Next-generation Sequencing Assay Research Articles

Molecular testing stratifies the risk of structural recurrence in high risk differentiated thyroid cancer: a retrospective cohort study

BackgroundHigh-risk differentiated thyroid cancer in 2015 American Thyroid Association risk stratification system (ATA-RSS) exhibits a significantly increased probability of recurrence and poor outcomes. This study aimed to investigate the molecular profiles of high-risk differentiated thyroid cancer and to assess the role of molecular testing in enhancing prognostic risk stratification.MethodsIn a single-center study conducted at Fujian Cancer Hospital, Fujian Province, China, a consecutive cohort of differentiated thyroid cancer patients identified as high-risk under 2015 ATA-RSS criteria were retrospectively assessed, spanning from November 1, 2019, to March 31, 2022. Molecular characterize groups were conducted using an 18-gene next-generation sequencing assay. Patients harboring mutations in the TERT promoter, TP53, or PIK3CA genes were categorized as the high molecular risk group, while all others were assigned to the non-high molecular risk group.ResultsAmong the 108 cases, 32 (29.6%) fell into the high molecular risk group, characterized by a significantly older mean age (57.8 vs. 42.6 years, p < 0.001), larger tumor size (3.1 cm vs. 2.0 cm, p = 0.003), a higher incidence of aggressive pathological subtypes (43.8% vs. 7.9%, p < 0.001), and an increased occurrence of distant metastasis (34.4% vs. 7.9%, p = 0.001). Over a median follow-up period of 32.5 months, this high-risk group demonstrated an elevated risk of local recurrence (32.1% vs. 9.5%, HR: 3.18, 95% CI: 1.15-8.78) and metachronous distant metastasis (38.1% vs. 2.9%, HR: 12.54, 95% CI: 2.60-60.41). Multivariate COX regression analysis confirmed that molecular characterize groups (HR: 5.77, 95% CI: 2.18-15.23, p < 0.001) and tumor size (HR: 1.32, 95% CI: 1.00-1.74, p = 0.047) independently predicted recurrence-free survival.ConclusionATA-RSS high-risk differentiated thyroid cancer often presents with late-hit genetic alterations, which are strongly associated with increased likelihood of structural recurrence. Molecular testing offers a precise approach to recurrence risk stratification in high-risk cases, enabling personalized follow-up and treatment strategies tailored to the specific prognostic profile.

Outcomes of Broader Genomic Profiling in Metastatic Colorectal Cancer: A Portuguese Cohort Study

Background: Colorectal cancer (CRC) is the third most diagnosed cancer globally and the second leading cause of cancer-related deaths. Despite advancements, metastatic CRC (mCRC) has a five-year survival rate below 20%. Next-generation sequencing (NGS) is recommended nowadays to guide mCRC treatment; however, its clinical utility when compared with traditional molecular testing in mCRC is debated due to limited survival improvement and cost-effectiveness concerns. Methods: This retrospective study included mCRC patients (≥18 years) treated at a single oncology centre who underwent NGS during treatment planning. Tumour samples were analysed using either a 52-gene Oncomine™ Focus Assay or a 500+-gene Oncomine™ Comprehensive Assay Plus. Variants were classified by clinical significance (ESMO ESCAT) and potential benefit (ESMO-MCBS and OncoKBTM). The Mann–Whitney and Chi square tests were used to compare characteristics of different groups, with significance at p < 0.05. Results: Eighty-six metastatic colorectal cancer (mCRC) patients were analysed, all being MMR proficient. Most cases (73.3%) underwent sequencing at diagnosis of metastatic disease, using primary tumour samples (74.4%) and a focused NGS assay (75.6%). A total of 206 somatic variants were detected in 86.0% of patients, 31.1% of which were classified as clinically significant, predominantly KRAS mutations (76.6%), with G12D and G12V variants as the most frequent. Among 33.7% RAS/BRAF wild-type patients, 65.5% received anti-EGFR therapies. Eleven patients (12.8%) had other actionable variants which were ESCAT level I-II, including four identified as TMB-high, four KRAS G12C, two BRAF V600E, and one HER2 amplification. Four received therapies classified as OncoKbTM level 1–2 and ESMO-MCBS score 4, leading to disease control in three cases. Conclusions: NGS enables the detection of rare variants, supports personalised treatments, and expands therapeutic options. As new drugs emerge and genomic data integration improves, NGS is poised to enhance real-world mCRC management.

