We characterized ESCC tumors for complex DNA alterations - LOH and CNV - and related these genomic alterations to gene expression. To our knowledge, this is the first report to comprehensively address the distribution of complex DNA alterations in ESCC and its relation to gene expression on a genome-wide scale.
Ninety percent of cases showed CNNLOH in their tumors and, over all cases, CNNLOH was found on every chromosome arm, indicating that it is a common phenomenon.
The frequency of CNNLOH observed here in ESCC was much less than has been reported in other cancers [3–19]. For example, in colon cancer and basal cell carcinoma nearly all LOH was associated with copy number neutral regions [3, 10]. In general, CNNLOH occurs with variable frequency in different genomic regions in tumors of different origin. There are several differences between the study reported here and previous studies which likely influenced the results. First, DNA from micro-dissected tumor and adjacent normal was used in the present study, while either cancer DNA without matched controls or cancer cell lines were used in most other reported studies. Second, we examined LOH and CN alterations using the same SNP array platform, while other studies used SNPs for LOH and CGH arrays for CN analyses. Third, the criteria for identifying LOH differed among the studies reported. Finally, the types of cancers studied previously differ from the present study which is the first report of CNNLOH in ESCC.
In previous LOH studies, we reported high-frequency LOH on several chromosome arms, including 3p, 4p, 4q, 9p, 9q, 13q, 17p, and 17q [23, 26, 27]. By integrating LOH and CN alteration data in the present study, we can now say that the LOH on 3p is primarily due to CN loss LOH, while the LOH on the other seven chromosome arms is predominantly due to CNNLOH.
Our results showed that CNNLOH can change expression levels of genes in ESCC, either increasing or decreasing them. We do not know why CNNLOH changes gene expression, but one possibility is that the two alleles may have different gene expression levels. For example, if allele A expression is greater than allele B, the expression level for the 3 genotypes would be ordered as AA > AB > BB. CNNLOH with retention of two B alleles (genotype BB) would then show lower expression than genotype AB. Conversely, CNNLOH with loss of the allele B would result in two copies of allele A and a higher level of expression than that of AB cells. Another possibility is that the two alleles have different expression due to different epigenetic states, with LOH resulting in copies with two extreme epigenetic states. A third possibility is that one allele harbors a mutation and subsequent LOH leads to a homozygous mutant. Several studies have shown that CNNLOH regions can harbor mutated genes. For example, JAK2 V617F, FLT3-ITD, AML1/RUNX1, WT1, and NPM1 mutations were all found in CNNLOH regions in AML . These various hypotheses merit testing in the future.
The study design in the present study has several important features: (i) we compared CN status between DNA from germ-line and micro-dissected adjacent normal tissue; (ii) we used micro-dissected DNA from tumor tissue; (iii) we assessed both LOH and CN alterations simultaneously using the same array platform; and (iv) we integrated complex DNA alterations and gene expression data on a genome-wide level using both high density SNP and expression arrays in the same cases. A noteworthy weakness of our study is the relatively small number of cases evaluated (including a particularly small number of cases with both LOH and RNA expression data to evaluate, due in part to the 500K chip mean heterozygosity of 27%), which limited our power to detect significant differences in loci between LOH and non-LOH groups. In addition, findings for ESCC from this high-risk region may not be generalizable to populations elsewhere in the world.
In summary, we investigated the distribution of complex DNA alterations in ESCCs at the genome-wide level and determined that CN neutral is the most common CN state in LOH, and that CNNLOH is a very common phenomenon overall. Importantly, we also showed that CNNLOH could alter the expression level of genes affected in ESCC.