The genetic landscape of chromosomal aberrations in 3776 Vietnamese fetuses with clinical anomalies during pregnancy.

Background: Copy number variation sequencing (CNV-seq) is a powerful tool to discover structural genomic variation, but limitations associated with its retrospective study design and inadequate diversity of participants can be impractical for clinical application. Aim: This study aims to use CNV-seq to assess chromosomal aberrations in pregnant Vietnamese women. Materials & methods: A large-scale study was conducted on 3776 pregnant Vietnamese women with abnormal ultrasound findings. Results: Chromosomal aberrations were found in 448 (11.86%) women. Of these, 274 (7.26%) had chromosomal aneuploidies and 174 (4.61%) carried pathogenic/likely pathogenic CNVs. Correlations were established between chromosomal aberrations and various phenotypic markers. Conclusion: This comprehensive clinical study illuminates the pivotal role of CNV-seq in prenatal diagnosis for pregnancies featuring fetal ultrasound anomalies.

De novo variants of dominant monogenic disorders in Vietnam detected by a noninvasive prenatal test: a case series.

Background: Noninvasive prenatal tests for monogenic diseases (NIPT-SGG) have recently been reported as helpful in early-stage antenatal screening. Our study describes the clinical and genetic features of cases identified by NIPT-SGG. Materials & methods: In a cohort pregnancy with abnormal sonograms, affected cases were confirmed by invasive diagnostic tests concurrently, with NIPT-SGG targeting 25 common dominant single-gene diseases. Results: A total of 13 single-gene fetuses were confirmed, including Noonan and Costello syndromes, thanatophoric dysplasia, achondroplasia, osteogenesis imperfecta and Apert syndrome. Two novel variants seen were tuberous sclerosis complex (TSC2 c.4154G>A) and Alagille syndrome (JAG1 c.3452del). Conclusion: NIPT-SGG and standard tests agree on the results for 13 fetuses with monogenic disorders. This panel method of screening can benefit high-risk Vietnamese pregnancies, but further research is encouraged to expand on the causative gene panel.

Developing and validating noninvasive prenatal testing for de novo autosomal dominant monogenic diseases in Vietnam.

Background: Over 60% of single-gene diseases in newborns are autosomal dominant variants. Noninvasive prenatal testing for monogenic conditions (NIPT-SGG) is cost-effective and timesaving, but not widely applied. This study introduces and validates NIPT-SGG in detecting 25 monogenic conditions. Methods: NIPT-SGG with a 30-gene panel applied next-generation sequencing and trio assays to confirm de novo variants. Diagnostic tests confirmed NIPT-detected cases. Results: Among 93 pregnancies with ultrasound findings, 11 (11.8%) fetuses were screened and diagnosed with monogenic diseases, mostly with Noonan syndrome. NIPT-SGG determined >99.99% of actual positive and negative cases, confirmed by diagnostic tests. No false-negatives or false-positives were reported. Conclusion: NIPT-SGG effectively identifies the fetuses affected with monogenic diseases, which is a promisingly safe and timely antenatal screening option for high-risk pregnancies.

Prevalence and genetic spectrum associated with hereditary colorectal cancer syndromes, the need to improve cancer risk awareness, and family cascade testing in Vietnam.

In Vietnam, colorectal cancer is one of the top diagnosed cancers, with 5-10% originating from inherited mutations. This study aims to define the mutation spectrum associated with hereditary colorectal cancer syndromes (HCCS) in Vietnam, evaluate the influence of genetic testing on carriers' awareness, and also investigate the barriers in familial testing. Genetic test reports were collected to identify HCCS cases, then cases underwent a survey investigating self-risk and familial-risk awareness, proactive cancer screening, and familial testing barriers. Participant characteristics, mutation prevalence, and results from the survey were descriptively analyzed and reported. Of all genetic test results, 3% (49/1632) were identified with mutations related to HCCS. Over 77% of them belonged to Lynch syndrome. PMS2 appeared to be the gene with the highest mutation frequency, while MLH1 was the lowest. 44% of cases further undertook cancer screening tests, and 48% of cases' families had uptake genetic testing. The biggest barrier of familial members for not taking genetic test was psychological reasons (fear, not being interested, or not feeling necessary). This study provided new evidence for HCCS mutation spectrum in Vietnamese population and the success in promoting cascade test in high-risk family members through financial and technical support. Also, study has suggested the needs of an innovative genetic testing process focusing on the quality of pre-and post-test consultancy, an increase in follow-ups, and the change in policy for permission of contacting relatives directly to improve the rate of cascade testing and proactive cancer screening.

