Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

Transcriptional regulation of the {\it neu\/} oncogene and its negative regulation by the c-{\it myc\/} oncogene

The first part of my research involved the characterization of the neu gene promoter. I subcloned a 2.2-kb sequence located upstream to the extreme 5$\sp\prime$ end of the neu gene, in front of the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Transfection of this construct into different cell lines and subsequent CAT assays demonstrated that this 2.2-kb fragment was functional as a promoter. A series of deletion constructs was engineered to study the contribution of different fragments to transcription. Subcloning of individual fragments was followed by a cotransfection competition experiment, which demonstrated the involvement of protein factors interacting with the promoter. A gel retardation assay was also performed to show the physical binding of protein factors to the promoter. The combined results suggested that both positively and negatively acting protein factors are involved in interacting with different regions of the promoter, contributing to the overall transcription activity. My findings provide an insight into the regulation of neu gene expression, which in turn provides the tools to understand the molecular mechanisms of overexpression of the neu gene in some breast cancer and ovarian cancer cell lines.^ In the second part of my research, I discovered that another oncogene, c-myc, was able to reverse the transformed morphology that was induced by the neu oncogene. Utilizing the promoter constructs that I made, I was able to show that the c-myc oncogene has a negative regulatory effect on the expression of the neu oncogene. Further studies suggested that c-myc is able to lower the effective concentration of a positive factor(s) that interact with a 139-bp fragment of the neu gene promoter. These findings may provide a direct evidence of the long suspected role of the c-myc gene in transcriptional regulation. The neu gene may very well be the first identified mammalian target gene that is regulated by the c-myc oncogene. Since c-myc is known to be stimulated by various mitogenic signals and the neu gene is likely to be a growth factor receptor, it is possible that c-myc, when stimulated by the signal transduction pathway of the neu gene, would function as a negative feedback regulator on the neu gene receptor. (Abstract shortened with permission of author.) ^

Repression of {\it neu} oncogene by E1A: Mechanisms and biological functions

The potential effects of the E1A gene products on the promoter activities of neu were investigated. Transcription of the neu oncogene was found to be strongly repressed by the E1A gene products and this requires that conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 140 base pair (bp) DNA fragment in the upstream region of the neu promoter. To further study if this transcriptional repression of neu by E1A can inhibit the transforming ability of the neu transformed cells, the E1A gene was introduced into the neu oncogene transformed B104-1-1 cells and developed B-E1A cell lines that express E1A proteins. These B-E1A stable transfectants have reduced transforming activity compared to the parental B104-1-1 cell line and we conclude that E1A can suppress the transformed phenotypes of the neu oncogene transformed cells via transcriptional repression of neu.^ To study the effects of E1A on metastasis, we first introduced the mutation-activated rat neu oncogene into 3T3 cells and showed that both the neu oncogene transformed NIH3T3 cells and Swiss Webster 3T3 cells exhibited metastatic properties in vitro and in vivo, while their parental 3T3 cells did not. Additionally, the neu-specific monoclonal antibody 7.16.4, which can down regulate neu-encoded p185 protein, effectively reduced the metastatic properties induced by neu. To investigate if E1A can reduce the metastatic potential of neu-transformed cells, we also compared the metastatic properties of B-E1A cell lines and B104-1-1 cell. B-E1A cell lines showed reduced invasiveness and lung colonization than the parental neu transformed B104-1-1 cells. We conclude that E1A gene products also have inhibitory effect on the metastatic phenotypes of the neu oncogene transformed cells.^ The product of human retinoblastoma (RB) susceptibility gene has been shown to complex with E1A gene products and is speculated to regulate gene expression. We therefore investigated in E1A-RB interaction might be involved in the regulation of neu oncogene expression. We found that the RB gene product can decrease the E1A-mediated repression of neu oncogene and the E1A binding region of the RB protein is required for the derepression function. ^

Autoregulation of the {\it neu\/} oncogene

The neu gene encodes a 185,000-Da membrane glycoprotein that is highly homologous to epidermal growth factor receptor. It is frequently overexpressed or amplified in human breast carcinomas and ovarian cancers, which correlates with a poor prognosis for patients. The importance of neu gene regulation is noted by the fact that many breast cancer cells overexpress the neu gene without proportional gene amplification. The mechanism for that is unclear. My initial finding of neu autoregulation led to a realization that defects in neu autoregulation pathway may contribute to neu overexpression in tumor cells. I have found in the nontransformed NIH 3T3 model system that (i) the neu gene product autorepresses its own promoter activity, (ii) the neu gene promoter contains a novel enhancer, (iii) neu autorepression is mediated through this enhancer by inhibition of the enhancer activity, and (iv) c-myc expression serves as an intermediate step downstream from the membrane bound neu-encoded receptor in this complicated feedback inhibition pathway.^ In addition, a part of my research is studying the neu-encoded receptor molecule. I have generated a construct coding the neu ligand-binding domain and demonstrated that (i) the neu ligand-binding domain is a secretory peptide, (ii) it inhibits the normal neu-associated tyrosine kinase but not activated neu-associated tyrosine kinase. My study provided experimental evidence for the mechanisms of neu gene activation. ^

