Squamous cell carcinoma is the most frequent histological type of cancer of the esophagus (ESCC) 1. It occurs predominantly in developing countries and is the sixth most common neoplasia in Brazil 2. ESCC generally has a poor prognosis because it is usually in an advanced stage at the time of diagnosis. Its epidemiological characteristics suggest the involvement of environmental factors in its etiology 345.

The mortality rate is still high despite the development of many therapeutic modalities in addition to surgical resection. Esophagectomy with lymph node dissection has significant morbidity and mortality rates 567. Therefore, it is important to understand the prognostic factors that determine the most appropriate surgical approach in order to select patients for target-directed therapies.

The family of epidermal growth factor receptors comprises four homologous molecules: EGFR, c-erbB-2, c-erbB-3 and c-erbB-4. Some reports have suggested that abnormal activation of kinase activity in these receptors has an important role in tumor development and progression in ESCC 89. Activation of these receptors induces cell migration and aggregation as well as hyperproliferation and epithelial cell differentiation 1. The importance of these receptors has been studied in many malignant neoplasms, but mainly in breast carcinoma, leading to specific treatments to inactivate them 10.

Gene amplification has been described in many tumor types and is associated with poor prognosis 11. When identified, gene amplification is an important indicator for more intensive treatment, mainly in breast and lung cancer 12131415. It is analyzed by fluorescence (FISH) or chromogenic (CISH) in situ hybridization to measure the gene copy number.

Studies have verified that the HER-2/neu (c-erbB-2) oncogene (located on chromosome 17) is amplified and overexpressed in many tumor types, and have compared these results with immunohistochemical expression and clinicopathological data using Tissue Microarray 1617181920 or conventional slides 212223. Amplification and overexpression of HER-2/neu (c-erbB-2) predict an especially poor prognosis in breast cancer.

Few studies have used FISH to evaluate HER-2/neu amplification in ESCC; these reports show low gene amplification frequencies in this tumor 2425. Mimura et al. (2005) found that 11% of cases showed HER-2/neu amplification, including all cases considered 3+ by immunohistochemical analysis and 3/6 (50%) of those considered 2+ 24. These authors also demonstrated that cases with gene amplification had lower survival rates. A study by Sunpaweravong et al. (2005) showed gene amplification in 2% of cases, but this factor was not associated with overall survival 26. Similar results were presented by Reichelt et al. (2007), in which 5% of the ESCC were positive for amplification of HER-2/neu; however, amplification did not influence prognosis 25.

Although recognition of gene amplification may be a key factor in therapeutic decision-making, there is a paucity of data regarding HER-2/neu amplification in ESCC. In this study, we detect gene amplification of HER-2/neu by fluorescence in situ hybridization and evaluate protein expression by immunohistochemistry in ESCC. We discuss the relationship between the clinicopathological features of this disease and the prognostic value of HER-2/neu amplification.

The TMA technique provides a high-throughput means of evaluating genetic alterations in a great number of tumor samples. Sections from TMA blocks can be used for FISH, allowing gene amplification to be evaluated in hundreds of tumor samples on a single slide.

Immunohistochemical analysis showed that 36.8% of the cases were positive for expression of c-erbB-2. The few studies reporting c-erbB-2 expression in ESCC show discrepant frequencies ranging from 0 to 64% 2728293031. This variability may have resulted from differences in immunohistochemical protocols, different antibody sources used by the different authors, or different criteria for evaluating expression.

We found no significant association between c-erbB-2 expression and the clinicopathological findings. Most reported studies agree with our results and report no influence of c-erbB-2 expression on ESSC prognosis 272832; however, a study performed by Lam et al. (1998) demonstrated a significant association between this protein and histological grade 29, and a recent study showed that c-erbB-2 overexpression is associated with lower rates of survival 33.

The reported frequencies of HER-2/neu amplification in ESCC vary from 2 to 11% of cases 242526. This variability may have resulted from differences in tissue preparation, probes, and the methods used to evaluate the alterations. Our study demonstrated gene amplification in 20% of cases, higher than any previously reported frequency 242526.

In this study, 199 cases were subjected to immunohistochemical evaluation; 47 were also suitable for FISH analysis. The remaining cases had complete loss of tumor cores from the TMA or fewer than 100 tumor nuclei were available for evaluation, or the samples were under- or over-digested, resulting in absence of hybridization signals and loss of cell and nuclear morphology.

