DNA methylation profiling to determine the primary sites of metastatic cancers using formalin-fixed paraffin-embedded tissues [ValidationSet]. DNA methylation profiling to determine the primary sites of metastatic cancers using formalin-fixed paraffin-embedded tissues [ValidationSet]

Accurate identification of the primary site of metastatic cancer is critical to guide the subsequent treatments. There is a significant portion of patients whose primary sites are initially classified as uncertain, and 3-9% of cancer patients are diagnosed with cancer of unknown primary (CUP) even after comprehensive diagnostic workups. Yet, a widely accepted molecular test is still not available. Here, we presented the combination of a novel DNA methylation sequencing-based method and an algorithm to predict the tissues of origin for metastatic cancers. The assay applied degraded DNA from formalin-fixed, paraffin-embedded (FFPE) tissues to generate reduced represent bisulfite sequencing libraries (FFPE-RRBS). Comparable DNA methylation metrics were obtained for the paired fresh frozen (FF) RRBS and FFPE-RRBS libraries and the FFPE-RRBS libraries of matched primary and metastatic cancer tissues. We generated and systemically evaluated 28 molecular classifiers built on four methylation evaluation methods and seven machine-learning approaches from a training data set of 498 primary cancer patients. Of those classifiers, the beta values-based (mean methylation) linear support vector (BELIVE) performed the best, achieving overall accuracies of 81-95% for identifying the primary sites of 215 metastatic cancer patients by utilizing the top-k predictions (k=1, 2, 3). The prediction accuracies ranged from 92% to 98% for 4702 patients with primary tumors in a cross-validation cohort. Lastly, BELIVE successfully identified the tissues of origin for approximately 81-93% of cases in a cohort of 68 patients initially diagnosed with CUP. Overall design: We sequenced RRBS libraries from 503 samples of primary tumor, 218 samples of metastatic tumor, and 68 samples of CUP patients.The assay was conducted using genomic DNA purified either from 10-20 mg of FF tissues or five to eight 5-10 µm FFPE tissue sections. Board-certified pathologists checked one H&E stained slide to ensure that tumor cells accounted for 10% or more cell population and that the necrosis area was less than 50%. NOTE FROM SUBMITTER: Raw data (FASTQ files) are controlled to protect patient privacy.