The HER-2/neu (also known as ERBB2) proto-oncogene, which encodes a trans- membrane growth factor receptor with tyrosine kinase activity, has become an important subject for human cancer re- search during the last decade. The impact of HER-2amplification and expression on prognosis (1–12) and on the response to cytotoxic (4,13–18) and hormonal (15,19–22) therapies in breastcancer patients have been studied intensively. Studies of HER-2 represent a paradigm of how genetic findings have led to the development of a gene-specific therapy: In September 1998, the U.S. Food and Drug Administration approved trastuzamab (Herceptin), a recombinant monoclonal antibody targeting Her-2, for the treat- ment of metastaticbreastcancer. Al- though trastuzumab binds to the Her-2 receptor with high affinity, the mecha- nism of action by which it causes tumor reduction is not understood. Despite the theoretic benefits of such a targeted treat- ment, not all patients respond favorably to trastuzamab treatment in practice. Among the patients with HER-2-positive meta- static breastcancer that is resistant to con- ventional cytotoxic treatment, only about 25% benefit from trastuzamab given in combination with cisplatin (23). The ge- netic features that distinguish the HER-2- positive breast cancers that respond to trastuzamab from those that do not remain unclear. However, it is possible that the extent of HER-2amplification and/or overexpression in the primary tumor dif- fers from that in the metastases. The HER-2 status of the primary tumor, which is removed from the patient, determines whether or not trastuzamab treatment is prescribed. But trastuzamab works by tar- geting the metastases that remain in the patient. If at least some of the multiple metastases of an HER-2-positive primarybreast tumor did not express HER-2, trastuzamab treatment would most likely not affect the course of the disease. A comprehensive, large-scale study compar- ing HER-2 gene copy numbers and pro- tein expression in primary tumors and in multiple different metastases derived from them has not been performed. To gain insight into the patterns of HER-2
9. Pegram MD, Slamon DJ. Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastaticbreastcancer: evidence for receptor-enhanced chemosensitivity. Semin Oncol. 1999; 26(4 Suppl 12):89-95. 10. Jimenez RE, Hussain M, Bianco FJ, Jr., Vaishampayan U, Tabazcka P, Sakr WA, Pontes JE, et al. Her-2/neuoverexpression in muscle-invasive urothelial carcinoma of the bladder: prognostic significance and comparative analysis in primary and metastatic tumors. Clin Cancer Res. 2001;7(8):2440-2447.
In brief, H & E-stained sections were made from primary tumor blocks to define two representative tumor regions and adjacent normal gastric tissues. Representative tumor regions were defined as tumor solid areas containing more than 75% cancer cells without necrosis. Normal gastric tissues were randomly selected adjacent to a tumor with a distance of more than 5 cm, avoiding the bleeding areas. Tumor typing and grading were performed according to Lauren and the World Health Organization (WHO) cri- teria. All patients with gastric cancer were staged using the seventh edition of the International Union Against Cancer Tumor-Node-Metastasis (TNM) staging system. Tissue cylinders (1.5 mm in diameter) were then punched from the defined regions of the block using a tissue micro- arrayer (Gentury, IL, USA) and brought into recipient paraffin blocks. Two sets of three paraffin-embedded tissue microarray (TMA) blocks were made. Sections of the resulting TMA blocks were transferred to glass slides.