One of the problems with the TMA technique for FISH is that fixatives such as formalin preserve the tissue structure by forming cross-bridges, which reduce the access of the probes to their targets. Another important challenge in FISH analysis is to standardize the tissue digestion on TMA: the hundreds of samples on one slide may differ in tissue type, fixation methods and age. Successful FISH analysis depends on a pretreatment protocol that is consistent in the use of digesting agents (sodium thiocyanate, pepsin or proteinase K) as well as appropriate conditions (concentration, temperature and time of incubation) to unmask the target DNA and optimize hybridization. The presence of hundreds of tissue cores on a TMA slide compounds the difficulty of optimization; a given set of conditions appropriate for some samples may over- or under-digest others.

Among the 47 cases suitable for FISH analysis, only those that were 3+ positive for immunohistochemistry showed HER-2/neu amplification (66.7%). The remaining 33.3% of the amplified cases were not suitable for immunohistochemical evaluation because there were no tumor cells in the cores or the tissue cores were completely lost from the TMA.

Mimura et al. (2005) found results similar to those of the present study, but as well as all the 3+ cases, 50% of their 2+ cases also showed gene amplification 24. Another study reported a significant association between gene amplification and protein overexpression in 70% of 3+ and in 30% of 2+ amplified cases 25. In contrast to these results, Sunpaweravong et al. (2005) found no significant association between gene amplification and immunohistochemical expression; the one case positive for amplification did not overexpress c-erbB-2 26.

With respect to overall survival, our data are in agreement with the findings of Mimura et al. (2005), showing significant differences in survival rates in cases with gene amplification 24. The significant association between HER-2/neu amplification and lower survival rate indicates a role for this gene alteration analysis in the prognosis of ESCC cases.

Our results did not show that c-erbB-2 expression has a prognostic value in ESCC. In contrast, Mimura et al. (2005) found that the survival rate in the HER-2-positive group was significantly worse than that in the HER-2-negative group. Despite the concordance between the FISH and immunohistochemistry results, only the presence of gene amplification seems to affect prognosis in ESCC. Unlike the previous report, FISH and immunohistochemistry analyses were performed on different numbers of cases (47 and 185, respectively) in the current study, which could have contributed to the discrepancy between the two studies 24. Further studies with more cases are necessary for a better understanding of the influence of this gene on ESCC progression.

Tumors showing c-erbB-2 overexpression but not gene amplification could occur because of transcriptional or post-transcriptional alterations, polysomy of chromosome 17 or even technical problems such as the high sensitivity of the immunohistochemical procedure. In the current study, the assessment of the number of copies of chromosome 17 showed that some 3+ cases could have resulted from polysomy 17. Transcriptional or post-transcriptional alterations were not evaluated in the present study; further studies employing other techniques such as RT-PCR would be required. Alternatively, the greater sensitivity of immunohistochemistry compared with FISH could also account for this result.

Nonetheless, our results corroborate previous reports that most c-erbB-2 overexpression is caused by gene amplification 14. There is a consensus that c-erbB-2 expression must be evaluated initially by immunohistochemistry and, if the results are not conclusive, FISH should be performed. Such a practice has been standard procedure to assist in making therapeutic decisions in breast and lung cancer cases 12131415.

Trastuzumab (Herceptin) prolongs survival in metastatic breast cancer patients whose tumours overexpress the HER2/neu protein 34. Similarly, patients with ESCC having HER-2/neu gene amplification might also benefit from treatment with Trastuzumab, since HER-2/neu amplification indicates a group of cases in which this type of treatment could improve the prognosis 2435.

Our results indicate that HER-2/neu gene amplification is an important prognostic indicator in ESCC. Further studies with more cases and including additional techniques are necessary to verify other molecular alterations involved in tumor progression, thus assisting in the development of new therapies for ESCC.

The authors declare that they have no competing interests.

YSK designed the study, evaluated the immunohistochemistry and FISH results and wrote the manuscript. JIN carried out all the immunohistochemistry and FISH reactions. JHTGF performed all the statistical analyses. RAS performed the surgical procedures and evaluated the clinical records. FAS designed the study, assisted in analysis of immunohistochemistry results, and wrote the manuscript. All authors read and approved the manuscript.