SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer based on bronchial aspirates

Abstract

Background: This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.

Methods: Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.

Results: Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).

Conclusions: Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.

Figure 1

Location and results of the DMH and real-time PCR assays used for discovery and confirmation of SHOX2 DNA methylation as a lung cancer biomarker. Tissue samples from 20 normal lungs and 35 lung tumors (14 adenocarcinoma, 11 squamous, 5 large, and 5 small cell lung carcinoma) were analyzed by DMH. Results were confirmed using a SYBR Green HM real-time PCR assay.

Figure 2

Analytical assay performance. Analytical performance of the quantitative real time PCR assay for quantifying SHOX2 DNA methylation. Different amounts of methylated DNA (3.1 - 10,000 pg) were spiked into a background of 50,000 pg unmethylated DNA. Number of replicates: Sixteen (0 and 3.1 pg), eight (6.2-200 pg) and three (1,250-10,000), respectively.

Figure 3

Clinical performance of the SHOX2 DNA methylation biomarker. Valid measurements were obtained from 523 patients (281 cases, 242 controls). A: SHOX2 DNA methylation values measured in cases (red) and controls (black). Low ΔΔCT indicate a low SHOX2 DNA methylation. A ΔΔCT = 0 refers to a sample showing the same methylation as the calibrator DNA (1%). B: Resulting sensitivity and specificity when using alternative cut-offs for patient stratification. C: Receiver Operating Characteristic (ROC) and the resulting Area Under the Curve (AUC).