TOPOIIα and HER-2/neu overexpression/amplification in Barrett’s oesophagus, dysplasia and adenocarcinoma

Rossi E, Villanacci V, Bassotti G, Donato F, Festa A, Cengia G, Grisanti S & Cestari R (2010) Histopathology57, 81–89 TOPOIIα and HER-2/neu overexpression/amplification in Barrett’s oesophagus, dysplasia and adenocarcinoma Aims: Topoisomerase IIα (TOPOIIα) and HER-2/neu are chromosome 17q genes coamplified in various cancers; no data exist for Barrett’s oesophagus (BO) and BO adenocarcinoma (ADC). The aim was to investigate gene amplification and protein overexpression of TopoIIα and Her-2/neu in non-dysplastic BO, dysplastic BO, Barrett ADC, and chromosome 17 aneusomy. Methods and results: Forty-four patients [18 BO, 13 BO with dysplasia (five low-grade dysplasia, eight high-grade dysplasia) and 13 ADC in BO] were evaluated by immunohistochemistry and fluorescence in situ hybridization (FISH). Genes (HER-2/neu and TOPOIIα) and chromosome 17 were evaluated by FISH. Patients with BO, dysplasia and ADC were compared. A significant association was found between TOPOIIα protein overexpression and TopoIIα gene amplification, chromosome 17 aneusomy, HER-2/neu gene amplification and HER-2 protein overexpression as well as between HER-2 protein and HER-2/neu gene, TopoIIα gene and aneusomy for chromosome17, and between the genes TOPOIIα and HER-2/neu. Gene amplification (HER-2/neu, TOPOIIα), protein overexpression (HER-2/TOPOIIα), and chromosome 17 aneusomy were associated with dysplasia or ADC. Most BO patients showed no amplification/overexpression/aneusomy for the above genes, proteins and chromosome, with no differences between dysplasia and ADC. Conclusions: HER-2/neu and TOPOIIα amplification/overexpression might discriminate between BO and dysplasia/ADC. Chromosome 17 aneusomy is associated with dysplasia or ADC in BO.


Introduction
HER-2 ⁄ neu and topoisomerase IIa (TOPOIIa) genes are both located on chromosome 17q, and they can be co-amplified in cancer. 1 Amplification of both genes has been reported in breast, prostatic, gastric, colorectal and pancreatic carcinomas. [1][2][3][4][5] HER-2 ⁄ neu oncogene encodes for the human epidermal growth factor receptor 2 and it is a well-known marker of poor prognosis in a variety of tumours. 6,7 There are two isoforms of mammalian topoisomerase II, a and b. DNA topoisomerase II catalyses a transient double-strand DNA break, which allows the passage of another DNA duplex through the break before the strands are resealed. TOPOIIa represents the target enzyme for specific anticancer drugs, such as anthracyclines, commonly used for a variety of both haematological and solid neoplasms, including leukaemias, lymphomas and breast cancer. In vitro studies have shown a correlation between the expression level of TOPOIIa in cancer cells and the sensitivity of those cells to topoisomerase inhibitors. 8,9 Some authors have suggested a concordance of HER-2 and TOPOIIa gene amplification in breast cancer, 3 while others have demonstrated that TO-POIIa amplification, identified by fluorescence in situ hybridization (FISH), may occur with or without HER-2 duplication and is often associated with TOPOIIa expression evaluated by immunohistochemistry. 1 In addition to the fact that amplification of HER-2 has become a valid biomarker to identify patients with breast cancer who respond to HER-2 protein targeting therapy, several recent clinical trials have found that HER-2-overexpressing breast cancers, 10 with or without HER-2 amplification, 11 are often responsive to anthracycline-based therapies. In fact, it has been proposed that HER-2 amplification in these tumours may be a marker of TOPOIIa amplification. 12 Recent studies have confirmed that patients with breast cancer with TOPOIIa gene amplification are more sensitive to TOPOIIa-based therapy. 13 How ever, it remains controversial whether gene amplification results in overexpression of the TOPOIIa protein. 9,14,15 Adenocarcinoma (ADC) of the oesophagus is currently the cancer with the fastest increasing incidence in the USA, and has replaced squamous cell carcinoma as the most common oesophageal malignancy. 16,17 In fact, an increase in relative and absolute numbers of ADCs of the lower third of the oesophagus has been observed in many Western countries. The most likely explanation for this finding seems to be the increasing prevalence of Barrett's oesophagus (BO) as a consequence of gastro-oesophageal reflux, which is becoming more common with increasing levels of obesity.
The present study was undertaken to investigate: (i) the role of amplification ⁄ overexpression of TOPOIIa and HER-2 ⁄ neu genes and proteins, (ii) the association between TOPOIIa amplification ⁄ overexpression, HER-2 ⁄ neu amplification ⁄ overexpression and chromosome 17 aneusomy, and (iii) the association between TOPOIIa and HER-2 ⁄ neu amplification ⁄ overexpression and chromosome 17 aneusomy and the presence of BO, low-grade (LGD) or high-grade dysplasia (HGD) and ADC.

