Androgen deprivation therapy has long been the first-line treatment for hormone-sensitive prostate cancer (HSPC). After progression to castration-resistant prostate cancer (CRPC), androgen receptor pathway inhibitors (AR...Androgen deprivation therapy has long been the first-line treatment for hormone-sensitive prostate cancer (HSPC). After progression to castration-resistant prostate cancer (CRPC), androgen receptor pathway inhibitors (ARPIs) are commonly used. Recently, combined therapy with androgen deprivation and an ARPI has been recommended for metastatic HSPC patients. Novel markers are urgently needed for monitoring this disease and for making therapeutic decisions. Plasma samples were collected from 140 patients with either metastatic HSPC (n = 72) or CRPC (n = 68) before the start of ARPI therapy. Digital PCR was used to assess AR gene amplification, while the expression levels of miR-375 were measured by quantitative PCR. Sixteen other clinical markers were also evaluated, including prostate-specific antigen (PSA), chromogranin A (CGA), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), C-reactive protein (CRP), lymphocyte-to-monocyte ratio, and platelet count. A multivariate analysis, adjusted for age and metastatic dissemination, identified miR-375 expression and lymphocyte-to-monocyte ratio to be the independent negative predictors of ARPI therapy failure in CRPC patients. Regarding the HSPC patients, this article reports the primary finding of the independent negative predictive value of platelet count, CRP, and CGA for the failure of combined androgen deprivation therapy and ARPI.
Oncogenic fusion detection is an essential part of clinical diagnosis and management of non-small-cell lung carcinoma. Numerous methods are available for detection of oncogenic fusions in the clinical laboratory, althoug...Oncogenic fusion detection is an essential part of clinical diagnosis and management of non-small-cell lung carcinoma. Numerous methods are available for detection of oncogenic fusions in the clinical laboratory, although RNA sequencing has rapidly gained prominence. Accordingly, however, multiple different RNA-sequencing assays exist, with diverse methods and varying performance characteristics. Here, a single-institutional clinical experience with a testing algorithm for non-small-cell lung carcinoma that uses amplicon-based DNA/RNA sequencing, followed by reflex hybridization-capture-based RNA sequencing if the initial testing is negative for oncogenic drivers, is reported. A total of 1211 non-small-cell lung carcinoma specimens were received for molecular testing, and 120 (approximately 10%) were reflexed for hybridization-capture-based RNA sequencing. Of the 120 cases tested, oncogenic fusions were identified in 9 and included clinically actionable fusions involving ALK, BRAF, NRG1, NTRK3, ROS1, and RET. None of these fusions was detected by the amplicon-based assay. Review of the 20,900 non-small-cell lung cancer cases in the American Association for Cancer Research Project Genie version 15.1 publicly available database (registration required) revealed that of the 1081 cases harboring fusions, 893 (82.6%) could theoretically be detected by the amplicon-based assay. Overall, this study shows that the addition of reflex hybridization-capture-based RNA sequencing could improve detection of rare and novel oncogenic fusions, maximizing patient eligibility for appropriate targeted therapies or clinical trials.
J Mol Diagn
· 2025 Jun · PMID 40122159
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Regulations, accreditation standards, and professional guidance require laboratories to use reference materials for assay development, validation, quality control, and proficiency testing of clinical genetic tests. There...Regulations, accreditation standards, and professional guidance require laboratories to use reference materials for assay development, validation, quality control, and proficiency testing of clinical genetic tests. There are, however, few publicly available reference materials for most genetic tests. To address this issue, the CDC's Genetic Testing Reference Material Program (GeT-RM), the Coriell Institute for Medical Research, and the genetic testing community have conducted 19 studies, including nine for pharmacogenetic (PGx) and human leukocyte antigen (HLA) testing, to generate characterized, renewable, and publicly available DNA samples for use as reference materials. Because new PGx alleles are frequently identified, and allele designations change over time, many samples were reanalyzed for the same gene(s) in subsequent GeT-RM studies. These studies used more comprehensive and sensitive methods and panels that examined additional single-nucleotide variants and/or star alleles to expand and update the consensus genotypes. Up-to-date information is available in two newly established resources: the GeT-RM Consolidated PGx and HLA Table and the GeT-RM PGx Search Tool. These resources contain all available PGx and HLA genotypes for 363 publicly available samples characterized during nine GeT-RM PGx or HLA studies for 34 genes/loci in a consolidated and searchable format.