Multimodal analysis of genome-wide methylation, copy number aberrations, and end motif signatures enhances detection of early-stage breast cancer.

Introduction: Breast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes. Methods: Here, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects. Results: We identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity. Discussion: Our findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.

Fragment length profiles of cancer mutations enhance detection of circulating tumor DNA in patients with early-stage hepatocellular carcinoma.

BACKGROUND: Late detection of hepatocellular carcinoma (HCC) results in an overall 5-year survival rate of less than 16%. Liquid biopsy (LB) assays based on detecting circulating tumor DNA (ctDNA) might provide an opportunity to detect HCC early noninvasively. Increasing evidence indicates that ctDNA detection using mutation-based assays is significantly challenged by the abundance of white blood cell-derived mutations, non-tumor tissue-derived somatic mutations in plasma, and the mutational tumor heterogeneity. METHODS: Here, we employed concurrent analysis of cancer-related mutations, and their fragment length profiles to differentiate mutations from different sources. To distinguish persons with HCC (PwHCC) from healthy participants, we built a classification model using three fragmentomic features of ctDNA through deep sequencing of thirteen genes associated with HCC. RESULTS: Our model achieved an area under the curve (AUC) of 0.88, a sensitivity of 89%, and a specificity of 82% in the discovery cohort consisting of 55 PwHCC and 55 healthy participants. In an independent validation cohort of 54 PwHCC and 53 healthy participants, the established model achieved comparable classification performance with an AUC of 0.86 and yielded a sensitivity and specificity of 81%. CONCLUSIONS: Our study provides a rationale for subsequent clinical evaluation of our assay performance in a large-scale prospective study.

High prevalence of maternal mosaic monosomy X in pregnant women in Vietnam.

OBJECTIVE: To demonstrate the prevalence of maternal mosaic monosomy X (MMXO) in a cohort of pregnant women in Vietnam. METHODS: All 105,594 singleton pregnant women undergoing noninvasive prenatal screening (NIPS) between January 2019 and February 2021 in Vietnam were analyzed by measuring discordance between size- and count-based z-scores for chromosome X (ChrX) to identify suspected cases of MMXO and validated by fluorescence in situ hybridization (FISH) on maternal blood. RESULTS: We identified 295 (0.279%) suspected MMXO cases. After FISH analysis, MMXO was confirmed in 125 cases (42.37%), revealing the MMXO prevalence of 0.118% (95% CI: 0.097-0.139%) in this cohort. CONCLUSION: We found a relatively high prevalence of MMXO in Vietnamese pregnant women and demonstrated a strong influence of MMXO on the ChrX z-score using a count-based method, resulting in false positives. The size-based method is not sensitive to MMXO and therefore achieves higher PPV.

Genetic landscape and personalized tracking of tumor mutations in Vietnamese women with breast cancer.

Breast cancer is the leading cause of cancer death in Vietnamese women, but its mutational landscape and actionable alterations for targeted therapies remain unknown. After treatment, a sensitive biomarker to complement conventional imaging to monitor patients is also lacking. In this prospective multi-center study, 134 early-stage breast cancer patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissues and paired white blood cells were sequenced to profile all tumor-derived mutations in 95 cancer-associated genes. Our bioinformatic algorithm was then utilized to identify top mutations for individual patients. Serial plasma samples were collected before surgery and at scheduled visits after surgery. Personalized assay tracking the selected mutations were performed to detect circulating tumor DNA (ctDNA) in the plasma. We found that the mutational landscape of the Vietnamese was largely similar to other Asian cohorts, showing higher TP53 mutation frequency than in Caucasians. Alterations in PIK3CA and PI3K signaling were dominant, particularly in our triple-negative subgroup. Using top-ranked mutations, we detected ctDNA in pre-operative plasma in 24.6-43.5% of the hormone-receptor-positive groups and 76.9-80.8% of the hormone-receptor-negative groups. The detection rate was associated with breast cancer subtypes and clinicopathological features that increased the risk of relapse. Interim analysis after a 15-month follow-up revealed post-operative detection of ctDNA in all three patients that had recurrence, with a lead time of 7-13 months ahead of clinical diagnosis. Our personalized assay is streamlined and affordable with promising clinical utility in residual cancer surveillance. We also generated the first somatic variant dataset for Vietnamese breast cancer women that could lay the foundation for precision cancer medicine in Vietnam.