Regulation of the {\it neu\/} oncogene by the GTG element binding proteins

A previous study in our lab has shown that the transforming neu oncogene ($neu\sp\*$) was able to initiate signals that lead to repression of the neu promoter activity. Further deletion mapping of the neu promoter identified that the GTG element (GGTGGGGGGG), located between $-$243 and $-$234 relative to the translation initiation codon, mediates such a repression effect. I have characterized the four major protein complexes that interact with this GTG element. In situ UV-crosslinking indicated that each complex contains proteins of different molecular weights. The slowest migrating complex (S) contain Sp1 or Sp1-related proteins, as indicated by the data that both have similar molecular weights, similar properties in two affinity chromatographies, and both are antigenically related in gel shift analysis. Methylation protection and interference experiments demonstrated these complexes bind to overlapping regions of the GTG element. Mutations within the GTG element that either abrogate or enhance complex S binding conferred on the neu promoter with lower activity, indicating that positive factors other than Sp1 family proteins also contribute to neu promoter activity. A mutated version (mutant 4) of the GTG element, which binds mainly the fastest migrating complex that contains a very small protein of 26-kDa, can repress transcription when fused to a heterologous promoter. Further deletion and mutation studies suggested that this GTG mutant and its binding protein(s) may cooperate with some DNA element within a heterologous promoter to lock the basal transcription machinery; such a repressor might also repress neu transcription by interfering with the DNA binding of other transactivators. Our results suggest that both positive and negative trans-acting factors converge their binding sites on the GTG element and confer combinatorial control on the neu gene expression. ^

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Transformation studies of a nontumorigenic rat prostatic epithelial cell line with activated {\it myc\/} and {\it neu\/} oncogenes

A cloned nontumorigenic prostatic epithelial cell line, NbE-1.4, isolated from Noble (nbl/crx) rat ventral prostate, was used to examine the potential role of activated myc and neu oncogenes in prostate carcinogenesis. Transfection of SV40 promoter/enhancer driven constructs containing either v-myc, truncated c-myc, or neu-T (activated neu) oncogenes was accomplished using calcium phosphate-mediated DNA transfer. Cells were cotransfected, as necessary, with pSV2neo, allowing for selection of positive clones using the antibiotic geneticin (G418). G418 resistant colonies were pooled in some cases or limiting dilution exclusion cloned in others as described. Transfection of NbE-1.4 cells with activated myc oncogenes resulted only in the partial transformation. These cells display an altered morphology and decreased dependence on serum factors in vitro; however, saturation density, soft agar colony formation and growth assay in male athymic nude mice were all negative. Transfection and overexpression of NbE-1.4 cells with an activated neu oncogene alone resulted in tumorigenic conversion. Cell transformation was evident following an examination of the altered cellular morphology, an increased soft agar colony formation, and an acquisition of a tumorigenic potential when injected s.c. into male athymic nude mice. neu-transformed NbE-1.4 cells displayed elevated activity of the neu receptor tyrosine kinase. Furthermore, qualitative changes in tyrosine phosphorylated proteins were found in neu transformed cell clones. These changes were associated with elevated expression of mRNAs for laminin $\beta$1, $\beta$2, and procollagen type IV. The expression of fibronectin and E-cadherin, which are often lost during tumorigenesis, did not correlate with the tumorigenic phenotype. Therefore, it appears that neu oncogene overexpression has been found to be associated with the transformation of rat prostatic epithelial cells, presumably through alterations in gene expression that regulate extracellular matrix. The possible interrelationship and functional significance between neu oncogene expression and the elevated extracellular matrix gene expression is discussed. ^

New molecular approaches to control rhabdomyosarcoma onset and metastasis in preclinical systems

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

Axl -Gas6 signaling counteracts adenovirus type 5 E1A-mediated cell growth suppression and proapoptotic activity