Prognostic Performance of the Next-Generation Sequencing-Based Multigene Assay in Early Breast Cancer Patients Treated According to the 21-Gene Assay Results.

Multigene assays guide treatment decisions in early-stage hormone receptor-positive breast cancer. OncoFREE, a next-generation sequencing assay using 179 genes, was developed for this purpose. This study aimed to evaluate the concordance between the Oncotype DX (ODX) Recurrence Score (RS) and the OncoFREE Decision Index (DI) and to compare their performance. We retrospectively collected tumor blocks from patients who underwent ODX and treatment between 2012 and 2022 at four tertiary hospitals and performed OncoFREE on these samples. Distant metastasis-free survival (DMFS) was compared using RS and DI, with score cut-offs of 25 and 20, respectively. Among 838 patients, a strong correlation was observed between RS and DI (Pearson correlation coefficient 0.83). At a median follow-up of 54 months, patients with high DI had significantly worse DMFS compared to those with low DI (log-rank p < 0.001, hazard ratio [HR] 5.73, 95% confidence interval [CI] 1.87-17.57; multivariable p=0.048, HR 3.45, 95% CI 1.01-11.76). In 513 patients aged ≤50 years, DMFS was significantly different as a function of DI (p=0.035, HR 3.98, 95% CI 1.00-15.89) but not RS (p=0.792). Among 376 patients aged ≤50 years who avoided chemotherapy based on low RS, 64 with high DI had worse DMFS (p=0.015, HR 5.91, 95% CI 1.17-29.78). OncoFREE showed strong concordance with ODX and effectively identified high-risk patients, particularly in younger individuals. It could be an affordable alternative to ODX for guiding treatment in hormone receptor-positive early breast cancer.

Integrated DNA estimation in tissue biopsy and detection in liquid biopsy by HBV-targeted NGS assay.

Integrated HBV DNA (iDNA) plays a critical role in HBV pathogenesis, particularly in predicting treatment response and HCC. This study aimed to use an HBV hybridization-capture next-generation sequencing (HBV-NGS) assay to detect HBV-host junction sequences (HBV-JS) in a sensitive nonbiased manner to detect and estimate the iDNA fraction in tissue biopsies and HBV genetics by liquid biopsy. HBV DNA from plasmid monomers, HBV-HCC cell line (SNU398, Hep3B, and PLC/PRF/5), tissue biopsies of patients with serum HBV DNA <4 log IU/ml, and matched urine and plasma of HBV patients were assessed by HBV-NGS. Junction-specific qPCR (JS-qPCR) assays were developed to quantify abundant HBV-JS. We demonstrated high coverage uniformity, reproducibility across all HBV genotypes A-D, and 0.1% sensitivity for detecting iDNA by the HBV-NGS assay. The sequence and structures of iDNA molecules from SNU398 and Hep3B are reported. An iDNA estimation model was developed using six abundant HBV-JS sequences identified from tissue biopsies by HBV-NGS assay and validated using total DNA of SNU398 and Hep3B cells. Furthermore, the utility of the HBV-NGS assay for HBV genetic analysis in liquid biopsies was explored using matched plasma-urine samples from three patients with serum HBV DNA levels ranging from high to undetectable. HBV-JS was detected in all body fluids tested, regardless of viral load. These findings suggest that the iDNA fraction in tissue biopsies from patients with limited or undetectable serum HBV DNA can be estimated using a robust HBV-NGS assay, and a sensitive HBV genetics liquid biopsy can be obtained. This study highlights the potential of NGS-based methods to advance HBV management.

Advantages of a genomic DNA-based NGS assay for detection of mutant NPM1 measurable residual disease in AML.