Tumor genomic profiling and personalized tracking of circulating tumor DNA in Vietnamese colorectal cancer patients.

Background: Colorectal cancer (CRC) is the fifth most common cancer with rising prevalence in Vietnam. However, there is no data about the mutational landscape and actionable alterations in the Vietnamese patients. During post-operative surveillance, clinical tools are limited to stratify risk of recurrence and detect residual disease. Method: In this prospective multi-center study, 103 CRC patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived somatic mutations in 95 cancer-associated genes. Our bioinformatic algorithm identified top mutations unique for individual patient, which were then used to monitor the presence of circulating tumor DNA (ctDNA) in serial plasma samples. Results: The top mutated genes in our cohort were APC, TP53 and KRAS. 41.7% of the patients harbored KRAS and NRAS mutations predictive of resistance to Cetuximab and Panitumumab respectively; 41.7% had mutations targeted by either approved or experimental drugs. Using a personalized subset of top ranked mutations, we detected ctDNA in 90.5% of the pre-operative plasma samples, whereas carcinoembryonic antigen (CEA) was elevated in only 41.3% of them. Interim analysis after 16-month follow-up revealed post-operative detection of ctDNA in two patients that had recurrence, with the lead time of 4-10.5 months ahead of clinical diagnosis. CEA failed to predict recurrence in both cases. Conclusion: Our assay showed promising dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies in CRC patients. This could lay foundation to empower precision cancer medicine in Vietnam and other developing countries.

Detection of maternal carriers of common α-thalassemia deletions from cell-free DNA.

α-Thalassemia is a common inherited blood disorder manifested mainly by the deletions of α-globin genes. In geographical areas with high carrier frequencies, screening of α-thalassemia carrier state is therefore of vital importance. This study presents a novel method for identifying female carriers of common α-thalassemia deletions using samples routinely taken for non-invasive prenatal tests for screening of fetal chromosomal aneuploidies. A total of 68,885 Vietnamese pregnant women were recruited and α-thalassemia statuses were determined by gap-PCR, revealing 5344 women (7.76%) carried deletions including αα/--SEA (4.066%), αα/-α3.7 (2.934%), αα/-α4.2 (0.656%), and rare genotypes (0.102%). A two-stage model was built to predict these α-thalassemia deletions from targeted sequencing of the HBA gene cluster on maternal cfDNA. Our method achieved F1-scores of 97.14-99.55% for detecting the three common genotypes and 94.74% for detecting rare genotypes (-α3.7/-α4.2, αα/--THAI, -α3.7/--SEA, -α4.2/--SEA). Additionally, the positive predictive values were 100.00% for αα/αα, 99.29% for αα/--SEA, 94.87% for αα/-α3.7, and 96.51% for αα/-α4.2; and the negative predictive values were 97.63%, 99.99%, 99.99%, and 100.00%, respectively. As NIPT is increasingly adopted for pregnant women, utilizing cfDNA from NIPT to detect maternal carriers of common α-thalassemia deletions will be cost-effective and expand the benefits of NIPT.

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.

Clinical and genetic features of congenital bile acid synthesis defect with a novel mutation in AKR1D1 gene sequencing: Case reports.

RATIONALE: Congenital bile acid synthesis defect (BASD) is a rare disease caused by mutations in the aldo-keto reductase 1D1 gene, which encodes the primary Δ4-3-oxosteroid 5ß-reductase enzyme. Early disease diagnosis is critical for early treatment with bile acid replacement therapy, with an excellent chance for recovery. In contrast, protracted diagnosis and treatment may lead to poor outcomes, including decompensated hepatic cirrhosis, liver transplant, and even death. PATIENT CONCERNS: Three clinical congenital bile acid synthesis defect cases in the Vietnamese population are herein reported. These pediatric patients presented with symptoms of prolonged postpartum jaundice and abnormal loose stool (mucus, lipids, and white). The clinical examinations showed hepatosplenomegaly. Urinalysis showed a very low fraction of primary bile acids and atypical 3-oxo-Δ4- bile acids in all three patients. DIAGNOSES: The patients were diagnosed with primary Δ4-3-oxosteroid 5ß-reductase deficiency. Next-generation gene sequencing revealed two homozygous mutations in the aldo-keto reductase family 1 member D1 gene. The first is a documented variant, c.797G>A (p.Arg266Gln), and the second is a novel mutation at c.155T>C (p.Ile52Thr). INTERVENTIONS: Immediately after diagnosis, patients were treated with oral chenodeoxycholate 5 mg/kg/d. OUTCOMES: The patients' symptoms, signs, and primary bile acids levels improved significantly. LESSONS: Clinicians should consider genetic disorders related to cholestasis for effective and life-saving treatment. A prompt genetic analysis by next-generation gene sequencing enables patients to access bile acid replacement therapy earlier, significantly improving short- and long-term outcomes.

