongest catalytic kinase activity and HER2-containing heterodimers have the strongest signaling activity. (8,12-14) HER2 exists in an open conformation exposing its dimerization domains making it the dimerization partner of choice among the family members. HER3 is activated by ligand (heregulin) binding but it lacks TK activity and, like HER2, must partner with another family member to be activated. Nevertheless, it has multiple docking sites for PI3K and when heterodimerized with HER2 is the most potent stimulator of the PI3K/AKT antiapoptosis pathway. (8,12,15,16) HER2 can also be activated by complexing with other membrane receptors such as insulin-like growth factor receptor I. (17) Even estrogen, working via the nongenomic activity of ER outside the nucleus, has been shown to activate HER2 signaling. (18) An aberrant form of HER2 missing the extracellular domain, so-called p95, is found in some breast cancers. (19,20) p95 is constitutively active because the external domain of these receptors acts as an inhibitor until they are bound by ligand. p95 can cause resistance to trastuzumab, an antibody that works by binding to a domain in the external domain of HER2. This domain is missing in p95. p95 is not detected by antibodies that target the external domain of HER2 for the same reason.

[FIGURE 1 OMITTED]

HER2 OVEREXPRESSION IN HUMAN CANCER

Normal tissues have a low complement of HER2 membrane protein. Overexpression of HER2 is seen in 20% of breast and in some ovarian and gastric cancers, and it confers worse biological behavior and clinical aggressiveness in breast cancer. (2,21) Breast cancers can have up to 25 to 50 copies of the HER2 gene and up to a 40- to 100-fold increase in HER2 protein resulting in 2 million receptors expressed at the tumor cell surface. (22) The differential in HER2 expression between normal tissues and tumors helps to define HER2 as an ideal treatment target. Trastuzumab, the first treatment targeting HER2, is well tolerated in patients and has little toxicity because its effects are relatively specific for cancer cells overexpressing HER2.

HER2 amplification is a relatively early event in human breast tumorigenesis, occurring in almost 50% of in situ carcinomas. (23,24) HER2 status is maintained during progression to invasive disease and to nodal and distant metastasis. (24-26) The fact that only 20% of invasive breast cancers are HER2 amplified suggests that many HER2-amplified in situ cancers never progress to the invasive stage. HER2 amplification defines a subtype of breast cancer with a unique signature of genes and this is maintained during progression. (27,28) Some tumors lose HER2 expression on treatment with trastuzumab, presumably by selection of a HER2-negative clone not killed by treatment. (29) Conversely, HER2 may become positive in some initially negative tumors over time, especially after endocrine therapy targeting ER. (30) Estrogen receptor has been shown to downregulate HER2 and, conversely, HER2 to downregulate ER expression, and, therefore, it is not surprising that blocking ER might upregulate HER2 and that blocking HER2 might upregulate ER. (31-35)

HER2-amplified breast cancers have unique biological and clinical characteristics (Table 2). They have increased sensitivity to certain cytotoxic agents such as doxorubicin, relative resistance to hormonal agents, and propensity to metastasize to the brain and viscera. (36,37) HER2-amplified tumors have an increased sensitivity to doxorubicin possibly due to coamplification of the topoisomerase-2 gene, which is near the HER2 locus on chromosome 17 and is the target of the drug. (38) Half of HER2-positive breast cancers are ER positive but they generally have lower ER levels, and many have p53 alterations. (39,40) These tumors have higher proliferation rates and more aneuploidy and are associated with poorer patient prognosis. (2,23) The poor outcome is dramatically improved with appropriate chemotherapy combined with the HER2-targeting drug trastuzumab.

HER2 DETECTION IN BREAST CANCER

HER2 testing has been problematic historically with error rates compared with a central, expert laboratory as high as 20% for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). (41,42) Errors are unacceptable considering that the test is used to select patients for potentially curative and expensive therapy.

