Multimodal analysis of ctDNA methylation and fragmentomic profiles enhances detection of nonmetastatic colorectal cancer.

Aims: Early detection of colorectal cancer (CRC) provides substantially better survival rates. This study aimed to develop a blood-based screening assay named SPOT-MAS ('screen for the presence of tumor by DNA methylation and size') for early CRC detection with high accuracy. Methods: Plasma cell-free DNA samples from 159 patients with nonmetastatic CRC and 158 healthy controls were simultaneously analyzed for fragment length and methylation profiles. We then employed a deep neural network with fragment length and methylation signatures to build a classification model. Results: The model achieved an area under the curve of 0.989 and a sensitivity of 96.8% at 97% specificity in detecting CRC. External validation of our model showed comparable performance, with an area under the curve of 0.96. Conclusion: SPOT-MAS based on integration of cancer-specific methylation and fragmentomic signatures could provide high accuracy for early-stage CRC detection.

A novel blood test for early detection of colorectal cancer. Colorectal cancer is a cancer of the colon or rectum, located at the lower end of the digestive tract. The early detection of colorectal cancer can help people with the disease have a higher chance of survival and a better quality of life. Current screening methods can be invasive, cause discomfort or have low accuracy; therefore newer screening methods are needed. In this study we developed a new screening method, called SPOT-MAS, which works by measuring the signals of cancer DNA in the blood. By combining different characteristics of cancer DNA, SPOT-MAS could distinguish blood samples of people with colorectal cancer from those of healthy individuals with high accuracy.

Combined Gap-Polymerase Chain Reaction and Targeted Next-Generation Sequencing Improve α- and ß-Thalassemia Carrier Screening in Pregnant Women in Vietnam.

Vietnam has a high thalassemia burden. We collected blood samples from 5880 pregnant Vietnamese women during prenatal health checks to assess thalassemia carrier frequency using combined gap-polymerase chain reaction (gap-PCR) and targeted next-generation sequencing (NGS). Thalassemia carriers were identified with prevalence of 13.13% (772), including 7.82% (460) carriers of α-thalassemia (α-thal), 5.31% (312) carriers of ß-thalassemia (ß-thal), and 0.63% (37) concurrent α-/ß-thal carriers. Deletional mutations (368) accounted for 80.0% of α-thal carriers, of which, --SEA (Southeast Asian) (n = 254; 55.0%) was most prevalent, followed by the -α3.7 (rightward) (n = 66; 14.0%) and -α4.2 (leftward) (n = 45; 9.8%) deletions. Hb Westmead (HBA2: c.369C>G) (n = 53) and Hb Constant Spring (Hb CS or HBA2: c.427T>C) (in 28) are the two most common nondeletional α-globin variants, accounting for 11.5 and 6.0% of α-thal carriers. We detected 11 different ß-thal genotypes. Hb E (HBB: c.79G>A) (in 211) accounted for 67.6% of ß-thal carriers. The most common ß-thal genotypes were associated with mutations at codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT), and codon 71/72 (+A) (HBB: c.217_218insA) (prevalence 0.70%, 0.68%, and 0.2%, respectively). Based on mutation frequencies calculated in this study, estimates of 5021 babies in Vietnam are affected with clinically severe thalassemia annually. Our data suggest a higher thalassemia carrier frequency in Vietnam than previously reported. We established that combining NGS with gap-PCR creates an effective large-scale thalassemia screening method that can detect a broad range of mutations.

Liquid biopsy uncovers distinct patterns of DNA methylation and copy number changes in NSCLC patients with different EGFR-TKI resistant mutations.

Targeted therapy with tyrosine kinase inhibitors (TKI) provides survival benefits to a majority of patients with non-small cell lung cancer (NSCLC). However, resistance to TKI almost always develops after treatment. Although genetic and epigenetic alterations have each been shown to drive resistance to TKI in cell line models, clinical evidence for their contribution in the acquisition of resistance remains limited. Here, we employed liquid biopsy for simultaneous analysis of genetic and epigenetic changes in 122 Vietnamese NSCLC patients undergoing TKI therapy and displaying acquired resistance. We detected multiple profiles of resistance mutations in 51 patients (41.8%). Of those, genetic alterations in EGFR, particularly EGFR amplification (n = 6), showed pronounced genome instability and genome-wide hypomethylation. Interestingly, the level of hypomethylation was associated with the duration of response to TKI treatment. We also detected hypermethylation in regulatory regions of Homeobox genes which are known to be involved in tumor differentiation. In contrast, such changes were not observed in cases with MET (n = 4) and HER2 (n = 4) amplification. Thus, our study showed that liquid biopsy could provide important insights into the heterogeneity of TKI resistance mechanisms in NSCLC patients, providing essential information for prediction of resistance and selection of subsequent treatment.

Ultra-Deep Massive Parallel Sequencing of Plasma Cell-Free DNA Enables Large-Scale Profiling of Driver Mutations in Vietnamese Patients With Advanced Non-Small Cell Lung Cancer.