Massively parallel sequencing uncovered disease-associated variant spectra of glucose-6-phosphate dehydrogenase deficiency, phenylketonuria and galactosemia in Vietnamese pregnant women.

BACKGROUND: Several inherited metabolic diseases are underreported in Vietnam, namely glucose-6-phosphate dehydrogenase deficiency (G6PDd), phenylketonuria (PKU) and galactosemia (GAL). Whilst massively parallel sequencing (MPS) allows researchers to screen several loci simultaneously for pathogenic variants, no screening programme uses MPS to uncover the variant spectra of these diseases in the Vietnamese population. METHODS: Pregnant women (mean age of 32) from across Vietnam attending routine prenatal health checks agreed to participate and had their blood drawn. MPS was used to detect variants in their G6PD, PAH and GALT genes. RESULTS: Of 3259 women screened across Vietnam, 450 (13.8%) carried disease-associated variants for G6PD, PAH and GALT. The prevalence of carriers was 8.9% (291 of 3259) in G6PD and 4.6% (152 of 3259) in PKU, whilst GAL was low at 0.2% (7 of 3259). Two GALT variants, c.593 T > C and c.1034C > A, have rarely been reported. CONCLUSION: This study highlights the need for routine carrier screening, where women give blood whilst receiving routine prenatal care, in Vietnam. The use of MPS is suitable for screening multiple variants, allowing for identifying rare pathogenic variants. The data from our study will inform policymakers in constructing cost-effective genetic metabolic carrier screening programmes.

Ultra-Deep Sequencing of Plasma-Circulating DNA for the Detection of Tumor- Derived Mutations in Patients with Nonmetastatic Colorectal Cancer.

Identification of tumor-derived mutation (TDM) in liquid biopsies (LB), especially in early-stage patients, faces several challenges, including low variant-allele frequencies, interference by white blood cell (WBC)-derived mutations (WDM), benign somatic mutations and tumor heterogeneity. Here, we addressed the above-mentioned challenges in a cohort of 50 nonmetastatic colorectal cancer patients, via a workflow involving parallel sequencing of paired WBC- and tumor-gDNA. After excluding potential false positive mutations, we detected at least one TDM in LB of 56% (28/50) of patients, with the majority showing low-patient coverage, except for one TDM mapped to KMT2D that recurred in 30% (15/30) of patients.

Circulating DNA methylation profile improves the accuracy of serum biomarkers for the detection of nonmetastatic hepatocellular carcinoma.

Aim: This study exploited hepatocellular carcinoma (HCC)-specific circulating DNA methylation profiles to improve the accuracy of a current screening assay for HCC patients in high-risk populations. Methods: Differentially methylated regions in cell-free DNA between 58 nonmetastatic HCC and 121 high-risk patients with liver cirrhosis or chronic hepatitis were identified and used to train machine learning classifiers. Results: The model could distinguish HCC from high-risk non-HCC patients in a validation cohort, with an area under the curve of 0.84. Combining these markers with the three serum biomarkers (AFP, lectin-reactive AFP, des-γ-carboxy prothrombin) in a commercial test, µTASWako®, achieved an area under the curve of 0.87 and sensitivity of 68.8% at 95.8% specificity. Conclusion: HCC-specific circulating DNA methylation markers may be added to the available assay to improve the early detection of HCC.

The early detection of liver cancer in high-risk populations can help people with the disease have a higher chance of survival and better quality of life. However, this is still a healthcare challenge. Current commercial blood tests measuring protein signatures in the blood have low accuracy due to increased levels of these proteins being detected in both liver cancer patients and patients with chronic liver diseases. In this study, we identified a set of signatures in DNA released by cancer cells into the bloodstream and used them as biomarkers to distinguish liver cancer patients from high-risk patients. We also demonstrated that adding those signatures to a commercial blood test currently used in clinics could improve the accuracy in detecting liver cancer patients.