In 2007, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) developed recommendations for HER2 testing performance to reduce assay errors. (43) Currently HER2 testing is carried out by several methods: IHC, FISH, chromogenic in situ hybridization (CISH), and silver-enhanced in situ hybridization (SISH). These tests are performed on tumor samples that are fixed in buffered formalinand embedded in paraffin. A new test to measure total HER2 as well as HER2 homodimers and heterodimers is also available as a predictor for efficacy of anti-HER2 therapies. (44)

HER2 Immunohistochemistry

Immunohistochemistry detects HER2 protein overexpression using monoclonal or polyclonal antibodies that bind to the protein. Currently in the United States, there are 2 US Food and Drug Administration (FDA)-approved methods for HER2 assessment: HerceptTest (Dako, Glostrup, Denmark) and HER2/ neu (4B5) rabbit monoclonal primary antibody (Ventana Medical Systems, Inc, Tucson, Arizona). Current evidence suggests that home-brew assays can be less accurate than these approved assays. (45) Accurate HER evaluation is determined not only by choice of antibody but also by other determinants such as tissue fixation, fixation time, and determination of thresholds for reporting positive results. According to the ASCO/CAP guidelines the optimal tissue handling requirements include that the time from tissue acquisition to fixation should be as short as possible, the formalin fixation in buffered formalin should range from 6 to 48 hours, and the time to fixation and duration of fixation should be recorded for each sample. (43) Additional IHC internal validation and quality assurance procedures, as well as external proficiency assessment, are recommended. (43) HER2 testing results by IHC fall into 3 categories: positive, equivocal, and negative (Table 3; Figure 2). Each of these results triggers different patient management. (43)

[FIGURE 2 OMITTED]

Several additional interpretation criteria for HER2 IHC are also recommended. These include the quality of the controls, the staining of the sample, the scoring of only the invasive carcinoma, and the status of adjacent normal tissue that should not stain. The report of the results must be standardized and include all of the previous items discussed including fixation time, IHC method used, result, and interpretation. (43) The recommendations do not specify the primary antibody, the antigen retrieval method, or the detection system, all of which can affect the result. It seems logical to use a method that has been validated on clinical samples and published in the literature.

HER2 Molecular Analysis

Fluorescence in situ hybridization or CISH for HER2 gene amplification has become an integral part of the diagnostic workup for patients with breast cancer. The principles of in situ hybridization are simple: DNA probes complementary to genomic sequences of interest are generated, labeled, and then hybridized to the target tissue. Fluorescence in situ hybridization/CISH methods can be applied to a wide range of samples: cell lines, frozen tissue, paraffin-embedded tissue, and microtissue arrays. Fluorescence in situ hybridization uses fluorescence microscopy, whereas CISH uses light microscopy.

HER2 FISH positive is defined as an average of more than 6 HER2 gene copies per nucleus for test systems without an internal control probe or HER2:CEP17 ratio of more than 2.2, where CEP17 is a centromeric probe for chromosome 17 on which the HER2 gene resides (Figure 3). The equivocal range for HER2 FISH assays is defined as HER:CEP17 ratios from 1.8 to 2.2 or average gene copy number between 4.0 and 6.0 for those systems without an internal control. (43) Negative HER2 FISH amplification is defined as HER2:CEP17 ratio of less than 1.8 or an average of fewer than 4 copies of HER2 gene per nucleus for systems without an internal control probe. Tumor samples with a limited amount of invasive cancer, tissue not fixed inbuffered formalinor fixed for more than 48 hours, and background autofluorescence render a specimen uninterpretable. The report of HER2 FISH results must follow the standardized form recommended by ASCO/CAP guidelines. (43)

Chromogenic in situ hybridization uses chromogens for signal identification by means of indirect labeling. (46) Chromogenic in situ hybridization has some advantages in the detection of gene amplification over FISH: (1) permanent staining, samples can be archived; (2) use of brightfield microscopy; (3) easy identification of the target cells; and (4) easy assessment of tumor heterogeneity. (46) Fully automated systems such as brightfield SISH (Ventana) should help in method standardization. Although CISH estimates of gene amplification correlate very well with FISH, CISH does not permit a determination of the actual gene copy number.

Both FISH and CISH protocols usually require 2 days to be completed, and the assays, like IHC, are difficult and can give erroneous results. Fluorescence in situ hybridization analysis requires specific training, a fluorescence microscope with multiband filters, a high-resolution digital camera, and manual counting, which is time consuming. Automated FISH scanning is being developed. Chromogenic in situ hybridization is less time consuming, is more easily read by pathologists due to similarities with IHC, and requires only brightfield microscopy. Chromogenic in situ hybridization methods such as the SPOT-Light HER2 CISH kit (Invitrogen, Carlsbad, California) are FDA approved.

Because of the advantages over IHC, some have considered in situ hybridization the method of choice for HER2 gene testing. (47) Two kits have been FDA approved--PathVysion (Vysis, Abbott Laboratories, Abbott Park, Illinois) and HER2 FISH Pharm Dx (Dako)--and Inform (Ventana) has been submitted for FDA approval. The first 2 are dual-color FISH tests and the latter uses only a probe specific for HER2. The first 2 use HER2:CEP17 ratios, and the third relies on the signal count of the HER2 probe. Other experts continue to recommend IHC with reflex to FISH in cases scored as 2+ by IHC. (43) Both strategies have pluses and minuses and require knowledge in their interpretation.

