Interrelationship between TP53 gene deletion, protein expression and chromosome 17 aneusomy in gastric adenocarcinoma

Background This study evaluates the existence of numerical alterations of chromosome 17 and TP53 gene deletion in gastric adenocarcinoma. The p53 protein expression was also evaluated, as well as, possible associations with clinicopathological characteristics. Methods Dual-color fluorescence in situ hybridization and immunostaining were performed in twenty gastric cancer samples of individuals from Northern Brazil. Results Deletion of TP53 was found in all samples. TP53 was inactivated mainly by single allelic deletion, varying to 7–39% of cells/case. Aneusomy of chromosome 17 was observed in 85% of cases. Chromosome 17 monosomy and gain were both observed in about half of cases. Cells with gain of chromosome 17 frequently presented TP53 deletion. The frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC. Immunoreactivity of p53 was found only in intestinal-type samples. The frequency of cells with two chr17 and two TP53 signals found was higher in samples with positive p53 expression than in negative cases in intestinal-type GC. Conclusion We suggest that TP53 deletion and chromosome 17 aneusomy is a common event in GC and other TP53 alterations, as mutation, may be implicated in the distinct carcinogenesis process of diffuse and intestinal types.


Background
Gastric cancer (GC) is the fourth most frequent malignancy and the second most common cause of cancer death in the World [1]. In the State of Pará (Northern Brazil), GC was the most common cause of cancer death in 2000. In Belém, State of Pará, the 5-year-survival rate is about 9-10% [2]. A better understanding of the biology of this neoplasia progression is crucial for the development of better tests to early neoplasia detection and also of new treatment strategies for GC.
Molecular events in the carcinogenesis of GC remain largely unknown [3]. A key feature in the pathogenesis of most GC, as in many other solid cancers, is chromosomal instability, resulting in gains and losses of parts or even whole chromosomes [4].
The TP53 tumor suppressor plays a pivotal role in the coordination of the repair process or in the induction of apoptosis. TP53 somatic alteration is described in approximately 50% of human cancers, including GC [25]. Deregulation of the TP53 pathway has been shown to involve mutations, loss of heterozygosity (LOH), increased expression of the TP53 inhibitor HDM2, or epigenetic silencing of the TP53 promoter [26,27].
The aim of this study was to investigate chr17 and TP53 numerical alterations in GC samples from Pará State by dual-color FISH technique. Immunostaining for p53 protein was also evaluated. These results were correlated with clinicopathological characteristics.

Samples
The study included 20 gastric adenocarcinoma samples. Samples of primary tumors submitted to surgical resection were obtained from João de Barros Barreto University Hospital (HUJBB). This study investigated cancer samples of patients from Pará State, where there is a mixed population composed of three main ethnic groups: Amerindian, African and European [28].
Patients' age, sex and tumor anatomical sites were obtained from tumor registries. The mean age of the twenty patients was 55 ± 14.67 years (range 24-77). The female/male ratio was 3:2. All samples were classified according to Laurén [29] and tumors were staged using standard criteria by TNM staging [30]. According to Laurén's classification, 6 were diffuse type (30%) and 14 were intestinal type (70%). Table 1 shows cases with their histopathological characteristics.
All patients had negative histories of exposure to either chemotherapy or radiotherapy prior to surgery; there was no other diagnosed cancer. An informed consent with approval of the ethics committee of HUJBB was obtained from the studied patients.

Fish
FISH was applied on cells fixed in methanol/acetic acid using recently made slides according to modified protocols [31]. The slides were washed in 2× saline sodium citrate (SSC)/0.5% NP-40 (pH 7.0) solution and dehydrated in 70%, 80% and 95% ethanol. To determine the chr17 and TP53 copy numbers, cells were hybridized with 10 μL dual-color direct labeled probe (Qbiogene ® , CA, USA) specific for chr17 α-satellite and TP53 gene region, labeled with fluorescein and rhodamine respectively. The probe applied to the slide under a glass coverslip. The probe and sample were denatured at 75°C for 5 minutes and. In situ hybridization occurred at 37°C in a moist chamber overnight. Post-hybridization washings were done and the nuclei were counterstained with DAPI/antifade. Molecular cytogenetic analysis was carried out under an Olympus BX41 fluorescence microscope with triple DAPI/FITC/ TRICT filter (Olympus, Japan) and the FISHView ® of Applied Spectral Imaging ® image analysis system (ASI Ldt., Israel). For each case, 200 interphase/metaphase nuclei were analyzed and were scored using the Hopman's criteria [32]. In our study, the cut-off level for interphase-FISH was 5%. To avoid misinterpretation due to technical error, gastric mucosal tissue (nonneoplastic) and normal lymphocyte nuclei were used as negative control.