Xia X, Cheng N, Liu Y
… +20 more, Yue D, Gao M, Hu C, Jiao K, Wang N, Zhu B, Chang X, Zeng M, Song J, Sun C, Yan C, Xi J, Lin J, Luo S, Wang Z, Lu J, Jones PL, Zhao C, Wu Q, Zhu W
Facioscapulohumeral muscular dystrophy (FSHD) is caused by pleiotropic contractions of the D4Z4 repeat array on chromosome 4q35 (FSHD1) or by mutations in repressive chromatin regulators of the D4Z4 loci (FSHD2), both re...Facioscapulohumeral muscular dystrophy (FSHD) is caused by pleiotropic contractions of the D4Z4 repeat array on chromosome 4q35 (FSHD1) or by mutations in repressive chromatin regulators of the D4Z4 loci (FSHD2), both resulting in epigenetic dysregulation at the D4Z4 array. DNA methylation of the D4Z4 repeat array has been proposed for diagnosis and prognosis of FSHD disease severity; however, further validation in larger populations is needed. Two hundred forty-seven clinically suspected FSHD cases were retrospectively analyzed with D4Z4 analysis by optical genome mapping or molecular combing and tested the DNA methylation levels for 75 patients and 49 healthy controls. A D4Z4 repeat length-dependent nonlinear increase was observed in both distal and global D4Z4 methylation levels. Distal D4Z4 methylation levels identified patients with FSHD1 with a sensitivity of 100% and a specificity of 97.96% at a cutoff value of 39.66% compared with controls. Distal FSHD1-like hypomethylation was also observed in one subject carrying a special D4Z4 rearrangement, resulting in a proximal contracted array. Clinically, distal methylation levels demonstrated a strong correlation with the age-corrected clinical severity score and onset age. Mediation analysis revealed that the influence of distal methylation on age-corrected clinical severity score was partially mediated by onset age. This study further confirms the distal 4qA D4Z4 methylation analysis as a valuable complement for differential diagnosis in patients with suspected FSHD, including those with complex structural variants.
The use of urine for cervical cancer screening is gaining international attention, although more data on the relative clinical accuracy of validated human papillomavirus (HPV) DNA tests on urine versus cervical samples a...The use of urine for cervical cancer screening is gaining international attention, although more data on the relative clinical accuracy of validated human papillomavirus (HPV) DNA tests on urine versus cervical samples are needed. This study primarily seeks to evaluate the clinical performance of Roche cobas 4800 and 6800 HPV Systems in first-void urine, collected at home, compared with clinician-collected cervical samples. Paired first-void urine (index test) and cervical samples (comparator test) from 499 females enrolled at five Belgian colposcopy clinics were analyzed with cobas HPV Systems. Colposcopy and histology of biopsies were used as reference test (trial registration number: NCT03064087). Sample processing protocols and clinical thresholds proposed by the manufacturer for cervical samples were also applied for first-void urine. In the total study population, HPV testing on first-void urine was similarly sensitive [ratio, 0.98; 95% CI, 0.93-1.02] and specific for cobas 4800 HPV (ratio, 1.00; 95% CI, 0.91-1.10) and cobas HPV for use on the cobas 6800 System (ratio, 0.96; 95% CI, 0.91-1.02; ratio, 1.01; 95% CI, 0.93-1.09) compared with cervical samples (P ≥ 0.05). Good to excellent HPV test agreements between paired samples were observed (κ = 0.68 to 0.87). In summary, HPV testing using cobas 4800 and 6800 HPV Systems was as accurate on first-void urine as on cervical samples collected by a clinician.
Copy number variants (CNVs), structural alterations in the genome involving duplication or deletion of DNA segments, are implicated in various health conditions. Despite their clinical significance, accurate identificati...Copy number variants (CNVs), structural alterations in the genome involving duplication or deletion of DNA segments, are implicated in various health conditions. Despite their clinical significance, accurate identification and interpretation of CNVs remain challenging, especially in the context of whole-exome sequencing (WES), which is commonly used in clinical diagnostic laboratories. Although WES offers economic advantages over whole-genome sequencing, it struggles with CNV detection because of technical noise introduced by laboratory and analytic processes. Manual curation of CNV calls generated by these tools is labor intensive and error prone. To address this, SeeNV, a command-line tool, is introduced to aid manual curation of CNVs at scale. SeeNV is one solution to these issues, developed in collaboration with and used by the Precision Diagnostics Laboratory at Children's Hospital Colorado. SeeNV generates static infographics for each CNV, incorporating sample and cohort sequencing coverage statistics, CNV population frequency, and, more, facilitating rapid and precise assessment. Using CNV calls identified in publicly available WES and whole-genome sequencing samples, users can rapidly and reliably curate CNV calls, needing only 4.3 seconds to curate a call, achieving 0.95 recall (analytical sensitivity) and 0.74 precision (positive predictive value). SeeNV is freely available for download on GitHub.