Patients and methods patient selection, clinical and endoscopic evaluation
The clinical records and histological specimens of 44 patients (six women and 38 men, age range 39-89 years) with a confirmed diagnosis of BO were analysed retrospectively. All patients underwent surveillance endoscopy at regular intervals or when clinically indicated at the Digestive Endoscopy Unit of the University of Brescia. Inclusion criteria were: a confirmed histological diagnosis of BO, oesophageal dysplasia (LGD and HGD) and ADC. Overall, specimens were obtained in 32 patients from biopsies and in 12 patients from mucosectomies.

pathological evaluation
Immediately after sampling, the specimens were fixed in 10% neutral-buffered formalin for 24 h, routinely processed in paraffin and stained with haematoxylin and eosin (H&E) and Alcian-periodic acid-Schiff for routine histological examination. H&E-stained slides from the resection specimens were evaluated for identification of the steps in cancer progression. ADC and precursor lesions were diagnosed according to the World Health Organization classification, 18 as previously reported. 19,20 We selected those slides with obvious areas showing BO (100% showed areas with BO not associated with dysplasia), LGD (in >90% of the areas), HGD (in >90%) and ADC (in >90%). The cases of dysplasia were not associated with an invasive carcinoma.
Serial 3-lm sections were cut for FISH and immunohistochemistry, and the first and last sections of each series were stained with H&E. Corresponding areas on sequential sections were thus investigated by the two methods and for both Topo IIa and Her-2 ⁄ neu.
HER-2 and TOPOIIa status was studied by immunohistochemistry and FISH on paraffin-embedded tissue. Numerical alterations of chromosome 17 [chromosome enumeration probe 17 (CEP17)] were also evaluated by FISH.
According to the recommendations of the manufacturer, tissue sections mounted on slides and stored at room temperature (25°C) were stained within 4-6 weeks from sectioning, in order to preserve the antigenicity, then the samples were counterstained with Mayer's haematoxylin. HER-2 oncoprotein expression was assessed by two investigators (E.R., V.V.), following the scoring system recommended by the manufacturer's instructions and the Food and Drug Administration (FDA) guidelines, according to the Hercep Test Ò criteria. 21,22 Immunoreactivity was scored as follows: 3+, complete and intense membranous reactivity of >10% of tumour cells; 2+, complete but moderate reactivity of >10% of cells; 1+, weak and incomplete reactivity in >10% of cells; and 0, no membranous reactivity, or reactivity in <10% of cells.
To evaluate TOPOIIa protein expression, formalinfixed, 3 lm thick paraffin-embedded tissues were cut, mounted on charged slides, and dried. For immunohistochemistry, slides were deparaffinized and rehydrated in graded solutions of ethanol and distilled water. Endogenous peroxidase was blocked by incubation with Peroxidase Block and Protein Block (NovoLink Polymer Detection System; Novocastra Laboratories, Newcastle, UK) at room temperature, both for 5 min. The immunohistochemical method involved sequential application of primary antibody to TOPOIIa (H-231, a rabbit polyclonal antibody raised against amino acids 1301-1531 of TOPOIIa of human origin; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:50 for 45 min, then post primary block (NovoLink Polymer Detection System; Novocastra Laboratories) was applied at room temperature for 15 min and a NovoLink Polymer (NovoLink Polymer Detection System; Novocastra Laboratories) for another 15 min. The immunoprecipitate was visualized by treatment with 3¢3-diaminobenzidine chromogen (NovoLink Polymer Detection System; Novocastra Laboratories) for 5 min and counterstained by haematoxylin (Dako). Immunoreactivity was considered positive for TOPOIIa when at least 10% of dysplastic ⁄ neoplastic cells were stained. We applied the same criteria proposed for the quantification of p53 in endometrial and breast carcinomas. 23,24 All samples were scored quantitatively and qualitatively at ·40 high-power fields (HPF) in every section (Nikon Eclipse E400, Tokyo, Japan) (Area of high-power field = 0.146 mm 2 ). TOPOIIa was considered positive when it could be recognized as a nuclear brown stain by immunohistochemistry. TOPOIIa gene was labelled by a locus-specific identifier probe for D17Z1 mixed with a probe specific for the centromeric region of chromosome 17 (17p11.1-q11.1) (LSI TOPO2A SO ⁄ CEP17 SG; Vysis Inc.). We applied the same protocol and scoring for both HER-2 and TOPOIIa.
Counterstaining of nuclei was performed using 4,6-diamidino-2-phenylindole. A special amplification pattern as Her-2 ⁄ neu 'signal clusters', usually with >10 confluent signals, was observed, as previously described. 25,26 Although gene amplification as 'homogeneously stained regions' was clearly evident, this pattern did not allow precise signal enumeration. Thus, the whole area of each neoplastic lesion present in the tissue section was independently evaluated by two investigators (E.R., V.V.) with fluorescence microscopy (Nikon Optiphot-2, Florence, Italy) equipped with selective filters for the fluorochromes used, in HPF (magnification 600·). FISH images were captured and elaborated using Genikon software (Nikon Instruments S.p.A, Florence, Italy). The Her-2 ⁄ neu gene locus was classified as amplified if there were more than twice the number of red (SO labelling) Her-2 ⁄ neu signals than green (SG labelling) centromere 17 signals (ratio >2:1) per cell nucleus. The presence of more than two nuclear red signals accompanied by the same number of nuclear green signals was considered to be indicative of aneusomy (in this case polysomy) of chromosome 17 (ratio 1:1). Following these criteria 23,27 the cell population of each HPF was classified as displaying disomy, polysomy or an amplification of the Her-2 ⁄ neu gene.
For TOPOIIa and HER-2 genes, control slides for FISH were bought from the same probe manufacturers (Vysis, FDA approved), while for immunohistochemical analysis for HER-2 protein control slides were provided by the kit used (Dako, FDA approved). For TOPOIIa immunohistochemical analysis (which is the only method not FDA approved) we used as control a slide of breast carcinoma previously shown to be positive with immunohistochemistry and also confirmed positive by FISH.

