Correlation of SHOX2 gene amplification and DNA methylation in lung cancer tumors

Abstract

Background: DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.

Methods: SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.

Results: A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference.

Conclusions: Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.

Figure 1

Response curve for the quantification of SHOX2 DNA methylation. Mixtures of bisulfite converted DNA from sperm and bisulfite converted artificially methylated DNA were used as template DNA. Each methylation mixture (0, 0.8, 1.6, 3.1, 6.2, 12.5, 25, 50, and 100%) was measured in five replicates.

Figure 2

SHOX2 DNA methylation in tumor tissue and morphologically normal adjacent tissue (NAT) from 55 lung cancer patients. Ninety-six percent (53/55) of patients exhibited a higher methylation value in tumor tissue as compared to the matched NAT. Fifty-five (30/55) of the NAT showed a low SHOX2 methylation of 0.01 to 0.3%. SHOX2 DNA methylation levels in three tumors were significantly higher than 100%.

Figure 3

SHOX2 RNA expression in tumor tissue and morphologically normal adjacent tissue (NAT) from lung cancer patients. Three qRT-PCR assays specific for both variants and variant a (NM_006884) and b (NM_003030), respectively, were used. Measurements were carried out in triplicates and normalized to SDHA and HPRT1 genes. Valid results were obtained for NAT and tumor tissues from 51 lung cancer patients.

Figure 4

Correlation of gene amplification and DNA methylation of the SHOX2 locus. SHOX2 copy numbers were determined by relating the total amount of SHOX2 copies (determined with real-time PCR) to the amount of total DNA as quantified by UV. Methylation of the SHOX2 locus was measured using a duplex PCR consisting of a HM assay for sensitive quantification of methylated SHOX2 copies and an ACTB reference assay. Means of a triplicate measurement are shown. Gene amplification and DNA methylation correlate highly (p = 0.0002).

Figure 5

Correlation of SHOX2 methylation in 55 tumors as determined using ACTB and SHOX2 as references. Left: Scatter plot of SHOX2 methylation. SHOX2 methylation was determined with an ACTB reference assay (x-axis) and a methylation unspecific SHOX2 reference assay (y-axis), respectively, for quantification of total DNA in a duplex PCR. Means of a triplicate measurement are shown. Right: Scatter plot of ranks. The methylation values as determined with both reference assays were ranked and the respective ranks were correlated.

Figure 6

Summary of comparative genomic hybridization. Abnormalities identified in tumor tissue from patient #1 as compared to the corresponding NAT. Chromosomal amplification is clearly demonstrated for 3q comprising the SHOX2 locus.