[FIGURE 3 OMITTED]

Several studies have examined the impact of the ASCO/ CAP guideline recommendations on HER2 interpretation. In the Mayo Clinic study, (48) 144 cases of invasive carcinoma with IHC of 3+ scored according to the older HercepTest guidelines (strong membrane staining of more than 10% cells) were identified. The slides were re-reviewed and results were recorded according to the new ASCO/CAP guidelines of 3+,2+,1+, and 0. Of the 144 cases, 3 cases were reclassified as 2+ by the HercepTest and removed from the study. Analysis was done on 141 cases, of which 12 were classified as 2+ according to the new ASCO/CAP guidelines. Of the 12 cases, 6 were HER2 FISH amplified (HER2:CEP17 ratio > 2.2), 4 cases were negative (ratio < 1.8), and 2 cases were equivocal (ratio, 1.8-2.2). One hundred nineteen tumors in this study showed 3+ staining by ASCO/CAP in a large proportion (70%) of the tumor. In 18 cases with 11% to 50% of the cells staining 3+ by IHC, 2 had nonconcordant HER2 FISH results and were not amplified. All but 2 of the 119 cases showing 3+ staining in more than 70% cells were gene amplified. This study shows that the 2007 ASCO/CAP guidelines improve the concordance between IHC and FISH. Other studies have shown that standardized fixation and adherence to the ASCO/CAP guidelines resulted in a greater correlation between HER2 IHC and HER2 FISH and showed fewer inconclusive FISH cases. (49)

Several studies have shown that more automated quantitative analysis for HER2 IHC can provide standardized scoring. (50-52) By using AQUA technology and immunohistochemical/immunofluorescence HER2 staining, 1 study showed that the average percent coefficients of variation across instruments, operators, and staining runs were 1.8%, 2.0%, and 5.1%, respectively. (50) Positive/ negative concordance rates ranged from 94.5% to 99.3% for all cases. Another study assessed the performance characteristics of the Automated Cellular Imaging System (ACIS III, Dako Corp, Carpenteria, California) for HER2 IHC analysis. (51) The automated score was compared with the manual score and to HER2 by FISH. The automated score yielded fewer 2+ cases than the manual score and had a higher positive predictive value for HER2 amplification.

Silver-enhanced in situ hybridization is a highly sensitive technique that requires fewer reagents than FISH or CISH (Figure 3). Like CISH, SISH staining is permanent and does not decay, allowing specimens to be archived. Unlike CISH, SISH allows quantitative scoring of gene copy number. In a recent study comparing HER2 FISH and HER2 SISH techniques in excisional biopsies, the results were identical in 50 of 53 cases (94%). (52) Two cases were SISH negative, FISH positive and 1 case was SISH positive, FISH negative. Silver-enhanced in situ hybridization results on the core biopsy were identical to the corresponding excisional biopsy in 89% of the cases. Four cases (7%) were SISH negative in the core but positive in the excision specimen and 2 cases were the other way around. These are comparable with FISH and validate the use of SISH for measurement of HER2.

Influence of Polysomy 17

By using HER2 FISH that evaluates HER2 gene copy number and the centromeric region of chromosome 17, the results can be categorized as (1) normal gene copy and ploidy status (2 signals of HER2 and 2 signals for chromosome 17), (2) HER2 amplification, (3) chromosome 17 polysomy, and (4) chromosome 17 polysomy with HER2 amplification. (53) Tumors with increased HER2 and chromosome 17 signals and with a ratio of less than 1.8 according to the ACSO/CAP guidelines are considered polysomy. These tumors have been associated with HER2-positive IHC but rarely with 3+. (54) Patients with chromosome 17 polysomic breast cancer in the absence of HER2 amplification or 3+ IHC are not candidates for targeted therapy, and they have not been considered eligible for adjuvant trastuzumab trials or trials comparing different HER2 therapies. (53) Recent studies have shown that true/ complete chromosome 17 polysomy is rare and that a CEP17 copy number of more than 3 in FISH analysis is due to concomitant gain or amplification of the centromeric region in addition to HER2. (55,56) Thus, patients may appear to have tumors with chromosome 17 polysomy but in reality have HER2 amplification with the amplicon extending to the centromere. These patients might be denied HER-targeted therapy due to misinterpretation of the FISH results. Further investigation is needed to confirm these data and to clarify the importance, if any, of polysomy 17 in breast cancer.