A correlation between serum HER-2/neu levels and tissue HER-2/neu status in metastaticbreastcancer has been examined. A serum ECD concentration of 16 µ g/L showed a sensitivity and specificity of approximately 90% and 80%, respectively, when IHC and FISH were the refer- ence standards. In contrast, another study determined a cut-off of 37 µ g/L with 62% sensitivity and 95% specificity for prediction of the tissue HER-2/neu status. The research- ers attributed this discrepancy to a difference in assays and proposed using a higher cut-off value for metastaticbreastcancer than for diagnosis of primarybreastcancer. A very high serum ECD concentration with negative tissue HER-2/ neu could be due to disease recurrence. 27 Trastuzumab is a
A point of utmost importance when assessing the utility of HER-2/neu as a prognostic factor is the technique of HER- 2/neu testing. Principally, gene-based assays such as Southern blot analysis or fluorecence in situ hybridisation (FISH) have to be distinguished from assays that assess the level of protein expression, such as western blot analy- sis or immunohistochemistry (IHC). When analysing the published studies of HER-2/neu as a prognostic factor with regard to the technique used, Ross and colleagues [14,15] found that those studies that applied FISH to the assess- ment of gene amplification found an association of HER-2/ neu with the prognosis of the patients, whereas studies that used IHC for the assessment of protein expression gave rather ambiguous results. Reasons for the apparently worse performance of IHC than that of FISH could be dif- ferences in the sensitivity of the applied antibodies  or the lack of a uniform scoring system in most of the older studies that used IHC. Standardisation of HER-2/neu test- ing has received considerably more attention in recent years owing to the advent of trastuzumab, a monoclonal antibody that affords prolonged survival in patients with metastaticbreastcancer . To improve the quality of IHC when testing for HER-2/neu status before starting a therapy with trastuzumab, a standardised scoring protocol was developed. On the basis of these findings, an algo- rithm incorporating FISH only in doubtful cases (2+ in IHC) was introduced.
The observation that morphologically similar neoplas- matic lesions of the breast can exhibit different biology has necessitated the identification of biological parame- ters that might improve risk assessment; the evaluation of HER-2/neu expression is a typical example . Indeed, several studies have demonstrated that HER-2/neu ampli- fication represents a prognostic and predictive marker; its expression is associated with early disease recurrence, rel- ative resistance to chemo- and/or hormonotherapy and short survival [2,10]. In addition it has been shown that genetic alterations of the HER-2/neu oncogene represent early events involved in breast carcinogenesis and tumour initiation, while their presence is observed in all stages of malignant development from in situ carcinomas to meta- static lesions . As a result, some researchers have maintained that HER-2/neuamplification and/or protein overexpression may also represent not only an important marker of prognosis but also a key indicator of the aetio- logical heterogeneity of breast carcinogenesis. [3,7-9]. On the other hand, the contribution of even well estab- lished breastcancer risk factors to the aetiology of carcino- genesis in the breast remains obscure, ill-defined and tenuous, mostly because of the existence of different path- ways for the initiation and the evolution of a breast tumour . In order to explain this incompatibility, sev- eral researchers have claimed that classification of tumours on the basis of HER-2/neuoverexpression or amplification may define a subset of breastcancer in which the net effect of a risk factor could be rather more obvious and its impact on breastcancer development more clear [3,7,8].
because metastatic tumor cells that originate from the HER-2 positive primary tumor prior to NAC may still harbor amplified HER2 gene. Various studies [16–18] have revealed certain discrepancies of HER2 expression between primary tumors and synchronous nodal metastases in the absence of chemotherapy, ranging from 0 to 9%. Little is known about the status of HER-2 gene of synchronous nodal metastases in a neoadjuvant setting, as confirmation of this oncogene in nodal lesions is crucial under the condition in which HER-2 gene sta- tus of the primary tumor converted from amplification to non-amplification after NAC . So, we shifted our attention to the pathologically confirmed metastatic le- sions within axillary lymph nodes (4/12). In order to optimize the patient’s clinical outcomes, the synchron- ous nodal metastases were submitted for IHC, only to find the nodal lesions shared the same negative HER-2 status with the primary tumor, which indicated that HER-2 protein expression of nodal lesions was also sup- pressed by NAC, as Oldham et al.  demonstrated that paclitaxel downregulated HER2 expression in MDA-MB 453 cells. Finally, FISH was performed and confirmed amplification of HER-2 gene of the synchron- ous nodal metastases. Then, the patient agreed to receive trastuzumab. This discrepancy between HER-2 gene and its oncogenic receptor as revealed by IHC and FISH, respectively, was in accordance with one study  indicating that the HER-2 expression at the tran- scriptomic level is not always parallel to that at the proteomic level based on ERBB2 mRNA analysis after neoadjuvant systemic therapy, thus emphasizing that conclusions drawn from proteomic level should be further investigated in genomic and transcriptomic level. We speculated that suppression of HER2 protein expres- sion of the nodal lesions in a neoadjuvant setting in this patient was possibly contemporary and the oncogenic receptor would soon be regained following a cessation of chemotherapy, due to the persistent amplification of HER-2 gene in nodal lesions.
