SHOX2 DNA methylation – validation of a lung cancer biomarker in bronchial lavage


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John Field1, Dimo Dietrich2, Christoph Kneip2, Olaide Raji1, Triantafillos Liloglou1, Anke Seegebarth2, Thomas Schlegel2, Nadja Flemming2, Sebastian Rausch2, Juergen Distler2, Michael Fleischhacker3, Volker Liebenberg4, Thomas Giles5, Martin Walshaw5, Chris Warburton6, Bernd Schmidt7
1The University of Liverpool, Liverpool, United Kingdom,2Epigenomics AG, Berlin, Germany,3Charite-Universitatsmedizin, Berlin, Germany,4Metanomics Health GmbH, Berlin, Germany,5Royal Broadgreen University Hospital Trust, Liverpool, United Kingdom,6Aintree University Hospital Trust, Liverpool, United Kingdom,7Universitaetsklinikum Halle- Saale, Halle, Germany

Background

DNA methylation has been shown to play a major role in carcinogenesis and cancer progression and the profiling of specific genes may provide clinically useful  biomarkers. In our previous case-control study on 500 patients, hypermethylation of SHOX2 in bronchial lavage has been shown to be a clinically useful tumor marker for identifying subjects with lung cancer, especially if histological and cytological findings after bronchoscopy are ambiguous. We have developed a diagnostic test for the SHOX2 DNA methylation biomarker which can be used in the clinical area, called the “Epi proLung BL Reflex Assay”. This study validates this biomarker in an independent patient dataset.

Method

The assay is composed of: “DNA Preparation Kit” for the preparation of bisulfite converted DNA; “Real-time PCR Kit” for the determination of SHOX2 DNA methylation; “Work Flow Control Kit” for controlling the detection process. This study describes validation of the biomarker in a blinded and randomized case control study comprised of 250 patients (125 cases, 125 controls).

Results

The  SHOX2 methyaltion assay was shown to be a robust and reliable diagnostic tool for identifying patients with lung cancer using Saccomanno-fixed bronchial lavage specimens. The assay detected patients with lung cancer sensitivity of 78% [CI = 69-86]. Specificity of the test was 96% [CI = 90-99] resulting in an accuracy (area under the ROC curve, AUC [95% CI]) of 0.95 [CI = 0.91-0.98].

Conclusion

The SHOX2 biomarker, measured in the new CE-marked IVD, Epi proLung BL Reflex Assay has been validated in a case control series with a clinically relevant sensitivity and specificity. This biomarker provides a method to accurately assess the SHOX2 DNA methylation status in bronchial lavage specimens from patients with suspected lung cancer and could be utilized in the diagnosis of lung cancer as an adjunct to existing clinical and pathological information.