Performance comparison of NextSeq and Ion Proton platforms for molecular diagnosis of clinical oncology.

PURPOSE: Next-generation sequencing is a powerful approach to detect genetic mutations with which cancer diagnosis and treatment can be tailored to the individual patient in the era of personalized and precision medicine. Ion Torrent Systems Ion Proton and Illumina NextSeq are 2 major targeted sequencing platforms; however, not much work has been done to compare these platforms' performance for mutation detection in formalin-fixed paraffin-embedded (FFPE) materials. METHODS: We benchmarked the performance by using a collection of FFPE samples from 23 patients with different cancers for NextSeq and Ion Proton platforms. We report analysis of sequencing in terms of average coverage depth, read length, and variant detection. RESULTS: Sequencing results by NextSeq and Ion Proton displayed near perfect coverage behavior (>99%) on target region. We analyzed the ability to call variants from each platform and found that Ion Proton sequencing can identify 89% of single nucleotide variants (SNVs) whose mutant allele frequency (MAF) is greater than or equal to 5% detected by the NextSeq pipeline in common analytical regions. The correlation coefficient of MAF for those common SNVs was 1.0046 (R2 = 0.973) between the 2 platforms. To call lower mutant frequency (5%-10%) mutations for NextSeq sequencing, coverage depth should be improved. The concordance of small insertions and deletions between these 2 pipelines was up to 100%. CONCLUSIONS: The 2 sequencing pipelines evaluated were able to generate usable sequence and had high concordance. They are proper for mutation detection in clinical application.

Detection of circulating tumor DNA in patients with advanced non-small cell lung cancer.

Circulating tumor DNA (ctDNA) isolated from plasma has great potential in identification of gene mutation in non-small cell lung cancers (NSCLC), which is a non-invasive technique and can avoid the inherent shortcomings of tissue biopsy. However the ability of NGS to detect gene mutation in plasma ctDNA has not been broadly explored. To assess the diagnostic ability of ctDNA for the total mutation profile, including single nucleotide variations (SNVs), insertions and deletions (indels) and gene rearrangements, we performed a targeted DNA sequencing approach to screen NSCLC related driver gene mutations in both tissue biopsies and matched blood plasma samples from 39 advanced NSCLC patients from China. The sensitivity of EGFR, KRAS, PIK3CA mutations and gene rearrangements detected in plasma ctDNA was 70.6%, 75%, 50% and 60%, respectively and the overall concordance of gene mutations between tissue DNA and plasma ctDNA was 78.21%. Our data provide evidence that ctDNA in plasma is likely to become an alternative source for cancer-related mutations profiling in advanced NSCLC patients and targeted sequencing of ctDNA offers a promising perspective on precise diagnostics and may serve as a feasible option for clinical monitoring of NSCLC patients.

Development and validation of an ultra-high sensitive next-generation sequencing assay for molecular diagnosis of clinical oncology.

Dramatic improvements in the understanding of oncogenes have spurred the development of molecular target therapies, which created an exigent need for comprehensive and rapid clinical genotyping. Next-generation sequencing (NGS) assay with increased performance and decreased cost is becoming more widely used in clinical diagnosis. However, the optimization and validation of NGS assay remain a challenge, especially for the detection of somatic variants at low mutant allele fraction (MAF). In the present study, we developed and validated the Novogene Comprehensive Panel (NCP) based on targeted capture for NGS analysis. Due to the high correlation between SNV/INDEL detection performance and target coverage, here we focused on these two types of variants for our deep sequencing strategy. To validate the capability of NCP in single-nucleotide variant (SNV) and small insert and deletion (INDEL) detection, we implemented a practical validation strategy with pooled cell lines, deep sequencing of pooled samples (>2000X average unique coverage across target region) achieving >99% sensitivity and high specificity (positive predictive value, PPV >99%) for all types of variations with expected MAF >5%. Furthermore, given the high sensitivity and that false positive may exist in this assay, we confirmed its accuracy of variants with MAF <5% using 35 formalin-fixed and paraffin-embedded (FFPE) tumor specimens by Quantstudio 3D Digital PCR (dPCR; Life Technologies) and obtained a high consistency (32 of 35 mutations detected by NGS were verified). We also used the amplification refractory mutation system (ARMS) to verify the variants with a MAF in a broad range of 2-63% detected in 33 FFPE samples and reached a 100% PPV for this assay. As a potential clinical diagnosis tool, NCP can robustly and comprehensively analyze clinical-related genes with high sensitivity and low cost.

Tyrosine phosphatase Shp2 mediates the estrogen biological action in breast cancer via interaction with the estrogen extranuclear receptor.

The extranuclear estrogen receptor pathway opens up novel perspectives in many physiological and pathological processes, especially in breast carcinogenesis. However, its function and mechanisms are not fully understood. Herein we present data identifying Shp2, a SH2-containing tyrosine phosphatase, as a critical component of extranuclear ER pathway in breast cancer. The research checked that the effect of Shp2 on the tumor formation and growth in animal model and investigated the regulation of Shp2 on the bio-effect and signaling transduction of estrogen in breast cancer cell lines. The results showed that Shp2 was highly expressed in more than 60% of total 151 breast cancer cases. The inhibition of Shp2 activity by PHPS1 (a Shp2 inhibitor) delayed the development of dimethylbenz(a)anthracene (DMBA)-induced tumors in the rat mammary gland and also blocked tumor formation in MMTV-pyvt transgenic mice. Estradiol (E2) stimulated protein expression and phosphorylation of Shp2, and induced Shp2 binding to ERα and IGF-1R around the membrane to facilitate the phosphorylation of Erk and Akt in breast cancer cells MCF7. Shp2 was also involved in several biological effects of the extranuclear ER-initiated pathway in breast cancer cells. Specific inhibitors (phps1, phps4 and NSC87877) or small interference RNAs (siRNA) of Shp2 remarkably suppressed E2-induced gene transcription (Cyclin D1 and trefoil factor 1 (TFF1)), rapid DNA synthesis and late effects on cell growth. These results introduced a new mechanism for Shp2 oncogenic action and shed new light on extranuclear ER-initiated action in breast tumorigenesis by identifying a novel associated protein, Shp2, for extranuclear ER pathway, which might benefit the therapy of breast cancer.