statistical analysis
The associations between TOPOIIa gene amplification ⁄ TOPOIIa protein overexpression and HER-2 ⁄ neu TopoIIa and Her-2 in Barrett's oesophagus 83 P-values <0.05 (two-tailed tests) were used to reject the null hypothesis. Immunohistochemistry for HER-2 protein was categorized at four levels based on the FDA's approved scoring system (0, 1, 2, 3); FISH for both TOPOIIa and HER-2 ⁄ neu genes was considered positive when amplified and negative when not amplified, and FISH for chromosome 17 was considerate positive or negative in the presence of chromosome aneusomy or disomy, respectively.

ethical considerations
Since this was a retrospective study, no individual patient identification was done and no study-driven clinical intervention was performed. Thus, a simplified Institutional Review Board approval for retrospective studies was obtained and no patient consent was necessary.

Results
The characteristics of the 44 subjects included in the study and the results of the overexpression ⁄ amplification of the genes investigated are shown in Table 1. HER-2 ⁄ neu gene copy number was higher because clusters were identified (>10 signals), whereas TOPOIIa copy number was lower because it was often possible to count the single spots. No deletions were seen in any of the 44 patients analysed. Almost perfect agreement (43 ⁄ 44, 98%) was found between FISH TOPOIIa and HER-2 ⁄ neu gene amplification, with the single exception of subject 22 (P < 0.001). A statistically significant association was found between immunohistochemical TOPOIIa protein overexpression and FISH TOPOIIa gene amplification (P < 0.001), FISH HER-2 ⁄ neu gene amplification (P < 0.001), immunohistochemical HER-2 protein overexpression (P < 0.001) and FISH chromosome 17 aneusomy (P = 0.03). A strong association was also found between immunohistochemical HER-2 protein overexpression and FISH HER-2 ⁄ neu gene amplification (P < 0.001), FISH TOPOIIa gene amplification (P < 0.001) and FISH aneusomy for chromo-some17 (P = 0.02). No association was found between FISH chromosome 17 aneusomy and either FISH HER-2 ⁄ neu or TOPOIIa gene amplification (P > 0.1 for both). Table 2 shows the distribution of the three groups of subjects according to each gene ⁄ protein assessment and chromosome aneusomy ⁄ disomy. Gene amplification (FISH HER-2 ⁄ neu and TOPOIIa), protein overexpression (HER-2, TOPOIIa) and chromosome 17 aneusomy were all associated with the presence of dysplasia or ADC with respect to BO (P < 0.05 for each comparison). Among subjects with BO, none showed HER-2 ⁄ neu or TOPOIIa amplification or chromosome 17 aneusomy, and almost none had moderate to complete membranous immunoreactivity for HER-2 protein expression (immunohistochemistry) (score 2 or 3) or >50% TOPOIIa protein expression (immunohistochemistry) (quartiles 3-4). On the other hand, 30-61% of subjects with dysplasia or ADC showed gene amplification and protein overexpression for both HER-2 ⁄ neu or TOPOIIa and chromosome 17 aneusomy. No significant differences in positivity were seen between dysplasia and ADC for any the Table 2. Distribution of subjects according to gene amplification, protein expression and histology  markers analysed. Representative images are shown in Figures 1 and 2.
With the single exception of case 22, all the cases of dysplasia and ADC showed co-amplification of TOPOIIa and HER-2 ⁄ neu genes (>50%). HER-2 ⁄ neu gene amplification was higher then TOPOIIa ( Figure 1D) because of the clustering (>10 signals) ( Figure 2B), as also reported in previously. 3,4,28 No deletions were seen in any of the 44 patients analysed. There was no significant association between protein ⁄ gene overexpression ⁄ amplification and age or gender (data not shown).