Mutations in the Nucleophosmin-1 (NPM1) gene are among the most common molecular aberrations in acute myeloid leukemia (AML). Various studies have established mutant NPM1 (mNPM1) as a faithful molecular measurable residual disease (MRD) marker with prognostic significance. Assessment of prognostic mNPM1 is included in the European LeukemiaNet (ELN) recommendations on MRD detection in AML. Due to recent advancements of promising drugs targeting mNPM1 AML, monitoring of mNPM1 MRD has gained interest, and is generally done by reverse transcriptase quantitative PCR (RT-qPCR). However, these RT-qPCR assays use cDNA as input, are based on gene expression levels of mNPM1, and are generally limited to specific mNPM1 gene variants. The main advantages of next-generation sequencing (NGS) using genomic DNA as input are stability, independence of gene expression levels, and the ability to detect any NPM1 variant in a single assay. Here we comprehensively investigated the applicability of NGS on DNA to detect mNPM1 MRD in a cohort of 119 (cDNA) and 310 (DNA) mNPM1 AML patients in complete remission after two cycles of induction chemotherapy. We demonstrate high correlations in levels and prognostic value between RT-qPCR/cDNA and NGS/DNA approaches, postulating NGS/DNA as an attractive alternative to RT-qPCR. We report that the 2% mNPM1/ABL1 threshold by RT-qPCR/cDNA corresponds to a NGS/DNA variant allele frequency (VAF) of 0.01%. The NGS/DNA threshold of >0.01% after two cycles of induction chemotherapy identifies significantly more AML patients with an increased relapse risk than current RT-qPCR/cDNA assays. The prognostic significance of mNPM1 MRD appears greatest in FLT3-ITD AML patients.

Targeted RNA sequencing in diagnostically challenging head and neck carcinomas identifies novel MON2::STAT6, NFATC2::NUTM2B, POC5::RAF1, and NSD3::NCOA2 gene fusions.

Although molecular tests developed for a growing list of oncogenic alterations have significantly aided in the classification of head and neck carcinomas, tumours in which prototypical histologic and immunophenotypic features are lacking or only partially developed continue to pose diagnostic challenges. Searching for known diagnostic and therapeutic targets by clinical next-generation sequencing (NGS) assays can often lead to new discoveries. We present our institutional experience in applying targeted RNA NGS in 36 head and neck carcinomas that were morphologically difficult to classify between 2016 and 2023. The patients ranged in age from 5 to 83 years (median, 64), with the majority of tumors occurring in the major salivary glands and the sinonasal tract. Overall, seven (19%) cases showed unusual gene rearrangements, including five novel alterations: MON2::STAT6 in a hard palate adenocarcinoma with mucinous features, POC5::RAF1 in apocrine intraductal carcinoma of the lacrimal gland, EWSR1::CDADC1 fusion in a basaloid carcinoma of the submandibular gland, NFATC2::NUTM2B in myoepithelial carcinoma, and NSD3::NCOA2 fusion in a peculiar high-grade carcinoma with a peritheliomatous growth pattern, and focal myogenic differentiation. Potential therapeutic actionability was identified in three cases (RAF1 and FGFR2 fusions). These findings broaden the current spectrum of gene rearrangements in head and neck carcinomas and support the utility of clinical NGS in identifying unusual, actionable alterations in diagnostically challenging cases.

Methodology of cfHPV-DNA Detection in Head and Neck Cancer: A Systematic Review and Meta-analysis.

We aim to compare the diagnostic accuracy of the different methodologies used in the detection of cell-free human papillomavirus (HPV) DNA in HPV-associated head and neck squamous cell carcinoma detection using bivariate analysis methods. Pubmed, Embase, and Scopus were queried using a broad search strategy to search for relevant studies. Test characteristics were extracted from 33 studies following literature screening, and underwent analyses utilizing a bivariate approach. Summary statistics were identified for each type of methodology, and forest plots and summary receiver operating characteristic curves were constructed. Bias was estimated using Deek's Funnel Plot and the QUADAS-2 tool. In terms of diagnostic accuracy, digital droplet polymerase chain reaction (ddPCR) based testing exhibited the highest diagnostics odds ratio at 138 (59.5, 318), followed closely by next-generation sequencing (NGS) at 120 (39.7, 362), then by polymerase chain reaction (PCR) at 31.4 (14.4, 68.6), and quantitative PCR at 8.74 (4.63, 16.5). NGS and ddPCR are comparable in overall diagnostic accuracy, bringing into question their relative roles in diagnosis and screening. Cost-effective ddPCR assays may serve as useful diagnostic and screening tests in the clinic with their low false positive rates and high sensitivity. However, NGS assays also offer high sensitivity and companion metrics, suggesting they may have a more precise role in disease monitoring. Importantly, assay development and benchmarking need further standardization to improve comparison between assays. Finally, saliva-based testing needs to be further investigated using NGS and ddPCR to further understand its limitations in disease detection and monitoring.