Immunohistochemical staining
Deparaffinized tissue sections (4 μm) were incubated with primary monoclonal antibody p53 (DO-7, dilution 1:50, DakoCytomation, CA, USA) and secondary antibody followed by streptavidin-biotin-peroxidase complex (DakoCytomation, CA, USA) as previously described [18]. Slides were visualized with diaminobenzidine-H 2 O 2 and counterstained with Harry's hematoxylin. The results were interpreted using the Ozturk's et al. criteria [33]. Positive p53 expression was defined as clear nuclear staining, whereas negative p53 immunostaining was considered when no positive cell was seen or rare cells were stained (less than 10% weakly stained tumor cells). A breast ade-nocarcinoma sample with known p53 immunoreactivity was used as positive control and a normal gastric mucosa as negative control. Two pathologists evaluated the immunostaining results independently.

Statistical analyses
Statistical analyses were performed using Fisher's exact test and Mann-Whitney test. P value < 0.05 was considered to be statistically significant.

FISH
Lymphocyte nucleus and normal gastric mucosa showed two signals to chr17 and TP53 in 97.5% and 96.5% of analyzed cells respectively. All cancer samples presented numerical alterations of chr17 and TP53 gene. Normal nuclei were observed in 50.7-88% of cells/case ( Table 1).
The main TP53 alteration observed was the single allelic deletion. This alteration was present in all cases, varying to 7-39% of cells/case ( Figure 1A). Chr17 monosomy observed in 45% of samples, ranging 5-12% of cells/case. Chr17 gain was also detected in 45% of cases. Chr17 trisomy was observed in 20% of the cases in a frequency up to 7.5% cells/case (case 19), in which 40% and 60% of these cases showed two and one single TP53 copy number respectively. Chr17 tetrasomy with two TP53 signals was detected in 35% of cases in a frequency up to 9% cells/ case. Four signals to chr17 and TP53 was observed in one case (case 2) in 13.5% of cells (Table 1).
The frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC (p = 0.026). Chromosome alterations were not associated with other clinicopathological characteristics.

p53 protein expression
In the present study, breast adenocarcinoma (positive control) showed p53 overexpression and the normal gastric mucosa (negative control) showed lack of p53 immunoreactivity.
Seventeen GC samples were analyzed by immunostaining. Immunoreactivity of p53 (positive) was found in 8 cases (47%) ( Figure 1B). All of these cases were intestinaltype (8 of 12 samples). Statistical analysis showed an association between intestinal-type GC and p53 expression (p = 0.0294). In our sample, p53 immunoreactivity was not associated with age, gender, location and TNM status (p > 0.05) ( Table 1).

Cells submitted to FISH (A) and immunohistochemistry (B) assays
found was higher in samples with positive p53 expression than in negative cases (p = 0.027).