Next-generation sequencing (NGS) allows the detection of multiple genetic targets in different tumor types. This study aimed to confirm the benefits of implementing in-house NGS testing for non-small-cell lung cancer (NS...Next-generation sequencing (NGS) allows the detection of multiple genetic targets in different tumor types. This study aimed to confirm the benefits of implementing in-house NGS testing for non-small-cell lung cancer (NSCLC) samples in molecular pathology laboratories. A multi-institutional study was conducted to evaluate the analytical performance, turnaround time, and feasibility of in-house NGS testing of 50 genes from 283 NSCLC samples. The first phase was a retrospective study with interlaboratory testing (21 samples), and the second phase was a prospective study with intralaboratory testing (262 samples). The retrospective study showed a 100% sequencing success rate for DNA and RNA, high interlaboratory concordance (95.2%), and a strong correlation (R = 0.94) between observed and expected single-nucleotide variant/insertion and/or deletion variant allele fraction. The prospective study showed a sequencing success rate of 99.2% for DNA and 98% for RNA. NGS identified 285 relevant variants (81.1% single-nucleotide variants/insertion and/or deletion variants, 9.8% copy number variants, and 9.1% gene fusions). Co-mutations with potential clinical relevance were detected in 20.5% of samples positive for the main oncogenic drivers in NSCLC. Additionally, 11% of samples wild type for the main oncogenic drivers carried alterations in other relevant genes. The in-house NGS testing had a median turnaround time from sample processing to molecular report of 4 days. This study demonstrates the advantages of implementing in-house NGS testing in molecular pathology laboratories.
Preimplantation genetic testing (PGT) is an essential tool for selecting embryos free of genetic abnormalities. However, current PGT methods often require separate platforms for aneuploidy (PGT-A), monogenic disorders (P...Preimplantation genetic testing (PGT) is an essential tool for selecting embryos free of genetic abnormalities. However, current PGT methods often require separate platforms for aneuploidy (PGT-A), monogenic disorders (PGT-M), and structural rearrangements (PGT-SR), leading to increased costs and operational complexity when multiple PGT tests are needed for a single embryo. Here, we present KaryoSeq, a low-pass whole-genome sequencing-based comprehensive PGT approach that integrates PGT-A, PGT-M, and PGT-SR into a single platform. An assistant decision-making system was constructed to pre-evaluate the required sequencing depth for specific genes or regions. Clinical validation of KaryoSeq was performed on 166 blastocyst samples from 31 families previously diagnosed by using conventional PGT methods. KaryoSeq achieved 100% concordance with traditional platforms using the Infinium Asian Screening Array in combination with low-coverage whole-genome sequencing (approximately 0.1×); it also offered improved whole-genome coverage, reduced variability, and efficient simultaneous analysis of PGT-A, PGT-M, and PGT-SR at a whole-genome sequencing depth of approximately 2× for most genes. In addition, KaryoSeq identified triploidy, uniparental disomy, parental origin of copy number variations, and maternal cell contamination, further enhancing its clinical utility and efficiency in PGT applications.