Biomarker analyses from the phase III randomized CLEAR trial: lenvatinib plus pembrolizumab versus sunitinib in advanced renal cell carcinoma.

In CLEAR, lenvatinib+ pembrolizumab (L+ P) significantly improved efficacy versus sunitinib in first-line treatment of patients with advanced renal cell carcinoma (aRCC). We report results from CLEAR biomarker analyses. Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) and next-generation sequencing assays (whole exome sequencing/RNA sequencing) were carried out on archival tumor specimens. For IHC-derived/RNA sequencing analyses, a continuous analysis was carried out adjusting by Karnofsky performance status (KPS) score for: PD-L1 combined positive score (CPS) versus best overall response (BOR)/progression-free survival (PFS); and each gene signature score [T-cell inflamed gene expression profile (TcellinfGEP)/non-TcellinfGEP signatures including proliferation and angiogenesis] versus BOR/PFS. Association between mutation status of RCC driver genes and PFS were analyzed for genes for which ≥20 patients per arm had oncogenic alterations. Association of molecular subtypes with outcome was evaluated with baseline KPS adjustments. The set of biomarkers evaluated and statistical significance criteria for PD-L1 CPS, gene signature scores, and molecular subtypes were prespecified. Within-arm analyses using continuous values showed no association between PD-L1 levels and BOR/PFS for either treatment. PFS hazard ratios between arms were similar regardless of the mutant or wild-type subgroups of RCC driver genes (VHL, PBRM1, SETD2, BAP1, KDM5C). No associations between PFS and gene signature scores were observed for L+ P. With sunitinib, high proliferation and MYC signature scores showed shorter PFS; high angiogenesis and microvessel density signature scores showed longer PFS. Six new molecular subtypes were defined. Tumors of patients with favorable/intermediate risk were enriched in angiogenesis and angiogenesis/stromal clusters; those with poor risk were enriched in proliferative and unclassified (low-TcellinfGEP/low-angiogenesis/low-proliferation) clusters. No association between molecular subtypes and PFS for L+ P/sunitinib was observed (after adjustment for KPS and gene signatures that were individually associated with PFS). Improvements in objective response rate and PFS for L+ P versus sunitinib in aRCC were observed consistently across a range of biomarker subgroups defined using RCC driver mutations, PD-L1, gene expression signatures, and molecular subtypes.

LOSS OF Y CHROMOSOME, AN ALARM TOWARDS AGGRESSIVE BLADDERCANCER IN MEN: ITS DETECTION AND DIAGNOSIS

In comparison to the other chromosomes, it is said that the Y chromosome is a bit poor in genes- being more than 50% of its sequence composed of repeated elements. Moreover, research says that the Y genes are in continuous decay probably due to the lack of recombination of this chromosome. Earlier assumptions carried a notion that the Y chromosome only played a role in guiding the development of the male sex organs in a foetus. However, detailed research studies over the past few years have revealed the fact that apart from only sex determination, the Y chromosome may actually protect men from aggressive bladder cancer. To examine the significance of Y chromosome losses in bladder cancer, fluorescence in situ hybridization (FISH) was used to determine its prevalence and associations with known parameters of malignancy. Detection of mosaic loss of Y chromosome (mLOY) is yet another important issue which is required for the proper diagnosis of the diseases in men. They usually include Next-generation sequencing (NGS) and Multiplex PCR-based assays.

The prevalence of optrA-carrying Enterococci in the vaginal micro-ecology of pregnant women in late pregnancy.