amplification of oncogenes and its impact on the course of breastcancer disease [4–7,10–32]. In a meta-analysis in which 29 studies were evaluated , c-myc amplification exhibited significant but weak associations with tumour grade, lymph node metastasis, negative progesterone receptor status and postmenopausal status. Furthermore c-myc amplification was significantly associated with risk for recurrence and death. However, studies in recent years have further shown that the c-myc gene participates in most aspects of cellular function, including replication, growth, metabolism, differentiation and apoptosis . Amplification of the oncogene HER-2/neu has also been shown to be indicative of poor prognosis in breastcancer. Studies revealed that the prognostic effect of HER-2/neu is stronger for survival than for recurrence [16,19,20,22, 27,29]. Furthermore, increased HER-2/neu levels in primary tumours were associated with a poor response to endocrine therapy [5,12,15,32].
Positive control tissue was used for ER. Normal breast epithe- lial cell nuclei are often used as an internal control with PgR. Uterine cervix tissue with stromal cells and basal squamous epithelial cells should show a distinct nuclear reaction with minimal cytoplasm staining for HER-2/neu. Human tumour control is Paget’s disease, which almost invariably shows gene amplification and 3 + protein overexpression for p53, breastcancer tissue, Ki-67, lymphoid tissue in tonsils, Bcl2, and tonsil lymphoid tissue EGFR. Placental tissues were used as positive controls. Negative controls included sections of breast tumor tissue incubated overnight in the respective blocking sera instead of the primary antibodies, following NordiQC IHC quality controls 2007.
analysis. This pattern of overexpression of Her-2 in large tumors has been demonstrated in other studies [27,28] while in some studies this association has not been found [24,29-32]. The prognostic value of grade is currently established. Hoff et al. demonstrated in their series that high-grade tumors were more likely to have a positive HER-2 status as low-grade tumors . Similarly, other studies have also reported the association between HER-2 positive status and a high histological grade [29-31]. This finding was also confirmed in our study. However, some studies have not demonstrated any cor- relation between high-grade status and HER-2 positive [24,34].The axillary lymph node involvement is a major prognostic factor. Most studies that have examined the prognostic role of HER-2 in N + patients showed that amplification of HER-2 gene or overexpression of Her-2/ neu protein is associated with pejorative results whether in univariate or multivariate analysis [35-37]. Our study found this association in univariate analysis, but disap- peared in multivariate analysis. Extra capsular spread and the vascular space invasion are two parameters that have been poorly studied in the literature and found to be as- sociated with overexpression of the protein Her-2/neu in our study. Despite the vast differences at the levels of the positivity of the Her-2, all the researchers report an in- verse correlation between HER-2 status and the hormone receptors [24,28-30,32,35,38-42]. Our data are consistent with those reported in the literature.