Galbraith K, Wu J, Sikkink K
… +17 more, Mohamed H, Reid D, Perez-Arreola M, Belton JM, Nomikou S, Melnyk S, Yang Y, Liechty BL, Jour G, Tsirigos A, Hermel DJ, Beck A, Sigal D, Dahl NA, Vibhakar R, Schmitt A, Snuderl M
J Mol Diagn
· 2025 May · PMID 40023492
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Chromosomal structural variants (SVs) are major contributors to cancer development. Although multiple methods exist for detecting SVs, they are limited in throughput, such as fluorescent in situ hybridization and targete...Chromosomal structural variants (SVs) are major contributors to cancer development. Although multiple methods exist for detecting SVs, they are limited in throughput, such as fluorescent in situ hybridization and targeted panels, and use RNA, which degrades in formalin-fixed, paraffin-embedded (FFPE) blocks and is unable to detect SVs that do not produce a fusion transcript. High-throughput chromosome conformation capture (Hi-C) is a DNA-based next-generation sequencing (NGS) method that preserves the spatial conformation of the genome, capturing long-range genetic interactions and SVs. Herein, a retrospective study analyzing 71 FFPE specimens from 10 different solid tumors was performed. Results showed high concordance (98%) with clinical fluorescent in situ hybridization and RNA NGS in detecting known SVs. Furthermore, Hi-C provided insight into the mechanism of SV formation, including chromothripsis and extrachromosomal DNA, and detected rearrangements between genes and regulatory regions, all of which are undetectable by RNA NGS. Lastly, SVs were detected in 71% of cases in which previous clinical methods failed to identify a driver. Of these, 14% were clinically actionable based on current medical guidelines, and an additional 14% were not in medical guidelines but involve targetable biomarkers. Current data suggest that Hi-C is a robust and accurate method for genome-wide SV analyses from FFPE tissue and can be incorporated into current clinical NGS workflows.
Unlike other cases of acute leukemia, the diagnosis of T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is uniquely based on morphology and flow cytometry. Although the genomic background has been broadly uncover...Unlike other cases of acute leukemia, the diagnosis of T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is uniquely based on morphology and flow cytometry. Although the genomic background has been broadly uncovered, the large spectrum of genes involved and the variability of the molecular mechanisms underlying gene deregulation have delayed the introduction of molecular cytogenetics into diagnostic flowcharts. To overcome these limitations and implement a genetic diagnosis of T-ALL/LBLs, a whole transcriptome expression assay (WTEa) was repurposed as a "priority test" to classify T-ALL/LBLs into the major genetic subtypes. A WTEa classifier based on a set of 312 probes on 215 T-ALL/LBLs was set up and applied, which properly assigned >95% of cases with subtype-defining alterations to the corresponding subgroups (ie, TAL/LMO, HOXA, TLX1, TLX3, BCL11B). It pinpointed cases that harbored cryptic alterations, such as noncoding mutations that generate new enhancer at TAL1 and LMO2 loci (8% of TAL/LMO), and duplications of noncoding element downstream BCL11B (BETA) (18% of BCL11B). It was also suitable to classify lymphoma cases for which only formalin-fixed embedded tissues were available, as confirmed in cases harboring TLX1 or TLX3 rearrangements, and distinguished new putative subtypes. WTEa offers a unifying tool to provide a genetic classification of T-ALL/LBLs. If introduced in multicenter prospective studies, it will facilitate evaluation of the clinical impact of genetic classification.
The National Academies of Sciences, Engineering, and Medicine issued a report on December 15, 2023, "Foundational Research Gaps and Future Directions for Digital Twins." This described the importance of using biomimetic...The National Academies of Sciences, Engineering, and Medicine issued a report on December 15, 2023, "Foundational Research Gaps and Future Directions for Digital Twins." This described the importance of using biomimetic digital twins and multiomics in research. These were incorporated in the current analysis of patients with rheumatoid arthritis (RA). Exome sequencing, genotype-phenotype ranking, and biomimetic digital twin analysis were used to identify five pathogenic and one likely pathogenic DNA variants in patient samples analyzed, which were absent from controls. The variants identified in these genes, P2RX7, HTRA2, PTPN22, FLG, CD46, and EIF4G1, play a role in the development of RA. Additionally, 3172 variants of unknown clinical significance (VUSs) were identified in patient samples, which were absent from controls. All VUSs appeared to be associated with RA. Hidden or dark data were identified from six genes. These genes, often found in patient samples, included HIF1A, HLA-DOA, PTGER3, HIPK3, TGFBR3, and HIF1A-AS3. VUSs identified in genes HIF1A, HLA-DOA, PTGER3, and HIPK3 were directly related to the pathogenesis of RA, whereas VUSs identified in genes TGFBR3 and HIF1A-AS3 were indirectly related. The current results suggest that biomimetic digital twins and multiomics can provide further insight into the development of RA. This may also potentially help with the process of reclassifying VUSs. The reclassification of VUSs will play a critical role in complex molecular diagnostics and drug development.