The colonization of Enterococcus in the female vagina leads to neonatal and pediatric enterococcal septicemia. Linezolid (LZD) is a kind of mainstream drug for treating multidrug-resistant Gram-positive infections. OptrA is the main LZD-resistance gene at Enterococci in human isolates. It is essential to explore the prevalence of optrA-carrying Enterococcus in vaginal secretions of late pregnant women and the drug resistance of optrA. From May to June 2023, this study recruited 340 volunteers in late pregnancy (35-40 weeks of pregnancy) to provide non-repetitive vaginal discharge samples. Luria-Bertani broth and florfenicol (10 µg/mL) were used to enrich bacteria. Enterococci was identified through time-of-flight mass spectrometry. Additionally, antimicrobial susceptibility, polymerase chain reaction, and next-generation sequencing assays were applied to this study. Fifty-four optrA-carrying Enterococcus strains were obtained, the proportion of the whole vagina of late pregnant women was 15.88% (54 out of 340), and Enterococcus faecalis account the highest proportion. All optrA-carrying Enterococcus were resistant to at least three drugs. Tetracycline, chloramphenicol, erythromycin, and LZD have higher bacterial resistance rates. Genetic environment analysis revealed that IS1216E, fexA, and erm(A) may synergistically exert multidrug resistance with optrA. It is necessary to strengthen the surveillance of optrA-carrying Enterococcus in pregnant women. This study provides scientific support for controlling hospital infections and managing antibiotic-resistant bacteria, and provides a scientific basis for rational clinical medication.IMPORTANCEThe disruption of cervicovaginal microbiota homeostasis is considered a key factor in causing imbalance in the microenvironment, leading to inflammation, transmission of infections, and illness. Enterococcus is considered a major cause of healthcare-related infections globally. It has resistance to multiple antimicrobial drugs, which pose significant challenges for clinical treatment. Therefore, it is crucial to assess the prevalence of optrA-carrying Enterococcus in vaginal secretions of late pregnant women and the drug resistance of optrA. This study detected 15.88% of optrA-carried Enterococci in 340 pregnant women. Furthermore, we found that optrA-carrying Enterococcus strains are highly resistant to tetracycline, chloramphenicol, erythromycin, and Linezolid. Additionally, genetic environment analysis revealed that IS1216E, fexA, and erm(A) may synergistically exert multidrug resistance with optrA. This study provides scientific support for controlling hospital infections and managing antibiotic-resistant bacteria and provides a scientific basis for rational clinical medication.

Metachromatic Leukodystrophy in Morocco: Identification of Causative Variants by Next-Generation Sequencing (NGS).

(1) Background: Most rare disease patients endure long delays in obtaining a correct diagnosis, the so-called "diagnostic odyssey", due to a combination of the rarity of their disorder and the lack of awareness of rare diseases among both primary care professionals and specialists. Next-generation sequencing (NGS) techniques that target genes underlying diverse phenotypic traits or groups of diseases are helping reduce these delays; (2) Methods: We used a combination of biochemical (thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry), NGS (resequencing gene panels) and splicing assays to achieve a complete diagnosis of three patients with suspected metachromatic leukodystrophy, a neurologic lysosomal disorder; (3) Results: Affected individuals in each family were homozygotes for harmful variants in the ARSA gene, one of them novel (c.854+1dup, in family 1) and the other already described (c.640G>A, p.(Ala214Thr), in family 2). In addition, both affected individuals in family 2 were carriers of a known pathogenic variant in an additionallysosomal disease gene, GNPTAB (for mucolipidosis III). This additional variant may modify the clinical presentation by increasing lysosomal dysfunction. (4) Conclusions: We demonstrated the deleterious effect of the novel variant c.854+1dup on the splicing of ARSA transcripts. We also confirmed the involvement of variant c.640G>A in metachromatic leukodystrophy. Our results show the power of diagnostic approaches that combine deep phenotyping, NGS, and biochemical and functional techniques.

Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting ERBB2 and Other Gene Amplifications in Solid Tumors.

Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10-30%, 31-60%, and 61-95% groups revealed significantly higher gene amplification detection rates in the 31-60% and 61-95% groups versus the 10-30% group (20.6% and 26.7% vs. 9.2%, p < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10-30% group versus the 61-95% group (0.11 vs. 0.52; p < 0.0001). To further investigate the analytic performance of OCAv3 in detecting ERBB2 amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed ERBB2 status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.

Low-Level MDM2 Amplification by FISH: An Institutional Experience With a Diagnostic Dilemma.

