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.

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.

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.

Severe hemolytic anemia in a Vietnamese family, associated with novel mutations in the gene encoding for pyruvate kinase.

BACKGROUND AND OBJECTIVES: Chronic hemolytic anemias are very frequent diseases in intertropical countries mainly caused by hemoglobin disorders. We studied a Vietnamese family in which a first child suffered from a severe transfusion-dependent anemia. The family requested an antenatal diagnosis during a second pregnancy. To characterize the molecular defect, we studied the family over three generations. DESIGN AND METHODS: Blood from family members was sampled for a full hematologic evaluation, including enzymatic dosage, and DNA analysis was performed for patients displaying pyruvate kinase deficiency (PK-R). Mutation research on the 11 exons of the PKLR gene was done using a scanning method and sequencing. Deletion was evidenced by a Sybergreen based quantitative real time polymerase chain reaction (PCR) and mapped using quantitative multiplex PCR of short fluorescent fragments spread along the whole sequence of the PKLR gene. RESULTS: Hematologic and molecular studies of this severe chronic anemia demonstrated the existence of two defects in the PKLR gene, a new mutation located on exon 7: c.948C->G (N316K) and a large deletion extending from exon 4 to exon 10. INTERPRETATION AND CONCLUSIONS: We describe a family in a south-east Asian country; the proband had severe transfusion-dependent chronic anemia caused by the association between two PKLR gene mutations, PK Saigon (N316K) and PK Viet del 4-10. Severe chronic anemia could be induced by various molecular defects mainly affecting the globin genes. However, even in populations in which hemoglobin diseases are frequent, enzymatic diseases should be considered.