Population-specific profiling of mutations in cancer genes is of critical importance for the understanding of cancer biology in general as well as the establishment of optimal diagnostics and treatment guidelines for that particular population. Although genetic analysis of tumor tissue is often used to detect mutations in cancer genes, the invasiveness and limited accessibility hinders its application in large-scale population studies. Here, we used ultra-deep massive parallel sequencing of plasma cell free DNA (cfDNA) to identify the mutation profiles of 265 Vietnamese patients with advanced non-small cell lung cancer (NSCLC). Compared to a cohort of advanced NSCLC patients characterized by sequencing of tissue samples, cfDNA genomic testing, despite lower mutation detection rates, was able to detect major mutations in tested driver genes that reflected similar mutation composition and distribution pattern, as well as major associations between mutation prevalence and clinical features. In conclusion, ultra-deep sequencing of plasma cfDNA represents an alternative approach for population-wide genetic profiling of cancer genes where recruitment of patients is limited to the accessibility of tumor tissue site.

Actionable Mutation Profiles of Non-Small Cell Lung Cancer patients from Vietnamese population.

Comprehensive profiling of actionable mutations in non-small cell lung cancer (NSCLC) is vital to guide targeted therapy, thereby improving the survival rate of patients. Despite the high incidence and mortality rate of NSCLC in Vietnam, the actionable mutation profiles of Vietnamese patients have not been thoroughly examined. Here, we employed massively parallel sequencing to identify alterations in major driver genes (EGFR, KRAS, NRAS, BRAF, ALK and ROS1) in 350 Vietnamese NSCLC patients. We showed that the Vietnamese NSCLC patients exhibited mutations most frequently in EGFR (35.4%) and KRAS (22.6%), followed by ALK (6.6%), ROS1 (3.1%), BRAF (2.3%) and NRAS (0.6%). Interestingly, the cohort of Vietnamese patients with advanced adenocarcinoma had higher prevalence of EGFR mutations than the Caucasian MSK-IMPACT cohort. Compared to the East Asian cohort, it had lower EGFR but higher KRAS mutation prevalence. We found that KRAS mutations were more commonly detected in male patients while EGFR mutations was more frequently found in female. Moreover, younger patients (<61 years) had higher genetic rearrangements in ALK or ROS1. In conclusions, our study revealed mutation profiles of 6 driver genes in the largest cohort of NSCLC patients in Vietnam to date, highlighting significant differences in mutation prevalence to other cohorts.

Evaluation of a Liquid Biopsy Protocol using Ultra-Deep Massive Parallel Sequencing for Detecting and Quantifying Circulation Tumor DNA in Colorectal Cancer Patients.

The identification and quantification of actionable mutations are critical for guiding targeted therapy and monitoring drug response in colorectal cancer. Liquid biopsy (LB) based on plasma cell-free DNA analysis has emerged as a noninvasive approach with many clinical advantages over conventional tissue sampling. Here, we developed a LB protocol using ultra-deep massive parallel sequencing and validated its clinical performance for detection and quantification of actionable mutations in three major driver genes (KRAS, NRAS and BRAF). The assay showed a 92% concordance for mutation detection between plasma and paired tissues and great reliability in quantification of variant allele frequency.

Characterization of Solanum melongena Thioesterases Related to Tomato Methylketone Synthase 2.

2-Methylketones are involved in plant defense and fragrance and have industrial applications as flavor additives and for biofuel production. We isolated three genes from the crop plant Solanum melongena (eggplant) and investigated these as candidates for methylketone production. The wild tomato methylketone synthase 2 (ShMKS2), which hydrolyzes ß-ketoacyl-acyl carrier proteins (ACP) to release ß-ketoacids in the penultimate step of methylketone synthesis, was used as a query to identify three homologs from S. melongena: SmMKS2-1, SmMKS2-2, and SmMKS2-3. Expression and functional characterization of SmMKS2s in E. coli showed that SmMKS2-1 and SmMKS2-2 exhibited the thioesterase activity against different ß-ketoacyl-ACP substrates to generate the corresponding saturated and unsaturated ß-ketoacids, which can undergo decarboxylation to form their respective 2-methylketone products, whereas SmMKS2-3 showed no activity. SmMKS2-1 was expressed at high level in leaves, stems, roots, flowers, and fruits, whereas expression of SmMKS2-2 and SmMKS2-3 was mainly in flowers and fruits, respectively. Expression of SmMKS2-1 was induced in leaves by mechanical wounding, and by methyl jasmonate or methyl salicylate, but SmMKS2-2 and SmMKS2-3 genes were not induced. SmMKS2-1 is a candidate for methylketone-based defense in eggplant, and both SmMKS2-1 and SmMKS2-2 are novel MKS2 enzymes for biosynthesis of methylketones as feedstocks to biofuel production.