It is widely accepted that a tumor arises from a single cell. However, how it progresses to an advanced stage is still being debated. Early studies of colorectal and pancreatic cancers led to a notion that the development and progression of these cancers are associated with accumulation of chromosomal aberrations, referred to as the multistep tumorigenesis model 
. For example, genomic aberrations of the APC, KRAS, SMAD4 and TP53 genes are involved in the adenoma-carcinoma sequence in the colon 
. However, such studies focused on only a proportion of tumor-related genes, and neglected the role of most other genes. Furthermore, this model was unable to evaluate the significance of intratumoral genomic heterogeneity for tumor development and progression. Meanwhile, recent studies have led to the establishment of another model, designated the clonal evolution model 
. In this model, a single clone evolves into several distinct subpopulations through the accumulation of diverse genetic abnormalities. The predominant population may be replaced by distinct subpopulations within a single tumor mass through the effects of environmental selection pressure and/or the stage of tumor progression. As a consequence, several genetically heterogeneous cell populations may coexist within a single tumor mass. Evidence of intratumoral genetic heterogeneity associated with clonal evolution has been obtained for a variety of solid tumors, including prostate cancer 
, Barrett's esophagus 
, ovarian cancer 
, cervical cancer 
, breast cancer 
, neuroblastoma 
, pancreatic cancer 
, and colorectal cancer 
. Interestingly, in a study of lethal metastatic prostate cancer, no CNAs specifically related to the site of metastasis were found 
. Similarly, in a study of high-grade serous ovarian carcinoma, there was no evidence for a relationship between acquisition of cisplatin resistance and specific CNAs 
. These results suggest that the multistep tumorigenesis model, in which specific aberrations play important roles in tumor development and progression, does not always represent the way in which tumors acquire their malignant character. In the present study, we initially hypothesized that acquisition of specific CNA(s) might be important for submucosal invasion. However, we were unable to find any CNAs that were more frequent in SM than in the paired MU sample. Furthermore, we also observed no significant difference regarding the number of CNAs in the paired MU and SM portions. However, we found that the majority of SMGCs were composed of clonally-related, but genetically distinct subpopulations, suggesting that clonal evolution may occur during the progression of gastric cancer. Taken together, although the number of cases examined was limited, our findings suggested that generation of genetically different subpopulations rather than acquisition of specific CNAs in the MU portion may be important for the process of submucosal invasion. On the basis of these findings, we propose a hypothetical model for the process of SM invasion and LN metastasis of early gastric cancer (). To confirm this hypothesis, further studies with larger samples will be required.
Hypothetical model for the submucosal invasion and lymph node metastasis in early gastric cancer.
Our data indicating that SMGCs are composed of genetically heterogeneous subpopulations are important in the context of gastric cancer research and treatment, because tumor heterogeneity makes the development of effective drugs difficult. Since genomic CNAs have an impact on gene expression profiles in various cancers 
, it is possible that each of the genetically distinct subpopulations within a single tumor may differ in both biological behavior and response to anticancer drugs, including molecular targeting agents. Cooke et al. have proposed that clarification of different genetic subpopulations within a single tumor would allow effective therapy employing a specific agent targeting a common genomic aberration or combined agents targeting unique genomic aberrations in each of the distinct subpopulations 
. This strategy may also applicable to the treatment of gastric cancer.
Among the 23 cases we analyzed, 15 showed a clonal relationship between the MU and SM portions. Furthermore, 13 of the latter 15 cases also showed differences in CNAs between the two regions, suggesting that clonal evolution frequently occurs in the early phase of gastric carcinogenesis. The relationship between the paired MU and SM samples in the other 8 cases without common CNAs remained unclear. Two possible explanations for this can be suggested. One is that tumors in the paired portions, which did not have common CNAs, developed independently. The other is that the paired portions shared other types of genetic aberrations, such as mutations and translocations, which cannot be detected by array CGH. In the latter case, next-generation sequencing might be useful for analyzing such relationships.
In this study, gains at 11q13, 11q14, 11q22, and 14q32, and amplification at 17q21, were more frequent in the SM portion of metastatic SMGCs than in those of non-metastatic SMGCs. Interestingly, gains at 11q13 and 14q32 are reportedly involved in liver metastasis of colon cancer 
. Therefore, these data suggest that gain at 11q13 and 14q32 may be involved in the metastasis of gastrointestinal cancers. Chromosome 17q21 harbors a potent oncogene, ERBB2. Association of ERBB2 expression with the clinicopathological features of gastric cancer has been investigated in several studies 
. However, the influence of ERBB2 overexpression on LN metastasis differed among those studies 
. In the present study, despite the limited number of SMGCs examined, all of those with ERBB2 amplification and overexpression showed lymph node metastasis. Further study using a larger number of SMGCs will be required to evaluate the significance of this tendency.