Protein Proximity HER2 Assay

The HERmark test (Monogram Biosciences, Inc, South San Francisco, California) is a new assay that uses the VeraTag system to identify total levels of a cellular protein or 2 similar or dissimilar proteins in close proximity. (44) The technology has been used to measure total HER2 protein and the amount of HER2 homodimers in breast cancer tissue. (57) For a total HER2, the technology uses 2 monoclonal antibodies specific for unique epitopes in the intracellular domain of the HER2 receptor protein. This results in the antibodies binding in close proximity to the same HER2 receptor proteins. One of the monoclonal antibodies is conjugated with a fluorescent reporter and the other to a photosensitizer molecule. The assay is done on paraffin-embedded tissues and photoactivation of the sample results in cleavage of the reporter, which is then quantified using electrophoresis. The amount of cleaved reporter is proportional to the concentration of HER2 in the tumor. In a study in metastatic breast cancer in HER2-positive patients treated with trastuzumab, the assay was superior to FISH and to IHC in predicting benefit from trastuzumab. (58) In a preliminary report from a recently completed adjuvant trial, the HERmark assay declassified 25% of cases originally classified as positive, equivocal, or negative when done by central IHC. (59) Correlation with outcome has not yet been performed. The assay is now being developed to detect HER3, various homodimer and heterodimer pairs, and other signaling molecules. Additional data are needed to know where this assay fits in the diagnostic evaluation of the breast cancer patient.

HER2-TARGETED THERAPY IN THE CLINIC

The need for accurate determination of HER2 status is illustrated by the excellent results of therapies targeting HER2 in the clinic. These strategies have been shown to benefit only tumors overexpressing HER2 protein and usually HER2 gene amplified. Tumors with negative staining and tumors that are 1+ or 2+, which is not gene amplified, do not benefit from HER2-targeted therapies.

Two drugs are currently FDA-approved for treatment of HER2-positive cancers. Trastuzumab is a humanized monoclonal antibody that recognizes the external domain of HER2. Its mechanism of action is still not totally clear, but it seems to have its greatest effects in tumors with increased HER2 homodimers. (57) Although it does not block autophosphorylation of HER2, it does inhibit HER2 downstream signaling. (60) In addition, it may disrupt the HER2/Src interaction, may cause internalization and downregulation of the receptor, and may enhance antibody-mediated cytotoxicity. In patients with meta-static breast cancer undergoing first-line treatment, about 25% of those patients with HER2-overexpressing tumors have an objective response to trastuzumab when used alone. (61)

Preclinically, trastuzumab has synergistic activity when combined with a variety of chemotherapy drugs, perhaps because it inhibits an important survival pathway making the cytotoxic effects of chemotherapy more potent. Doxorubicin may be more effective because of coamplification with HER2 of its target topoisomerase 2 on chromosome 17. (62) In clinical trials, trastuzumab increased the objective response rate and time to progression when combined with doxorubicin, cyclophosphamide, or paclitaxel. (63) The high rate of cardiac toxicity when trastuzumab was combined with doxorubicin-containing regimens demanded that trastuzumab only be used sequentially with regimens containing anthracyclines. A variety of phase II and phase III studies confirmed the effectiveness of trastuzumab combined with a variety of chemotherapeutic agents, some showing an overall survival advantage. (63) Trastuzumab is also beneficial when combined with endocrine therapy in patients with ER-positive and HER2-positive tumors. (64)

More impressive results were observed when trastuzumab was combined with chemotherapy in the adjuvant treatment of patients with primary breast cancer following surgery. Several randomized trials using different chemotherapy regimens combined with trastuzumab all showed significant improvements in disease-free survival and overall survival with reductions in the risk of recurrence ranging from 40% to 50%. (65-68) Because prior studies of chemotherapy alone had shown a 40% to 50% reduction in the risk of recurrence, the addition of trastuzumab to chemotherapy in patients with HER2-overexpressing tumors has resulted in a marked improvement in the outcome of these patients with a tumor subtype noted for its aggressive behavior and poor prognosis in the absence of therapy.

A subset analysis of one of the large adjuvant trials performed by the National Surgical Adjuvant Breast and Bowel Project showed an advantage for trastuzumab even in patients found at central laboratory testing to be HER2 negative. (69) These investigators have done an exhaustive analysis of these patients to confirm that they are indeed not HER2 amplified. Whether this retrospective analysis on a small number of patients originally deemed HER2 positive in the local laboratory and found on central laboratory testing to be HER2 negative is merely a statistical fluke or whether it reflects real biology of the tumor remains to be clarified. Until then HER2-targeted therapy is reserved for patients whose tumor overexpresses the protein or is amplified for the gene.