Clinical trial. Between August 1996 and August 1998, 19 patients with breast or ovarian cancer were enrolled in a phase I HER-2/neu peptide–based vaccine trial approved by the University of Washington’s Human Subjects Division and the United States Food and Drug Administration. Eligibility was dependent upon subjects (a) being diagnosed with stage III/IV breast or ovarian cancer and having been treated for their pri- mary and metastatic disease according to recommend- ed disease-appropriate standards with surgery, chemotherapy, radiation therapy, or combined modal- ity, (b) having a white blood cell count greater than 3.5 dl/ml, (c) showing HER-2/neu protein overexpression in the primary tumor or metastasis, (d) being off immunosuppressive drugs and chemotherapy for at least 30 days before enrolling, and (e) being HLA-A2 positive. Patients were tested for immune competence responsiveness to a minimum of two of seven recall antigens by skin testing with Multitest CMI (Pasteur Merieux Connaught Labs, Institut Merieux, Lyon, France). All patients signed a protocol-specific consent and received monthly vaccinations with three 15–amino acid (15-aa) HER-2/neu–derived peptides, p369–p384, p688–p703, and p971–p984, containing within each the putative HLA-A2–binding motifs p369–p377 (6), p689–p697 (7), and p971–p979 (18). Five hundred micrograms of each peptide (1.5 µg total peptide dose) were solubilized in 10 mM sodium acetate (pH 4.0) and admixed with 125 µg rhuGM-CSF (kindly supplied by Immunex Corp., Seattle, Washing- ton, USA) as an adjuvant. The vaccine preparation was divided into two intradermal injections administered to the same draining lymph node site monthly for 6 months. Subjects underwent peripheral blood draws or a leukapheresis before and 30 days after each vaccina- tion for immunologic monitoring.
In contrast to breastcancer where IHC for HER-2/neu is usually homogenous, tumour heterogeneity is more common in gastric cancer. Heterogeneous staining can sometimes be seen within one gland. The most important reason for heterogeneous staining, however, is that up to one third of gastric cancers are of mixed intestinal/diffuse type. Strong staining is often seen in areas of an intestinal type of differentiation, while areas of diffuse types of differentiation are negative. (34) Signet ring cell carcinomas are almost always negative. An identical heterogeneous pattern of staining is identified at the DNA level (amplification). Because of heterogeneity, the 10% cut-off level for positivity, which is required in breastcancer, is omitted in gastric cancer. Positivity in gastric cancer specimens is thus independent of the percentage of stained cells and it is sufficient to have a cohesive group of cells displaying HER-2/neu positivity. The heterogeneity of HER-2/neuoverexpression / amplication is difficult to explain from the biologic point of view the mechanisms leading to silencing HER-2/neu expression in an area of a tumor with homogeneous HER-2/neuamplification, is at present unknown. (32)
Seven cases with the score of 3+ by IHC were FISH negative for gene amplification (>2.2). Earlier studies have shown similar results 23,27-29 . Although there is good correlation between HER-2 gene amplification and protein overexpression, many studies 23 also have shown that 3 to 15 per cent of breast carcinomas over- express the HER-2 protein without gene amplification and a small subset of breast carcinomas amplify the HER-2 gene without overexpression. Various theories have been proposed to account for these discrepancies. Increased receptor expression in breastcancer without genetic alterations has been reported in approximately 10 per cent of cases, probably caused by transcriptional or post-translational activation. Other proposed explanations for the phenomenon of overexpression without amplification include artifactual high sensitivity of immunohistochemical assays, single copy overexpression of the HER-2 gene at the messenger RNA (mRNA) transcription level, or gene amplification below the level of detection of FISH assays 30,31 . The presence of chromosome 17 polysomy is not uncommon in breast carcinomas and has been suggested to account for overexpression without amplification 23 . We found that average chromosome 17 copy number when compared to average HER-2/neu copy number, was significantly higher in FISH interpreted as polysomy of chromosome 17, and this could be falsely interpreted as HER2/