Background: MD M2 (murine double minute-2) amplification via fluorescence in-situ hybridization (FISH) is the gold standard test used for confirming the diagnosis of atypical lipomatous tumor/well differentiated liposarcoma and dedifferentiated liposarcoma. It is also used as a screening test in high grade spindle cell or pleomorphic neoplasms. MDM2 FISH is considered positive for amplification when the MDM2/CEP12 ratio is greater than 2; however, a ratio between 2 and 3 is considered a "low-level" amplification and raises the possibility of a false positive result; thus, posing a diagnostic dilemma. Another molecular modality, next generation sequencing (NGS) assay, can help in such situations in confirming or excluding the M D M 2 amplification status. Confronted by a number of neoplastic specimens at our institution with "low-level" MDM2 amplification via FISH, we aimed to assess the specificity of fluorescence in situ hybridization (FISH) in such tumors by comparing their NGS assay results and determine an accurate MDM2 amplification status that further aids in diagnosis. Methods: Tumors with "low-level" MDM2 amplification via FISH, defined as MDM2/CEP12 ratio between 2 and 3, and harboring a high grade and/or pleomorphic morphology were retrospectively retrieved from our institutional archives from the last five years. The retrieved specimens were evaluated for concordant retrospective Oncomine v3 analysis. Oncomine v3 is an institutional NGS assay that covers 161 genes and assesses for DNA mutations, RNA fusions, and copy number alterations including MDM2 gene gain or amplification. The tumors with Oncomine v3 results were compared and the FISH specificity was calculated. Results: Twenty-seven high grade and/or pleomorphic tumors with "low-level" MDM2 amplification were retrieved. Eight out of twenty-seven tumors had Oncomine v3 performed on them. The tumors correlated to neoplasms from different lineage types including undifferentiated melanoma, sarcomatoid squamous cell carcinoma, leiomyosarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, and high-grade poorly differentiated pleomorphic neoplasm. All 8 tumors had a low-level MD M2 amplification ratio ranging between 2.09 and 2.84. Seven out of eight had no MD M2 copy number alteration. One only (leiomyosarcoma) demonstrated MD M2 copy number gain (∼5 copies) that did not qualify as amplification due to the "6 copy number" gain cutoff. TP53, CDKN2A/B, PIKC3, and PTEN alterations were the most common genetic aberrations detected by Oncomine v3. Conclusion: We demonstrated the absence of MDM2 amplification via Oncomine in all our 8 "low-level" MDM2 FISH amplification specimens confirming the FISH results as "false positive" with a corresponding FISH specificity rate of 0%. Laboratory measures and utilizing NGS assay when needed, could be implemented when encountering such problematic "low-level" MDM2 amplification specimens to avoid misdiagnosis and misuse of targeted therapy. Future studies are needed to better characterize and investigate such findings.

Abstract A022: Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring

Abstract Monitoring minimal residual disease (MRD) provides invaluable information for disease progression and therapeutic decision making. However, there are many challenges to reliably detect key mutations or biomarkers, such as the accessibility, turnaround time, and sensitivity of current methods. High sensitivity is crucial due to the limited DNA available in certain sample types such as liquid biopsy. Next generation sequencing (NGS) is capable of highly sensitive detection but is dependent on high sample input and read depth, which is not always feasible due to the variable nature of circulating tumor DNA (ctDNA) in blood or plasma. In addition, NGS is not an economical nor rapid option for recurrent longitudinal monitoring. Digital PCR (dPCR) offers the sensitivity required for MRD monitoring but has yet to be widely adopted for this application. While real-time and qPCR are commonly used in oncology research, RT-PCR does not have the sensitivity required for early detection of key MRD mutations. Here, we present novel patent-pending technology that enhances the sensitivity of RT-PCR, allowing this common platform to be used to reliably detect low abundant mutations. We performed comparisons between GT Molecular’s patent-pending technology paired with RT-PCR and commercially available research use only dPCR and NGS assays to assess the sensitivity this new technology provides for RT-PCR. Cell-free DNA (cfDNA) was extracted from real patient or contrived plasma samples using the QIAamp Circulating Nucleic Acid Kit and analyzed using RT-PCR and dPCR assays targeting KRAS or ESR1 mutations. Samples with KRAS or ESR1 mutations were tested at variant allele frequencies (VAFs) varying between 1%-0.01% to evaluate the ability of GT Molecular’s new technology to detect low abundant mutations using RT-PCR. KRAS and ESR1 mutations were reliably detected with RT-PCR at VAFs as low as 0.01%, demonstrating sensitivity comparable to dPCR. Samples demonstrating positivity for KRAS or ESR1 mutations via dPCR were also positive via RT-PCR. With this patent-pending technology, RT-PCR is now capable of achieving the sensitivity required for MRD monitoring, providing critical information from liquid biopsy samples at a fraction of the cost. Citation Format: Mary Stischer, Nick Allen, Stephanie Barbari, Sarah Kane. Novel technology affords real-time PCR the sensitivity required for minimal residual disease monitoring [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A022.

