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Purpose: There have been needs to improve the sensitivity of liquid biopsy. This report aims to report the analytical and clinical validation of a next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assay. Materials and Methods: Analytical validation was conducted in vitro by evaluating the limit of detection (LOD), precision, and specificity for various genomic aberrations. The real-world performance in non-small cell lung cancer (NSCLC) was assessed by comparing the results of AlphaLiquid100 to the tissue-based results. Results: The LODs with 30 ng input DNA were 0.11%, 0.11%, 0.06%, 0.21%, and 2.13 copies for detecting single nucleotide variants, insertions, deletions, fusions, and copy number alterations (CNA), respectively. Quantitatively, single nucleotide variants/insertions and deletions, fusions, and CNAs showed a good correlation (R2=0.91, 0.40, and 0.65; y=0.95, 1.06, and 1.19) to the manufacturer's values, and per-base specificities for all types of variants were near 100%. In real-world NSCLC (n=122), key actionable mutations in NSCLC were detected in 60.7% (74/122) with the ctDNA assay. Comparative analysis against the NGS-based tissue results for all key mutations showed positive percent agreement (PPA) of 85.3%. For individual genes, the PPA was as high as 95.7% for epidermal growth factor receptor (EGFR) mutations and 83.3% for ALK translocations. AlphaLiquid100 detected drug-sensitive EGFR mutation at a variant allele frequency as low as 0.02% and also identified an EGFR mutation in a case where tissue sample missed. Blood samples collected post-targeted therapies revealed additional acquired mutations. Conclusion The AlphaLiquid100 ctDNA assay demonstrates robust analytical validity, offering clinically important information for NSCLC patients. [ABSTRACT FROM AUTHOR]

Purpose Circulating tumor DNA (ctDNA) is emerging as a valuable non-invasive tool to identify tumor heterogeneity and tumor burden. This study investigated ctDNA dynamics in metastatic colorectal cancer patients treated with regorafenib. Materials and Methods In this prospective biomarker study, plasma cell-free DNA (cfDNA) samples obtained at baseline, at the first response evaluation after 2 cycles of treatment, and at the time of progressive disease were sequenced using a targeted nextgeneration sequencing platform which included 106 genes. Results A total of 285 blood samples from 110 patients were analyzed. Higher baseline cfDNA concentration was associated with worse progression-free survival (PFS) and overall survival (OS). After 2 cycles of treatment, variant allele frequency (VAF) in the majority of ctDNA mutations decreased with a mean relative change of -31.6%. Decreases in the VAF of TP53, APC, TCF7L2, and ROS1 after 2 cycles of regorafenib were associated with longer PFS. We used the sum of VAF at each time point as a surrogate for the overall ctDNA burden. A reduction in sum (VAF) of = 50% after 2 cycles was associated with longer PFS (6.1 vs. 2.7 months, p=0.002), OS (11.3 vs. 5.9 months, p=0.001), and higher disease control rate (86.3% vs. 51.1%, p < 0.001). VAF of the majority of the ctDNA mutations increased at the time of disease progression, and VAF of BRAF increased markedly. Conclusion Reduction in ctDNA burden as estimated by sum (VAF) could be used to predict treatment outcome of regorafenib. [ABSTRACT FROM AUTHOR]

Purpose This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines. Materials and Methods We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. Results One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3- PLA2G15 FT was found in two. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. Conclusion These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation. [ABSTRACT FROM AUTHOR]

