PURPOSE A circulating tumor DNA (ctDNA) test to detect plasma Epstein-Barr viral DNA can be used to screen for early nasopharyngeal cancers; however, the reported sensitivity of viral ctDNA tests to detect human papillomavirus (HPV)-associated cancers is modest. We assessed the utility of droplet digital polymerase chain reaction (ddPCR) to detect early-stage HPV-associated cancers using sequential HPV16 and HPV33 assays that account for HPV subtype distribution and subtype sequence variants. PATIENTS AND METHODS We collected plasma specimens from 97 HPV-positive patients with oropharyngeal squamous cell carcinoma and eight patients with HPV-positive anal squamous cell carcinoma, each with locoregionally confined disease. Negative controls included samples from seven patients with HPV-negative head and neck cancers and 20 individuals without cancer. RESULTS Of 97 patients with nonmetastatic, locoregionally confined oropharyngeal squamous cell carcinoma, 90 patients had detectable HPV16 ctDNA and three patients had HPV33 ctDNA, indicating an overall sensitivity of 95.6%. Seven of eight patients with early anal cancer were HPV16 ctDNA positive. No HPV ctDNA was detected in 27 negative controls, indicating 100% specificity. HPV16 ctDNA was detected in 19 of 19 patients with low-volume disease, defined as patients with a single, asymptomatic positive lymph node (N1) or an isolated T1–2 asymptomatic primary tumor. HPV16 ctDNA levels directly corresponded to tumor responses to chemoradiation and surgery. CONCLUSION With an updated understanding of HPV subtypes and sequence variation, HPV ctDNA by ddPCR is highly sensitive and specific, identifying HPV16 and HPV33 subtypes in a similar distribution as reported in major genomic profiling studies. The detection of small tumors indicates that HPV16 and HPV33 ctDNA ddPCR could be readily used in early detection screening trials and in disease response monitoring, analogous to Epstein-Barr virus DNA.

591 The human genome is adorned with methylated cytosine residues that function in the epigenetic guidance of cellular differentiation and development. Cellular interpretation of this epigenetic mark is incompletely understood and tissue specific patterns of DNA methylation vary with age, can be altered by environmental factors, and are often abnormal in human disease. Aberrant DNA methylation is a common means by which tumor suppressor genes (TSGs) are inactivated during carcinogenesis (Baylin, Herman, Graff, Vertino and Issa 1998; Laird and Jaenisch 1996; Singal and Ginder 1999). Unlike genetic mechanisms of gene inactivation, such as gene deletion and mutation, the epigenetic silencing of TSGs by promoter hypermethylation is potentially reversible. This has led to the broad interest of cancer biologists in the study of DNA methylation. Method: We developed a method for genome-wide analysis of DNA methylation by using a recombinant protein containing a methyl-CpG binding domain (MBD) to enrich methylated DNA fragments that are then identified by massively parallel sequencing using the SOLiD sequencer (ABI). We generated ∼15-million sequence tags per specimen and wrote custom R-language algorithms to develop an analytical platform with which to study DNA methylation. We used this technology to study the pharmacodynamics of DNA methylation in acute myelogenous leukemia (AML) cells following exposure to the hypomethylating agent, 5-aza-2'-deoxycytidine (decitabine). We compared DNA methylation patterns before and after decitabine treatment with transcriptional activity revealed by microarrays (Illumina) and quantitative PCR. We found that Sequence Tag Analysis of Methylation Profiles (STAMP) permits highly reproducible, genome-wide identification of DNA methylation density at near base-pair resolution. This method is cost effective and can be extended, without modification, to any mapped genome. Results: STAMP analysis revealed patterned DNA methylation at all scales across the genome: from whole chromosomes to individual genes. We found that densely methylated elements (DMEs) of the human genome are often highly conserved or closely associated with gene coding regions and promoters. We identified distinct patterns of DNA methylation surrounding the transcription start and termination sites of all genes. These methylation patterns are associated with transcriptional activity of neighboring genes. Interestingly, genes with a densely methylated transcription start site (TSS) have little methylation in the surrounding regions whereas genes with little or no methylation at the TSS have disproportionately higher methylation within their gene bodies. In untreated cells, we detected ∼75,000 DMEs (false discovery rate <0.01) with a median length ∼600 bp and with 75% being less than 960bp. The longest DMEs extend up to ∼24000 bp and are composed of microsatellite clusters. The majority of the DMEs are not classic CpG islands (CGI) but are GC-rich regions (median 57% GC) with a greater than expected incidence of CpG dinucleotides (median CpG observed/expected 0.49): results that suggest the definition of a CGI excludes the majority of the methylated human genome. Although the pattern of DNA methylation was qualitatively similar in cells treated with decitabine, we found that the density of methylation was generally lower and fewer DMEs (∼50,000) were identified. Decitabine treatment led to increased expression of ∼800 genes involved in cell cycle control, apoptosis and cellular differentiation whereas the ∼50 genes with downregulated expression were most commonly involved in RNA metabolism. Distinct pre-treatment DNA methylation patterns were associated with, and tended to predict, the transcriptional activity following treatment with decitabine. Summary: We developed and utilized a powerful new technology to uncover the genome-wide effects of decitabine on DNA methylation patterns in AML. We found that although decitabine induces genome-wide DNA hypomethylation, its effect on transcription depends upon the pattern of DNA methylation prior to treatment. The STAMP methodology leverages the power and flexibility of massively parallel sequencing with the high selectivity of the MBD for its natural ligand, methyl-CpG. This assay permits robust, unbiased and highly sensitive whole-genome identification of methylated DNA segments. Disclosures: No relevant conflicts of interest to declare.

Background:The TCGA classification has a definite guiding and suggestive effect on the treatment and prognosis of endometrial carcinoma. However, TCGA typing is based on the results of multi-omics studies such as gene mutation, gene expression and gene methylation, which is expensive and not suitable for clinical widespread application.Methods: A cohort of 88 endometrial cancer (EC) patients were enrolled. Targeted NGS of more than 450 gene panel on their tumor tissues (fresh or FFPE) and matched leucocyte controls was carried out by OrigiMed (Shanghai, China), a CAP accredited and CLIA certified laboratory. Subsequently, the molecular typing was established according to POLE mutation, microsatellite status and copy number variation (CNV) in proper order.Results: The percentages of POLE, MSI-high, CNV-low and CNV-high groups were 6.9% (n¼6), 10.3% (n¼9), 72.4% (n¼63), and 10.3% (n¼9), and the mean TMB of them 3.46, 11.80, 60.92 and 177.10 muts/Mb (P<0.001), respectively. Furthermore, there was no significant difference in the diagnostic age among the four groups (P¼0.24), and TMB in stage I was significantly higher than that in stage II-IV (P¼0.028). Alterations of POLE, TP53, BRCA1/2, TERT, ATM and APC were positively correlated with TMB, and KMT2C and MYC were negatively correlated with TMB (P < 0.05). TP53 mutation was positively correlated with CNV (P¼0.012), and was more likely to appear in the elderly (P¼0.06). Besides, Most of IV stage were enriched in CNV-low (25%, n¼4) and CNV-high groups(68.75%, n¼11), and most of I and III stages were enriched in POLE and MSI-high groups.Conclusions: We firstly constructed four types of EC (POLE, MSI-high, CNV-low and CNV-high) based on targeted NGS, and made clinical suggestions on immunotherapy, clinical stage and age.Legal entity responsible for the study: The authors.