Laboratory validation of a clinical metagenomic next-generation sequencing assay for respiratory virus detection and discovery

Tools for rapid identification of novel and/or emerging viruses are urgently needed for clinical diagnosis of unexplained infections and pandemic preparedness. Here we developed and clinically validated a largely automated metagenomic next-generation sequencing (mNGS) assay for agnostic detection of respiratory viral pathogens from upper respiratory swab and bronchoalveolar lavage samples in <24 h. The mNGS assay achieved mean limits of detection of 543 copies/mL, viral load quantification with 100% linearity, and 93.6% sensitivity, 93.8% specificity, and 93.7% accuracy compared to gold-standard clinical multiplex RT-PCR testing. Performance increased to 97.9% overall predictive agreement after discrepancy testing and clinical adjudication, which was superior to that of RT-PCR (95.0% agreement). To enable discovery of novel, sequence-divergent human viruses with pandemic potential, de novo assembly and translated nucleotide algorithms were incorporated into the automated SURPI+ computational pipeline used by the mNGS assay for pathogen detection. Using in silico analysis, we showed that after removal of all human viral sequences from the reference database, 70 (100%) of 70 representative human viral pathogens could still be identified based on homology to related animal or plant viruses. Our assay, which was granted breakthrough device designation from the US Food and Drug Administration (FDA) in August of 2023, demonstrates the feasibility of routine mNGS testing in clinical and public health laboratories, thus facilitating a robust and rapid response to the next viral pandemic.

PATH-51. CENTRAL NERVOUS SYSTEM METASTASES ARE GENOMICALLY DIVERGENT FROM THEIR RESPECTIVE EXTRACRANIAL SITES

Abstract INTRODUCTION Next-generation sequencing (NGS) has become standard of care in establishing the diagnosis and guiding treatment of cancers. Our understanding of genomic evolution of brain metastases (BM) and leptomeningeal disease (LMD) from solid tumor malignancies remains limited. Here we present updated data from our pilot study. METHODS We prospectively collected clinical data and NGS of seventeen patients undergoing craniotomy for symptomatic BM from solid tumor malignancies or cerebrospinal fluid (CSF) sampling for LMD. Matched systemic and CNS analyses were performed using a research-based NGS assay. When extracranial disease tissue was unavailable, liquid NGS served as a surrogate. RESULTS CNS metastases were present at initial diagnosis in 10/17 and 11/17 patients had not received systemic therapy prior to CNS tissue sampling. The predominant cancer type was NSCLC (n=11), followed by breast cancer (n=3), ovarian high grade serous carcinoma (n=1), GEJ adenocarcinoma (n=1), and basosquamous carcinoma of skin (n=1). The mean tumor mutational burden in extracranial samples was 3.65 (SD 3.34; SEM 0.97), in contrast to CNS samples 21.63 (SD 26.47; SEM 7.64). Concordant mutations in matched sampling had an increase in CNS variant allele frequency (VAF) (27.8% vs 17.3%; arithmetic difference = 10.5, p=0.054) and CNS gene copy number alterations (CNA) (2.4 copies vs 0.8 copies; arithmetic difference = 1.6, p=0.171). Known driver alterations in EGFR, KRAS and PIK3CA were retained. LMD was present in 4/17 patients, with two present at enrollment and two developing during the study. CONCLUSIONS Matched sequencing reveals CNS metastases to harbor a significantly higher TMB, retention of primary driver alterations with a trend towards higher VAF and CNA of concordant mutations. While limited by small sample size, these data indicate genetic evolution in CNS metastases regardless of prior treatment status.