bersome and more economical; the signal intensity is permanent; and pathologists are familiar with IHC signals and are able to correlate findings with the underlying tumor histomorphology. CISH allows observers to select fields of invasive ductal carcinoma, avoiding foci of intraductal car- cinoma for which Her-2/neu has a different clinical signifi- cance. CISH is a specific, sensitive, and easily applicable method for the detection of Her-2/neu gene amplification, and it can be used together with IHC for the evaluation of patients with breast carcinoma . Ross and colleagues recently reviewed the current status of Her-2/neu testing, and they concluded that CISH is a more convenient gene- based technique that is 'waiting in the wings' . In this present study, the concordance between 3+ IHC and CISH-amplified cases was 100% (40 of 40), denoting all gene amplified cases to be overexpressing the Her-2/ neu protein. In contrast, the agreement on 2+ IHC and CISH-amplified cases was only 45%, which is lower than previously reported (93%) . The 13.75% (22 of 160) IHC-positive/CISH non-amplified tumors in this study, all of which had 2+ IHC scores, is higher than the 6% false pos- itive result by Zhao and colleagues , although compa- rable to the 17% result (IHC CB11 versus FISH) obtained by Tubbs and colleagues . Apparently, we find that the 2+ IHC score is equivocal when compared with CISH. This subset might benefit from further CISH reflex testing. All negative IHC cases were CISH non-amplified, showing complete agreement of all negative results. The 86.25% overall concordance between IHC (using CB11 antibody) and CISH in this study is lower than the previous reports (94–96%) [24,30]. We concur with Sapino and colleagues that gene amplification analysis can be avoided in all 0, 1+, and 3+ IHC cases because results are predictable from IHC and are completely concordant with CISH . The utility of CISH is therefore in further testing Her-2/neu alter- ation on equivocal cases in IHC, which remains the primary screening method. The combined IHC–CISH algorithm provides an economical and comprehensive Her-2/neu data to guide clinicians in mapping treatment options. The low-amplified CISH category (6–10 signals) was the most difficult to interpret, requiring an accurate enumera- tion of gene copy. Signal clustering, more probably a result of intrachromosomal amplification of homogeneously stain- ing regions, was immediately apparent in highly amplified cases and was easily evaluated [23,31].
significantly lower rate of liver and bone metastases. 9 There is a slight decrease in expression of these bio-markers in the metastatic tumours. This effect may be due to tumour heterogeneity, a well-known fact, in anticancer chemo sensitivity, and may be reflected in hormonal receptor status of metastaticbreast carcinoma. Neoplastic cells from high-grade tumours may also loose estrogen and progesterone receptors during the process of metastasis. HER-2/neu expression, however, remains almost same in primary and metastaticbreast carcinomas. 10,11 Metastaticbreastcancer is an important area of research for both researchers and clinicians because MBC has a poor prognosis. The present study was conducted to compare the expression of these bio-markers between primary and metastaticbreast carcinoma.
The primary antibodies used in this study are presented in Table 2. Immunohistochemistry was performed on paraffin sections 5 µm thick. The alkaline phosphatase anti-alkaline phosphatase method was used for antibody demonstra- tion. Antigen retrieval was carried out with 0.01 M citrate buffer at pH 6.1 for 20 min (both phospho-Akt antibodies), for 40 min (phospho-ERK1/2) and for 20 min (FAK, phos- pho-Src Tyr-215 and phospho-Src Tyr-416) in a hot water bath (95°C). Antibodies were incubated overnight in a humidified chamber at 4°C. Positive controls were included in each staining series. No significant staining was observed in the negative controls using mouse immu- noglobin replacing the primary antibody. The DAKO Her- ceptTest™ (DakoCytomation, Glostrup, Denmark) and a HER2/ErbB2 polyclonal antibody at a 1:50 dilution (rabbit polyclonal antibody; Cell Signaling Technology, Beverly, MA, USA) were used for the detection of HER2 protein expression. The estrogen receptor (ER) status of the tumors was determined using a monoclonal anti-human antibody as previously described .
