Cell-free DNA-based liquid biopsy is emerging as a revolutionary new method in individualized cancer treatment and prognosis monitoring, although detecting early-stage cancers using cell-free DNA remains challenging, partially because of the undefined biological background of cell-free DNA. We investigated somatic mutations in the cell-free DNA of 259 cancer-free individuals with a median age of 47 years using an endogenous barcoding duplex method with an ultralow base error rate (2 × 10⁻⁷) and compared the variant allele frequencies of these mutations between the cell-free DNA and the corresponding blood cell DNA. Sixty percent (155/259) of the samples showed at least one nonsynonymous mutation on either of two similar target panels covering 508 and 559 cancer-related genes. For individuals older than 50 years of age, the positive rate increased to 76%. Most cell-free DNA mutations were also present at similar variant allele frequencies in the paired blood cell DNA. The most frequently mutated genes were driver genes of hematologic malignancies, including DNMT3A, TET2, AXSL1, and JAK2. However, the other 58.4% (192/329) of the mutations were likely “passenger mutations” of clonal hematopoiesis, including mutations in NOTCH2, FAT3, EXT2, ERBB4, and ARID2, which are driver genes of solid tumors. Hematopoietic clone-derived mutations, including “driver mutations” and “passenger mutations”, are prevalent in the cell-free DNA of both healthy individuals and cancer patients and may be a potential source of false positives in the liquid biopsy. Our results also suggest the ineffectiveness for distinguishing clonal hematopoietic mutations of low variant allele frequency (≤0.1%) from tumor-derived mutations using conventional next-generation sequencing of blood cell DNA. However, an error correction model with an ultralow error rate and high coverage depth is required for blood cell DNA sequencing, which is difficult and costly to achieve with current technologies.

Background: To better understand the pathogenesis of cervical cancer (CC), we systematically analysed the genomic variation and human papillomavirus (HPV) integration profiles of cervical intraepithelial neoplasia (CIN) and CC. Methods: We performed whole-genome sequencing or whole-exome sequencing of 102 tumour-normal pairs and human papillomavirus probe capture sequencing of 45 CCs, 44 CIN samples and 25 normal cervical samples, and constructed strict integrated workflow of genomic analysis. Results: Mutational analysis identified eight significantly mutated genes in CC including four genes ( FAT1 , MLL3 , MLL2 and FADD ), which have not previously been reported in CC. Targetable alterations were identified in 55.9% of patients. In addition, HPV integration breakpoints occurred in 97.8% of the CC samples, 70.5% of the CIN samples and 42.8% of the normal cervical samples with HPV infection. Integrations of high-risk HPV strains in CCs, including HPV16, 18, 33 and 58, also occurred in the CIN samples. Moreover, gene mutations were detected in 52% of the CIN specimens, and 54.8% of these mutations occurred in genes that also mutated in CCs. Conclusion: Our results lay the foundation for a deep understanding of the molecular mechanisms and finding new diagnostic and therapeutic targets of CC.

Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells

Single cancer cell–sequencing studies currently use randomly selected cells, limiting correlations among genomic aberrations, morphology, and spatial localization. We laser-captured microdissected single cells from morphologically distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of more than 100 such cells. Two major subclones coexisted in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor, with additional copy number changes, including chr8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events led us to assess whether other clonal and subclonal genomic aberrations preexisting in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in 5 breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chr1p, chr8q, chr9p, chr12q, and chr20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chr1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.

Targeted next generation sequencing in Chinese colorectal cancer patients guided anti-EGFR treatment and facilitated precision cancer medicine Targeted next generation sequencing in Chinese colorectal cancer patients guided anti-EGFR treatment and facilitated precision cancer medicine

Motivation: Sequencing of cell-free DNA (cf-DNA) has enabled Noninvasive Prenatal Testing (NIPT) and”liquid biopsy” of cancers. However, while the aneuploidy and point mutations were focused on by most of NITP and liquid biopsy studies, detecting sub-chromosome CNVs that affect a few to dozens of megabases was rarely reported, likely attributable to the difficulty in accurately identifying them, especially for those present in a small fraction of cf-DNA. Results: We developed a somatic CNV detection tool (SCDT), for detecting sub-chromosome CNVs in cf-DNA using whole genome sequencing (WGS) data or off-target reads in target sequencing data. Additional to using control samples for correcting genome position specific bias, two GC correction steps were performed, which regressed GC content of DNA fragments and that of genome bins, respectively. After GC correction, the coefficients of variation of copy ratios approximated the lower boundary of theoretical values, suggesting removing of almost all systematic errors. Finally, CNVs were detected by a piecewise least squares fitting based segmentation algorithm, which outperformed other segmentation methods. We applied SCDT on simulated and real maternal plasma samples, and target cf-DNA sequencing of 118 normal individuals and 240 cancer patients, and demonstrated high sensitivity and specificity. Availability: SCDT is available at https://github.com/Martiantian/Somatic_cnv_detect_tool . Contact: zhuhongmei@genomics.cn Supplementary Information: Supplementary data are available at Bioinformatics online