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].
Gastrointestinal tract tumors are notorious for being difficult to be analyzed by standard cytogenetic techniques [
5‐
9]. Fluorescence
in situ hybridization (FISH) assay allows rapid detection of numerical genetics aberrations in interphase nuclei in tumor cells. FISH assay should be used to evaluate cell-to-cell heterogeneity in gene or loci copy number and detect small subpopulations of genetically aberrant cells [
10]. FISH studies have shown numerical aberrations 1, 7, 8, 9, 17, 20, X and Y to be common in GC [[
7,
11‐
19], see also review [
20]]. There are some studies in literature concerning
TP53, located at chromosome 17p13.1, and chromosome 17 (chr17) copy number alterations by FISH assay in GC [[
21‐
24], see also review [
20]].
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.
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‐
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 intestinal-type 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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RRB and MACS designed the study. ASK, ACG, DQC, ADS, EML, MFL were involved in data collection, literature searches, genetic and statistical analysis. MHGF, SHBR, SD were involved in pathological analysis. PPA recruited patients and was responsible by samples collection. ASK wrote the first draft of the manuscript. All authors listed have contributed to all subsequent drafts, and have approved the final manuscript.