J Mol Diagn
· 2025 May · PMID 39954999
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The widespread adoption of next-generation sequencing technology in molecular pathology has enabled us to interrogate the genome as never before. The huge quantities of data generated by sequencing, the enormous complexi...The widespread adoption of next-generation sequencing technology in molecular pathology has enabled us to interrogate the genome as never before. The huge quantities of data generated by sequencing, the enormous complexity of human and microbial genetics, and the need for fast answers demand increasing use of automation as we diagnose disease and guide patient treatment. Much of this automation is based on tools that fall under umbrellas that have come to be known as machine learning and artificial intelligence. This review outlines some of the broad ideas that underpin these complex computational methods. It discusses the roles of pathologists and data scientists in generating new tools and factors to keep in mind when adopting these systems for use in molecular pathology. It pays special attention to regulatory and professional society guidance for validating them in individual institutions and to possible sources of bias. Finally, it briefly discusses ongoing efforts in computer science that may dramatically impact artificial intelligence in the future.
Godfrey TE, Kintsurashvili E, Rasic G
… +3 more, Kaur J, D'Amato C, Meltzer RH
J Mol Diagn
· 2025 Apr · PMID 39952465
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Detection and analysis of circulating tumor DNA (ctDNA) as a biomarker for cancer is a promising approach. Applications for ctDNA analysis include screening, diagnosis, treatment selection, treatment monitoring, minimal...Detection and analysis of circulating tumor DNA (ctDNA) as a biomarker for cancer is a promising approach. Applications for ctDNA analysis include screening, diagnosis, treatment selection, treatment monitoring, minimal residual disease detection, and recurrence monitoring. Detection of ctDNA is challenging and requires highly sensitive methods. Approaches such as digital PCR are appropriate when only a small number of targets is being interrogated, whereas next-generation sequencing (NGS) is typically used when more targets are being analyzed. There are several NGS methods available, some of which are published and can be implemented in laboratories with the required expertise while other, commercial approaches are proprietary and are only available as a service. Of the published methods, most use some kind of unique molecular identifiers (or barcodes) to facilitate NGS error correction and detection of rare mutations at mutant allele frequencies of <0.1%. However, incorporation of barcodes and amplification of the resulting libraries are not trivial and typically require multiple steps and considerable hands-on time by an experienced molecular biologist. Herein, a novel approach for switched temperature amplicon barcoding was used, in which barcoding and library amplification were performed in the same tube using a two-stage PCR protocol with no additional manipulation. Total hands-on time was 10 to 15 minutes for reaction setup; the library was then cleaned and was ready for sequencing.
The TruSight Oncology 500 (TSO500) High-Throughput Assay is a genomic profiling assay, supported by a bioinformatic analysis pipeline to evaluate somatic single-nucleotide variations/deletions/insertions, gene amplificat...The TruSight Oncology 500 (TSO500) High-Throughput Assay is a genomic profiling assay, supported by a bioinformatic analysis pipeline to evaluate somatic single-nucleotide variations/deletions/insertions, gene amplification, microsatellite instability, tumor mutational burden (TMB), gene fusion, and splice variants in solid tumors. This study outlines the approach used by the Genomics Laboratory at the Mayo Clinic to evaluate the technical performance of TSO500. The assessment involved 104 DNA and 223 RNA samples extracted from >20 tumor types. The assay demonstrated robust performance using 40 ng of input DNA and RNA, with slightly improved results observed at 60 ng of input DNA. Tumor percentage significantly influenced assay performance, with all variants being detected at 93% and 85% and above at tumor percentage >50% and >20%, respectively. Precision exceeded 93% across all variant types, including single-nucleotide variations and deletions/insertions with a variant allele frequency of ≥5%. Accuracy was ≥97% for all variant types except for TMB, which was 83.3% when compared with the reference method. Most discordant TMB cases had scores in the range of 8 to 12 mutations per megabase. Overall, the TSO500 assay demonstrated strong performance and reliable accuracy in detecting the evaluated markers.