Discussion
Topoisomerases are nuclear enzymes that regulate cellular processes such as replication and transcription; in addition, these enzymes represent a major molecular target for drugs of the anthracycline class or chemotherapeutic agents. 9 Clinical and in vitro evidence supports the concept that in breast cancer the association between HER-2 amplification and response to anthracycline-based chemotherapy is not a direct effect of HER-2 overexpression but the result of co-amplification of the TOPOIIa gene. 29 Some studies have shown that increased expression of TOPOIIa in BO is associated with HGD, 30 as well as the fact that HER-2 is overexpressed in oesophageal ADC 31 and this overexpression may predict early transition from dysplasia to ADC in BO. 32 However, to the best of our knowledge the expression of TOPOIIa and HER-2 has not been previously investigated in BO patients.
In the present study we examined HER-2 ⁄ neu and TOPOIIa gene ⁄ protein by FISH and immunohistochemistry, respectively, and we also investigated chro-  Table 1). B, Area of high-grade dysplasia, where the cells are positive for TOPOIIa (case 30, Table 1). C,D, Fluorescence in situ hybridization for TOPOIIa. C, A normal oesophagus displays two signals for TOPOIIa (red spots) and for chromosome17 (green spots). D, Low-grade dysplasia with gene amplification (patient 22, Table 1). mosome 17 status and histological features (BO, LGD, HGD and ADC).
In agreement with previous studies on breast carcinomas, 3 we confirmed that TOPOIIa is rarely amplified in the absence of HER-2 amplification, that it is co-amplified with HER-2 in dysplasia (LGD and HGD) and ADC, and that the HER-2 gene copy number, because of clustering, was higher than the TOPOIIa copy number. Moreover, statistically significant associations were found between gene amplification and protein expression for both HER-2 and TOPOIIa, between HER-2 and TOPOIIa gene amplification and between HER-2 and TOPOIIa protein expression, confirming the results of previous studies regarding pancreatic and gastric cancers. 2,4 Chromosome 17 aneusomy was found to be associated with TOPOIIa protein overexpression but not with each gene amplification. No deletions for TOPOIIa and ⁄ or HER-2 ⁄ neu were seen in any of our patients, whereas monoallelic deletions were found in other pathological conditions, i.e. breast cancer. 3 We investigated the associations between the above-mentioned markers and pathological findings. We found HER-2 ⁄ TOPOIIa gene amplification ⁄ protein overexpression and chromosome 17 aneusomy (polysomy) in a high proportion of patients with dysplasia (LGD and HGD) or ADC, but in almost none of the patients with BO, suggesting involvement of these factors in cancer development. It is well known that BO is associated with gastrointestinal acid reflux, and it has been suggested that acid reflux could introduce mutations in oesophageal cells due to acid pH-induced DNA damage. 33 However, acid reflux also causes inflammatory responses known to contribute to carcinogenesis. 34 Recent clinical studies have shown that TOPOIIa gene amplification is a more specific predictor than TOPOIIa expression assessed by immunohistochemistry and HER-2 gene amplification for clinical response to TOPOIIa inhibitors in breast cancer. 35 For this reason, FISH testing for TOPOIIa status in addition to HER-2 evaluation may be useful in the characterization of Barrett's pathology and progression to dysplasia. Furthermore, ascertaining TOPOIIa status might be useful to select patients for combination therapy: a trial could be designed to investigate whether patients with TOPOIIa amplification may be treated with HER-2 targeting drug together with a cytotoxic drug, such as TOPOIIa-inhibitor.
In conclusion, these findings suggest that the investigation of these markers might be useful in characterizing the evolution from BO to dysplasia and ADC. These potential markers might also contribute to deciding alternative therapeutic approaches, as suggested by some preliminary data. 36 A B Figure 2. A,B, Case 29. HER-2 analysed respectively by immunohistochemistry and fluorescence in situ hybridization. A, All the areas of dysplasia are positive for the membranous stain which identifies HER-2 receptor. B, The same area of dysplasia. HER-2 gene amplification (red spots) is strong and shows typical clusters; chromosome17 aneusomy is present in all the nuclei with more than two green signals.