Discussion
Aneusomy is one of the most common findings in human cancer. Chromosome copy number changes encompass a continuum ranging from sporadic events to a change of chromosome numbers with each cell division. Although aneusomy can be detected at early stages of transformation and even in certain premalignant lesions, the degree of numerical chromosomal aberrations generally increases with tumor progression, and tumors with aggressive clinical behavior are more likely to be aneusomy than less malignant tumors. Aneusomy has also been found to be associated with poor treatment outcome in cancer patients [34].
Regarding chr17 and TP53 copy number, we observed normal nuclei in 50.7-88% of cells/case. This result corroborates our previous conventional and molecular cytogenetic studies, which demonstrated that chr17 aneusomy is not a frequent in GC samples of individual from Northern Brazil [7,35,36].
There are few studies in literature concerning chr17 and TP53 copy number alterations. Our findings corroborated Kobayashi et al. [21] that found deletion of TP53 in 39% of 67 tumors and all of these samples showed chr17 polysomy. Takahashi et al. [22] also observed that p53 signal count was lower than the chromosome 17 signal count in 1 of 3 intestinal-type GC.
Gomyo et al. [23] demonstrated 3 or 4 signals for chr17 in 46% of 13 intestinal-type GC samples and 77% of these cases showed TP53 deletion by FISH assay. In our sample, 45% of all cases presented 3 or 4 signals for chr17 and TP53 deletion was detected in all cases.
Suzuki et al. [24] observed an increased of chr17 polysomy frequency and the incidence of TP53 deletion ranged from 55% to 90% in ten GC samples. They also described that TP53 deletion was significantly higher in intestinal than in diffuse-type cancers. However, in our sample we found TP53 deletion in up to 49% cells/case and the frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC. Inconsistencies regarding the frequency of TP53 deletion in GC between our study and Suzuki et al. [24] may be suggestive of distinct gastric carcinogenesis pathways in different ethnic composition or differences in stage when the analysis was done. It is widely reported that differences between carcinogenesis processes can be the result of distinct environmental and genetic factors.
Suzuki et al. [24] also observed that chr17 monosomy was present in 70% of 10 cases and the most frequent pattern in these cases was the combination of one copy of chr17 and one of TP53. On the other hand, in our sample we observed chr17 monosomy in 45% of 20 cases (cut-off level of 5%) and the more frequent pattern was the combination of two copies of chr17 with one TP53 copy by cell.
In the present study, chr17 tetrasomy with two TP53 signals was frequently observed. We also could observe that chr17 gain tended to be more frequently found in tumors with higher extension (T3 or T4 stages). This finding suggests that tetrasomy event is a subsequent step after gene deletion, which could justify the higher frequency of cells with two copies of chr17 and one TP53 copy and also the tendency of increased level of chr17 gain in tumors with higher extension. Galipeau et al. [37] suggested that increased polysomy level is associated with inactivation of the TP53 in Barrett's esophagus in vivo, supporting our hypothesis.
Williams et al. [38] described that TP53 deletion was the most common aberration in gastritis, intestinal metaplasia, dysplasia e GC by FISH assay. The author suggested that this abnormality may exist in the initiation and progression to gastric cancer.
TP53 deletion, as well as chromosome 17 aneusomy, was observed in all analyzed samples, despite Laurén's histopathologic types. However, differential p53 expression was detected between these groups.
Increased immunostaining of p53 can depend on either increased synthesis of wild-type protein or accumulation of mutated protein in the cell, since the antibody recognizes both types of the protein [39]. In the present study, we observed an increased frequency of immunostained nuclei and the greater staining intensity in 47% of GC samples, as compared to normal gastric mucosa. We suggest that the p53 overexpression may be related to the mutated type of this protein. The frequency of p53 overexpression in GC has been described varying from 19% to 57.5% of cases [23,[40][41][42][43][44] and some studies also described TP53 mutations related with its protein overexpression [23,41].
In the present study, only intestinal-type GC presented p53 immunoreactivity. Our research suggests that, beside TP53 loss by allelic deletion or chr17 aneusomy, a mutation in the remaining TP53 allele may exist in intestinaltype GC samples, which would explain the protein immunoreactivity. On the other hand, two possibilities might be considered to the absence of immunoreactivity in diffuse-type GC: this absence was not due to mutations in TP53 gene or an eventual mutation in this gene would not interfere in the protein accumulation. In both situations the immunoreactivity cannot be detected.
The p53 expression was also associated with a higher frequency of cells with two chr17 and two TP53 signals in intestinal-type GC. We hypothesize that these cells may present TP53 with mutations and this event could be occurring earlier than allelic deletion in intestinal-type gastric carcinogenesis. Further investigations concerning TP53 mutations and expression should be done in larger samples, also including early GC specimens.

Conclusion
Our findings showed that TP53 deletion and chromosome 17 aneusomy are common events in GC. Our results also suggest that LOH is an important TP53 alteration in GC. However, other TP53 alterations than allelic deletion may be implicated in the carcinogenesis process.