Genetic landscape of recessive diseases in the Vietnamese population from large-scale clinical exome sequencing.

Accurate profiling of population-specific recessive diseases is essential for the design of cost-effective carrier screening programs. However, minority populations and ethnic groups, including Vietnamese, are still underrepresented in existing genetic studies. Here, we reported the first comprehensive study of recessive diseases in the Vietnamese population. Clinical exome sequencing data of 4503 disease-associated genes obtained from a cohort of 985 Vietnamese individuals was analyzed to identify pathogenic variants, associated diseases and their carrier frequencies in the population. A total of 118 recessive diseases associated with 164 pathogenic or likely pathogenic variants were identified, among which 28 diseases had carrier frequencies of at least 1% (1 in 100 individuals). Three diseases were prevalent in the Vietnamese population with carrier frequencies of 2-12 times higher than in the world populations, including beta-thalassemia (1 in 23), citrin deficiency (1 in 31), and phenylketonuria (1 in 40). Seven novel pathogenic and two likely pathogenic variants associated with nine recessive diseases were discovered. The comprehensive profile of recessive diseases identified in this study enables the design of cost-effective carrier screening programs specific to the Vietnamese population.

Pathogenic Variant Profile of Hereditary Cancer Syndromes in a Vietnamese Cohort.

BACKGROUND: Hereditary cancer syndromes (HCS) are responsible for 5-10% of cancer cases. Genetic testing to identify pathogenic variants associated with cancer predisposition has not been routinely available in Vietnam. Consequently, the prevalence and genetic landscape of HCS remain unknown. METHODS: 1165 Vietnamese individuals enrolled in genetic testing at our laboratory in 2020. We performed analysis of germline mutations in 17 high- and moderate- penetrance genes associated with HCS by next generation sequencing. RESULTS: A total of 41 pathogenic variants in 11 genes were detected in 3.2% individuals. The carrier frequency was 4.2% in people with family or personal history of cancer and 2.6% in those without history. The percentage of mutation carriers for hereditary colorectal cancer syndromes was 1.3% and for hereditary breast and ovarian cancer syndrome was 1.6%. BRCA1 and BRCA2 mutations were the most prevalent with the positive rate of 1.3% in the general cohort and 5.1% in breast or ovarian cancer patients. Most of BRCA1 mutations located at the BRCA C-terminus domains and the top recurrent mutation was NM_007294.3:c.5251C>T (p.Arg1751Ter). One novel variant NM_000038.6(APC):c.6665C>A (p.Pro2222His) was found in a breast cancer patient with a strong family history of cancer. A case study of hereditary cancer syndrome was illustrated to highlight the importance of genetic testing. CONCLUSION: This is the first largest analysis of carrier frequency and mutation spectrum of HCS in Vietnam. The findings demonstrate the clinical significance of multigene panel testing to identify carriers and their at-risk relatives for better cancer surveillance and management strategies.

Genetic profiling of Vietnamese population from large-scale genomic analysis of non-invasive prenatal testing data.

The under-representation of several ethnic groups in existing genetic databases and studies have undermined our understanding of the genetic variations and associated traits or diseases in many populations. Cost and technology limitations remain the challenges in performing large-scale genome sequencing projects in many developing countries, including Vietnam. As one of the most rapidly adopted genetic tests, non-invasive prenatal testing (NIPT) data offers an alternative untapped resource for genetic studies. Here we performed a large-scale genomic analysis of 2683 pregnant Vietnamese women using their NIPT data and identified a comprehensive set of 8,054,515 single-nucleotide polymorphisms, among which 8.2% were new to the Vietnamese population. Our study also revealed 24,487 disease-associated genetic variants and their allele frequency distribution, especially 5 pathogenic variants for prevalent genetic disorders in Vietnam. We also observed major discrepancies in the allele frequency distribution of disease-associated genetic variants between the Vietnamese and other populations, thus highlighting a need for genome-wide association studies dedicated to the Vietnamese population. The resulted database of Vietnamese genetic variants, their allele frequency distribution, and their associated diseases presents a valuable resource for future genetic studies.

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.

Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients.

The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen's kappa coefficient of 0.85 with 95% CI = 0.72-0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90-0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.