Lapatinib is a new drug that works by a different mechanism to inhibit HER2 signaling (Figure 1). (70) Lapatinib is a small molecule TK inhibitor that blocks the kinase activity of HER1 and HER2. This drug has been shown to cause remissions in patients whose tumors are resistant to trastuzumab and it may be more effective when given together with trastuzumab. (71,72) Lapatinib should still be effective in patients whose tumors express high levels of HER2 p95 and are trastuzumab resistant. This shortened form of HER2 has lost the extracellular domain to which trastuzumab binds, but lapatinib would be expected to still block the TK activity. Lapatinib, like trastuzumab when combined with certain chemotherapy drugs in patients with metastatic disease, increases the patient benefit compared with the chemotherapy drug alone. (73) Several trials of metastatic disease as well as in the adjuvant setting are now underway evaluating chemotherapy and lapatinib as a single agent or combined with trastuzumab. The combinations are of particular interest given the dramatic affects of combined HER-targeted therapies in preclinical xenograft models. (74) Several other drugs are under development for HER2-overexpressing breast cancer. Among them are an irreversible dual TK inhibitor similar to lapatinib, a monoclonal antibody that blocks the dimerization domain on HER2 to prevent heterodimerization with other members of the HER receptor family, and an antibody-drug conjugate that links trastuzumab with a cytotoxic agent. (75,76)

SUMMARY AND CONCLUSION

The HER2 oncogene is an important member of the HER family of membrane TKs. The gene is amplified in a subset of breast cancers in which the HER network is the major driver of tumor cell proliferation and survival. Targeting this pathway therapeutically has remarkably improved the outlook of patients with these aggressive breast cancers, but treatment is only effective in patients whose tumors express high levels of the protein or are amplified for the gene. The pathologist's role in accurately assessing the HER2 status of a tumor is critical for successful treatment. Diagnostic assessment of HER2 by IHC or gene amplification should follow established guidelines and procedures to avoid misclassification of patients who might not otherwise receive life-saving treatment.

This manuscript was supported in part by grants P50 CA58183 (Breast Cancer SPORE) and P01 CA30195 from the National Cancer Institute, Bethesda, Maryland.

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Carolina Gutierrez, MD; Rachel Schiff, PhD

Accepted for publication September 20, 2010.

From the Department of Pathology (Dr Gutierrez) and the Lester & Sue Smith Breast Center (Dr Schiff), Baylor College of Medicine, Houston, Texas.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Carolina Gutierrez, MD, Baylor College of Medicine, Department of Pathology, One Baylor Plaza, BCM 600, Houston, TX 77030 (e-mail: mgutierr@bcm.edu).
Table 1. HER Family of Membrane Receptors

 Tyrosine Kinase
Receptor Activity Major Ligands

HER1 (EGFR) Yes EGF, amphiregulin,
 TGF-a
HER2 Yes None
HER3 No Heregulin
HER4 Yes Heregulin

Abbreviations: EGF, epidermal growth factor;EGFR, epidermal growth
factor receptor;TGF-a, transforming growth factor a.

Table 2. Characteristics of HER2-Amplified
Breast Cancer

Increased proliferation rates
High histologic and nuclear grade
Low ER and PR levels
More aneuploidy
Propensity to metastasize to CNS and viscera
Relative resistance to endocrine therapy
Increased sensitivity to doxorubicin; coamplification of
 topoisomerase 2
Response to HER2-targeted therapy

Abbreviations: CNS, central nervous system; ER, estrogen receptor; PR,
progesterone receptor.

Table 3. HER2 Testing by Validated Immunohistochemistry Assay

Status Score Significance Reflex
 HER2 FISH

Positive 3+ Uniform intense membrane No
 staining of > 30% of invasive
 tumor cells

Equivocal 2+ Complete membrane staining, Yes
 nonuniform or weak in
 intensity, in at least 10% of
 the cells, or intense complete
 membrane staining in 30% or
 less of tumor cells

Negative 1 + Weak or incomplete membrane No
 staining in any proportion of
 tumor cells

Negative 0 No staining No

Abbreviation: FISH, fluorescence in situ hybridization.
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Author:Gutierrez, Carolina; Schiff, Rachel
Publication:Archives of Pathology & Laboratory Medicine
Date:Jan 1, 2011
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