This study demonstrates the analytical and clinical validity of the approved (United States and Japan) plasma-based Guardant360 companion diagnostic (CDx) test for selecting patients with human epidermal growth factor re...This study demonstrates the analytical and clinical validity of the approved (United States and Japan) plasma-based Guardant360 companion diagnostic (CDx) test for selecting patients with human epidermal growth factor receptor 2 (HER2 [ERBB2])-mutated (HER2m) non-small-cell lung cancer (NSCLC) for trastuzumab deruxtecan (T-DXd) treatment. Concordance between the Guardant360 CDx test and the plasma-based AVENIO ctDNA Expanded Kit Assay (AVENIO), as well as the tissue-based clinical trial assays (CTAs) was investigated. Clinical utility was assessed by comparing T-DXd clinical efficacy results of patients in DESTINY-Lung01/02 who tested positive for HER2 mutations using the Guardant360 CDx test to benchmark efficacy results from DESTINY-Lung01/02. Finally, concordance between the Guardant360 CDx test and the tissue-based Oncomine Dx Target (ODxT) test was explored. High concordance was observed between the Guardant360 CDx test versus AVENIO [positive percent agreement (PPA), 98.8%; negative percent agreement (NPA), 91.5%] and CTAs (DESTINY-Lung01 Cohort 2-PPA, 91.0%; NPA, 100%; DESTINY-Lung02 arm 1-PPA, 86.0%; NPA, 100%). Confirmed objective response rates were similar in patients with HER2m NSCLC identified by the Guardant360 CDx test and by CTAs. There was a high level of agreement between the Guardant360 CDx test and the ODxT test. The Guardant360 CDx test demonstrated analytical and clinical validity for identifying patients with HER2m NSCLC for T-DXd therapy; results support plasma-based testing when tissue-based testing is not feasible.
This article examines the frequency distribution of tier 1 pharmacogenetic variants of the Association for Molecular Pathology Pharmacogenomics Working Group Recommendations in two large (>1000 individuals) cohorts of th...This article examines the frequency distribution of tier 1 pharmacogenetic variants of the Association for Molecular Pathology Pharmacogenomics Working Group Recommendations in two large (>1000 individuals) cohorts of the admixed Brazilian population, and in patients from the Brazilian Public Health System enrolled in pharmacogenetic trials. Three tier 1 variants, all in DPYD, were consistently absent, which may justify their noninclusion in genotyping panels for Brazilians; 13 variants had frequency ≤1.0%, and the remaining 21 variants ranged in frequency from 1.2% (NUDT15∗3) to 76.4% (CYP3A5∗3). The frequency of some CYP2C9, CYP2D6, CYP3A4, and VKORC1 variants differed significantly across the three major race/color categories of the Brazilian Census (White, Brown, and Black), as a consequence of different proportions of individual European and African ancestry. However, it is recommended that selection of variants for inclusion in pharmacogenetic testing panels and implementation of pharmacogenetic-informed dosing guidelines for Brazilians should not be determined by race/color categories. Native Americans (0.4% of the Brazilian population), virtually absent from the study cohorts, display wide interethnic diversity in frequency of some tier 1 variants (eg, NUDT15∗3 and TPMT∗3A) and/or differ markedly from non-Indigenous people in frequency of some variant alleles (eg, CYP2C19∗17). Collectively, the data support the notion that population diversity must be taken into account on the design and implementation of pharmacogenetic testing panels.
Previous studies have reported that mitochondrial DNA copy number (mtDNA-CN) of blood was associated with a series of aging-related diseases. However, it remains unknown whether mtDNA-CN can be a potential biomarker of a...Previous studies have reported that mitochondrial DNA copy number (mtDNA-CN) of blood was associated with a series of aging-related diseases. However, it remains unknown whether mtDNA-CN can be a potential biomarker of acute aortic syndromes (AASs). The mtDNA-CN in blood of 190 male patients with AAS and 207 healthy controls were detected by standardized real-time quantitative PCR-based assay. The mtDNA sequencing data of blood and myocardial muscle in 134 individuals were used to analyze mtDNA somatic mutations in blood. mtDNA-CN in peripheral blood was negatively correlated with age of individuals. Further analysis based on next-generation sequencing data demonstrated numbers and heteroplasmy of mtDNA mutations were positively correlated with age. Remarkably, mtDNA-CN of patients with AAS was lower than that of healthy controls. Logistic regression also showed that mtDNA-CN was independently associated with risk of AAS. During follow-up, patients with the lowest mtDNA-CN quartile had a hazard ratio of 2.543 for all-cause-mortality and 1.964 for composite end points compared with the other patients. Moreover, multivariate Cox regression indicated that lowest mtDNA-CN quartile was independently associated with all-cause mortality in patients with AAS. Our study demonstrated a negative correlation between mtDNA-CN and age. Moreover, lower mtDNA-CN in peripheral blood was significantly associated with higher risk and worse prognosis of AAS. It provided crucial evidence supporting the potential of mtDNA-CN as a novel biomarker of AAS.