The adenovirus type 5 E1A (abbreviated E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the EGF receptor, Her-2/Neu, Src, and Axl are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (Proc. Natl. Acad. Sci, 93, 5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. RT-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified as Axl by analyzing the Alu I-digested RT-PCR products. We isolated the DNA fragment of interest, and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl (ip1-E1A-Axl). The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in the control cells ip1-E1A and protected the Axl-expressing cells from serum deprivation-induced apoptosis. Further study showed that Akt is required for Axl-Gas6 signaling to prevent ip1-E1A-Axl cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis, and thereby contributes to E1A's anti-tumor activities. ^

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation ( FISH). Participating laboratories were blinded to these test results. Oestrogen receptor ( ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH ( kappa coefficient=1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good ( kappa coefficient=0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

Mechanisms of HER2/{\it neu\/} proto-oncogene overexpression in breast cancer cells

Overexpression and amplification of HER2/neu have been documented in many primary tumors, most notably in breast. Not only do approximately 30% of breast cancer patients carry tumors that overexpress the gene, but those that do generally have shorter overall and disease-free survival times than patients with tumors expressing low levels of HER2/neu. Thus, overexpression of HER2/neu plays an important role in the pathogenesis of breast cancer. We have examined the mechanisms that result in HER2/neu overexpression in breast cancer by using, as a model system, established breast cancer cell lines that express much higher levels of HER2/neu mRNA than normal breast tissue while maintaining a near normal HER2/neu gene copy number. Nuclear run-on experiments indicate that the breast cancer cell lines MDA-MB453, BT483, and BT474 have an increased HER2/neu gene transcription rate. By using HER2/neu promoter-CAT constructs, we have found that the enhanced HER2/neu transcription rate in MDA-MB453 cells is due to activation of the gene in trans, while the enhanced transcription rate in BT483 cells is due to activation of the gene in either trans or cis. In BT474 cells, transcriptional upregulation is primarily due to gene amplification. Since the levels of increased transcription are not as high as the levels of HER2/neu mRNA in any of these three lines, post-transcriptional deregulation that increases HER2/neu expression must also be functioning in these cells. The half-life of HER2/neu mRNA was measured and found to be equivalent in these lines as in a control. Thus, the post-transcriptional deregulation is not increased stability of the HER2/neu transcript.^ Much work has been performed in characterizing the altered trans-acting factor involved in increased HER2/neu transcription in MDA-MB453 cells. Using promoter deletion constructs linked to a reporter gene, the region responsive to this factor was localized in the rat neu promoter. When human HER2/neu promoter constructs were used, the homologous sequence in the human promoter was identified. Furthermore, a number of protein/DNA complexes are detected when these promoter regions are used in gel mobility shift assays. UV-crosslinking experiments indicate DNA-binding proteins of roughly 110 kDa, 70 kDa, and 35 kDa are capable of interacting with the human promoter element. ^

An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

Expressão de HER-2/neu e p53 em adenocarcinoma de esôfago e cárdia

A incidência de adenocarcinoma de esôfago e cárdia tem aumentado nas últimas décadas por razões ainda não conhecidas. São doenças da civilização ocidental. A incidência de adenocarcinoma de esôfago e carcinoma de cárdia ultrapassou a de carcinoma epidermóide de esôfago. O desenvolvimento da biologia molecular e descoberta de oncogenes e genes supressores de tumores permitiu novos achados e melhor entendimento das características moleculares dos carcinomas de esôfago e cárdia. Novos genes envolvidos em ciclo celular, apoptose e reparo de DNA são agora alvo importante de estudos da patogênese destes tumores. HER-2/neu é um oncogene expresso in diversas neoplasias e relacionado à pior prognóstico. P53 é igualmente importante estando mutado em 50%-70% das neoplasias sólidas com implicações clínicas para muitos tumores. Este trabalho determina a frequência de p53 e HER-2/neu através de imunohistoquímica utilizando anticorpos policlonais e monoclonais DAKO anti-HER-2/neu e p53 respectivamente. Foram selecionados 22 casos de adenocarcinoma de esôfago e carcinoma de cárdia do departamento de cirurgia. HER-2/neu foi positivo em 47.7% dos casos, média entre dois observadores. P53 foi positivo em 36.6% dos casos. A correlação entre os escores de HER-2/neu e p53 foi estabelecida usando o coeficiente de correlação de Spearman que mostrou um resultado negativo –0.27 para o primeiro observador que não foi significante. Para o segundo observador, a correlação foi a mesma -0.27 e não significante, mostrando que o aumento na expressão de HER-2/neu não está relacionada com aumento de expressão de p53. Nós concluímos que a expressão de HER-2/neu neste grupo de neoplasias, necessita de investigações futuras e que mesmo estando alterado com muitos outros oncogenes em outros trabalhos, p53 não está correlacionado com aumento de expressão de HER-2/neu nesta série de casos.