SARCP, a Clinical Next-Generation Sequencing Assay for the Detection of Gene Fusions in Sarcomas: A Description of the First 652 Cases

An amplicon-based targeted next generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 of 652 (36.5%) of cases, including 83 distinct fusions in the 238 fusion positive cases, 10 of which had not been previously described. Among the 238 fusion positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR ITDs were identified in 6 of 485 (1.2%) patients. Among the 138 genes in the panel, 66 were involved in one or more fusions and 72 were not involved in any fusions. There was little overlap between the genes involved as 5’-partners (31 different genes) and 3’-partners (37 different genes). This study demonstrates the clinical utility of a next generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas.

Clinical Significance of Perioperative Minimal Residual Disease Detected by Circulating Tumor DNA in Patients With Lung Cancer With a Long Follow-up Data: An Exploratory Study

IntroductionMolecular residual disease detected by circulating tumor DNA (ctDNA) has been reported to be predictive of patients’ outcomes in various types of cancers after curative intent treatment. Nevertheless, additional detailed information regarding the association of longitudinal ctDNA detection with long-term follow-up in lung cancer is needed. Here, we report on a cohort of patients with NSCLC who underwent definitive surgery and ctDNA analysis in the pre-operative, adjuvant, and surveillance settings. MethodPlasma samples were collected from 46 patients with clinical stage II-III NSCLC before surgery (N = 46), after surgery (N = 45), and every six months until two years thereafter (N = 78). A clinically validated, personalized, tumor-informed 16-plex polymerase chain reaction–next-generation sequencing assay was used for the detection and quantification of ctDNA in retrospectively analyzed plasma samples. ResultsCirculating tumor DNA was detected in the first postoperative (within 51 days after surgery) plasma samples in 13% (6/45) of patients (landmark analysis). All of them had disease recurrence within a median of 9.1 months. These patients had shorter recurrence-free and overall survivals than those without detectable ctDNA at a landmark time point (p < 0.01) and in multivariate analyses (p < 0.03). Longitudinally (considering all postoperative follow-up time points), ctDNA was detected in 13 patients, all of whom experienced disease recurrence (positive predictive value = 100%). Three patients who had central nervous system–only metastases did not have detectable ctDNA. ConclusionsThe presence of ctDNA post-surgery or during surveillance identifies patients with NSCLC at high risk of recurrence. Serial testing is important to detect disease recurrence earlier (lead-time: 3.2 months).

The potential role of next-generation sequencing in identifying MET amplification and disclosing resistance mechanisms in NSCLC patients with osimertinib resistance.

Osimertinib, one of the third-generation EGFR-tyrosine kinase inhibitors (TKIs) designed to target EGFR T790M mutation, significantly improves the prognosis of lung cancer. However, drug resistance still happens and MET amplification is responsible for one of the main causes. Fluorescence in situ hybridization (FISH) is the gold standard for MET amplification detection, but fundamentally limited by observer subjectivity. Herein, we assessed the value of next-generation sequencing (NGS) method in MET amplification detection in non-small cell lung cancer (NSCLC), as well as revealed the mutation profiling of NSCLC patients with osimertinib resistance to provide some valuable clues to the mechanisms of resistance. A total of 317 cancer tissue samples from 317 NSCLC patients at time of progression following osimertinib were submitted to NGS and only 96 tissues were tested by FISH simultaneously. With FISH results as gold standard, enumeration algorithm was applied to establish the optimal model for identifying MET amplification using gene copy number (GCN) data. The optimal model for identifying MET amplification was constructed based on the GCN of MET, BRAF, CDK6 and CYP3A4, which achieved a 74.0% overall agreement with FISH and performed well in identifying MET amplification except polysomy with a sensitivity of 85.7% and a specificity of 93.9%. The inconsistency between NGS and FISH occurred mainly in polysomy subtype, while MET GCN ≥ 5 could be reliably recognized by NGS. Moreover, the most frequently mutated genes in NSCLC patients with osimertinib resistance were EGFR (59.94%), followed by TP53 (43.85%), NRG1 (9.46%), PIK3CA (6.31%), and ATM (5.36%). The known resistance mechanisms, including MET amplification, EGFR (C797S, L718Q/R), TP53, CDK4, CDK6, CDKN2A, BRAF, KRAS, NRAS and PIK3CA mutations were also disclosed in our cohort. NGS assay can achieve a high concordance with FISH in MET amplification detection and has advantages in portraying various genetic alterations, which is of worthy in clinical promotion.

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.