be very low ( > 1%); therefore, minor differences in antibody sensitivity can alter hormone receptor status. Current ASCO CAP guidelines require the hormone receptor status to be reported as positive or negative based upon a semiquantita- tive score of both the number of invasive tumor cells staining positively and the intensity of the stain. This system should better predict responses to hormone therapy and be easier and more cost-effective. Identifying the HER-2/neu-driven tumor has been vexing. Current guidelines define a breast tumor as HER-2/neu positive using either IHC or FISH. 11 Recent
The pathologists challenge is to identify breast cancers with abnormally high cellular HER2 expression or gene amplification, at least in many of the invasive cancer cells. We must not to misidentify normal p185 expression or gene copy number in invasive cancer as abnormal, nor should we mis-interpret HER2 from in situ cancer cells. These and other variables have been considered in detail, in the current guidelines for HER2 testing (7- 10). For IHC, cells with low to normal HER2 expression typically show only rare cells with membranous positivity, staining which is weak in intensity, or incomplete staining of the cytoplasmic membrane. A new cut point for positivity has recently been enacted, at 30% of invasive cancer cells. Benign breast epithelium, stroma and inflammatory cells should not express HER2 by IHC and if they do, the case should be considered
Currently, treatment of MBC patients with HER-2/neu positive tumors is based on HER-2/neu status derived from the primary tumor, which was generally removed many years previously and stored as paraffin-embedded blocks. In a 2005 report by Zidan and coworkers  it was pointed out that HER-2/neu status of the primary tumor may not accurately reflect the HER-2/neu status of the metastatic tumor, and that this should be taken into account when making treatment decisions. Those investigators demon- strated 14% discordance between primary and metastatic tumors by IHC. Twelve per cent (7/58) of the patients were HER-2/neu positive in the metastatic tumor yet negative in the corresponding primary tumor. Interestingly, three of the seven patients who were HER-2/neu negative in the primary tumor but positive by IHC in the metastatic tumor responded to trastuzumab-based therapy. The references list of that report directed us to several other papers comparing HER-2/ neu status, as determined using IHC and FISH, between primarybreast tumor and metastatic tumor from the same patient. Evidence supporting the observation that a primarybreast tumor can be HER-2/neu negative while the metastatic tumor can positive is illustrated below with a few examples. Edgerton and coworkers , employing IHC and FISH, reported 20% discordance between the primary and metastatic tumor, which was due to normal HER-2/neu expression in the primary tumor and HER-2/neu over- expression in the metastatic tumor. Gancberg and colleagues  compared HER-2/neu status of the primarybreast tumor with that of at least one distant metastatic tumor in 107 patients using both IHC and FISH. There was a 6% (6/100) rate of discordance with IHC between the primary and metastatic tumor. In the six cases of discordance, there was greater HER-2/neu staining in the metastatic tumor tissue than in the primary tumor tissue. By FISH analysis, 7% (5/68) of the cases were discordant. Three of the five discordant patient specimens exhibited amplification in the metastatic tumor but not in the primary tumor. It was also reported that if all metastatic tumor sites were included in the analysis, then the HER-2/neu positive metastatic tumors with a corres- ponding negative primary tumor were more frequent than the converse, suggesting that HER-2/neu expression in primary tumors might represent an underestimation of reality. In another study , 80 paired primary tumors and metastatic tumors from the same patients were evaluated for HER-2/neu
DNA was prepared from tail samples by incu- bating tails overnight at 50°C in 500 l Tail Buffer (17 mM Tris [pH 7.5], 17 mM EDTA, 170 mM NaCl, 0.85% SDS and 0.2% proteinase K added immediately prior to use). After proteins were pelleted in 250 l 6M NaCl and cen- trifuged, DNA was precipitated in 95% ethanol and resuspended in 100 l water. The DNA was electrophoresed through a 0.8% agarose gel af- ter complete digestion with BamHI restriction enzyme. The gel was blotted overnight in 0.4 N sodium hydroxide onto a Genescreen Plus membrane (NEN Research Products, Boston, MA). The filter was then rinsed for 10 min in 2 SSC (0.30 M sodium chloride, 0.030 M sodium citrate) and crosslinked by ultraviolet light. Filters were prehybridized for at least 1 hr at 42°C in hybridization solution (50% for- mamide, 5 SSC, 5% dextran sulfate, 20 mM sodium phosphate, 1 Denhardt’s reagent, 0.5% sodium dodecyl sulfate and 20 g/ml sonicated herring sperm DNA) before probe was added. An 800-basepair Pst I fragment of SV40 polyA vector (18) was labeled with [∝ 32