Purpose RNA editing generates protein diversity by altering RNA sequences in coding regions without changing the overall DNA sequence. Adenosine-to-inosine (A-to-I) RNA editing events have recently been reported in some types of cancer, but they are rare in human colorectal cancer (CRC). Therefore, this study was conducted to identify diverse RNA editing in CRC. Materials and Methods We compared transcriptome data of 39 CRC samples and paired adjacent tissues from The Cancer Genome Atlas database to identify RNA editing patterns in CRC, focusing on canonical A-to-I RNA edits in coding sequence regions. We investigated nonsynonymous RNA editing patterns by comparing tumor and normal tissue transcriptome data. Results The number of RNA edits varied from 12 to 42 per sample. We also observed that hypoand hyper-RNA editing patterns were distinguishable within the samples. We found 10 recurrent nonsynonymous RNA editing candidates in nine genes (PDLIM, NEIL1, SRP9, GLI1, APMAP, IGFBP7, ZNF358, COPA, and ZNF587B) and validated some by Sanger sequencing and the inosine chemical erasing assay. We further showed that editing at these positions was performed by the adenosine deaminase acting on RNA 1 enzyme. Most of these genes are hypoedited in CRC, but editing of GLI1 was increased in cancer tissues compared with normal tissues. Conclusion Our results show that nonsynonymous RNA editing patterns can be used to identify CRC patients and could serve as novel biomarkers for CRC. [ABSTRACT FROM AUTHOR]

Purpose Epigenetic alterations of specific genes have recently been identified as diagnostic biomarkers for human cancers. However, there are currently no standardized epigenetic biomarkers for drug sensitivity in human gastrointestinal cancer. Therefore, the aim of this study is to identify a novel epigenetic biomarker in gastrointestinal cancer. Materials and Methods Using bisulfite sequencing and pyrosequencing analysis, DNA methylation patterns of gastric, colon primary tissues and their cancer cells were analyzed, and histone modifications were analyzed using chromatin immunoprecipitation assay. In addition, cancer cells were exposed to cisplatin and treated with a DNA methyltransferase inhibitor. Results We report that in human gastric and colon cancers, latrophilin 2 (LPHN2) is silenced by epigenetic modifications, including CpG island methylation and aberrant histone modifications. We also confirmed that LPHN2 was silenced by DNA hypermethylation in primary gastric and colon tumor tissues compared to their normal counterparts. Interestingly, we found that cancer cells with methylated LPHN2 showed higher sensitivity to cisplatin. Also, 5-aza- 2!-deoxycytidine combined with cisplatin decreased the cytotoxicity of cisplatin in cancer cells with methylated LPHN2. In addition, LPHN2 knockdown in cancer cells with high LPHN2 expression sensitized these cells to the anti-proliferative effects of cisplatin. Conclusion In human gastrointestinal cancer, we found that LPHN2 is regulated by epigenetic modifications, and that cancer cells with lower LPHN2 expression show higher sensitivity to cisplatin. Therefore, the methylation status of LPHN2 is a potential novel epigenetic biomarker for cisplatin treatment in human gastric and colon cancers. [ABSTRACT FROM AUTHOR]

An oral medication of a molecular targeted drug, lapatinib, is taken regularly to maintain the drug concentration within the desired therapeutic levels. To alleviate the need for such cumbersome administration schedules in several drugs, advanced drug delivery systems (DDSs), which can provide time-controlled and sustained drug release, have recently received significant attention. A biodegradable synthetic polymer, such as polycaprolactone (PCL), is usually used as a carrier material for DDSs. In this paper, lapatinib powder-entrapped, PCL microstructures were fabricated with a precise X-ray lithography-based method. In vitro experiments on HER2 positive-human gastric cancer derived NCI-N87 cells were performed to appraise the drug release characteristics of the fabricated DDSs. The in vitro results indicate that after the X-ray lithography process, the lapatinib powder is still working well and show time- and dose- dependent drug release efficiencies. The cell growth inhibition characteristics of one hundred 40-µm sized microstructures were similar to those of a 1 µM lapatinib solution for over 144 h. In conclusion, the developed lapatinib-entrapped PCL microstructures can be used in molecular targeted delivery and sustained release as effective cancer-targeted DDSs. [ABSTRACT FROM AUTHOR]

Background: Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulinlike growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer. Methodology/Principal Findings: In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumabsensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity. Conclusion and Significance: The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells. [ABSTRACT FROM AUTHOR]

Background: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated. Methodology and Principal Findings: In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib. Conclusions and Significance: These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine. [ABSTRACT FROM AUTHOR]