Purmorphamine We performed binominal classifications case vs

We performed binominal classifications (case vs. control) based on logistic regression (using 63 methylation markers plus plasma-cfDNA concentration as additional predictor) to compare the diagnostic performance of our selected methylation markers with that of previously published markers. We saw the best discriminative power when comparing cancer to healthy samples yielding an AUC=0.91 (95%CI: 0.84–0.96) with a sensitivity of 0.82 (95%CI: 0.61–0.97) and specificity of 0.89 (95%CI: 0.47–0.98). Comparable accuracy using cfDNA analysis from serum or Purmorphamine samples was previously observed in two studies addressing cancer vs. healthy (a) via measurement of global hypomethylation by massively parallel bisulfite sequencing for detection of lung, breast and nasopharyngeal cancer Chan et al., 2013, or (b) via vimentin hypermethylation using methyl BEAMing for diagnosis of colon cancer Li et al., 2009. The most widely used serum protein marker for lung cancer, the carcino-embryonic antigen (CEA) Okamura et al., 2013 and the best cfDNA methylation-based lung cancer assay Kneip et al., 2011, however, do not achieve a comparable discriminative power. In view of the increased cancer risk in ILD and COPD, and given the lack of suitable blood tests for both diseases, we then applied our multiplexed minimal invasive testing for identification of ILD and COPD. For ILD we achieved classification results (Fig. 2C) comparable to the of cfDNA methylation analysis in various cancer types Egger et al., 2012. Reports on minimal invasive DNA methylation in COPD show similar classification rates as observed in this study, however they concentrate on sputum samples Bruse et al., 2014, a matrix which is not available at all times in COPD Han et al., 2010. We then asked whether our approach was useable for differential diagnosis of all three pulmonary diseases. We identified four top markers (HOXD10, PAX9, PTPRN2, and STAG3) capable of effectively discriminating lung cancer, ILD and COPD. Of these, PAX9 and PTPRN2 had a particular specificity for lung cancer (Fig. 4A), while HOXD10 and STAG3 were well-qualified to discriminate all three diseases from healthy (Fig. 4B). To our best knowledge, changes of cfDNA methylation within these four loci have not been reported in plasma samples of patients with lung cancer, ILD or COPD. The results of our simulated prospective sample classification allow for two clinical strategies: (a) separation of cancer from non-cancer with sensitivity of 87.8% (95%CI: 0.67–0.97) and a specificity of 90.2%, (95%CI: 0.65–0.98), and (b) discrimination of cancer from ILD with a specificity 88% (95%CI: 0.57–1) and from COPD with a specificity of 88% (95%CI: 0.64–0.97) (Fig. 5). The classification results further suggest that multiplexed cfDNA methylation profiling allows for the capture of possibly interconnected phenotypes in spite of different clinical diagnoses. This can be deduced on the distribution pattern across prediction probabilities from correctly classified samples (Supplemental Fig. S12). The flexibility of multiplexed MSRE enrichment is likely to allow for the introduction of further marker panels as well as the combination with SNP analysis improving the overall diagnostic capacity of both approaches. Moreover, plasma or serum samples, often referred to as ‘liquid biopsies’ have several advantages over tissue sampling: (a) they are easily accessible, (b) are not subject to biopsy bias Crowley et al., 2013 and (c) can be repeatedly drawn from the same patient Dawson et al., 2013; Thierry et al., 2014; Murtaza et al., 2013. Thus, application of multiplexed MSRE enrichment to plasma or serum samples is ideally suited for clinical monitoring of disease progression, particularly as our method requires just 400μl of plasma.
Contributors
Declaration of Interests
Role of the Funding Source
Acknowledgments This work was part of the project RESOLVE, funded by the European Commission under FP7-HEALTH-F4-2008, Contract no. 202047 (http://resolve.punkt-international.eu/).