Next-generation DNA Sequencing Research Articles

Clinical utility of an ultra-sensitive ctDNA NGS assay for the detection and monitoring in HR-positive, HER2-negative breast cancer.

e13076 Background: Breast cancer is the most diagnosed cancer in women globally, mainly as hormone receptor HR-positive, HER2-negative. While endocrine therapies have markedly improved clinical outcomes, the threat of disease progression persists. This study introduces an ultra-sensitive circulating tumor DNA (ctDNA) Next-Generation Sequencing (NGS) assay that was designed for in-depth genomic profiling of breast cancer patients undergoing endocrine therapy, aimed at detecting treatment resistance and monitoring disease progression. Methods: In this ongoing investigation, 46 patients with advanced HR-positive, HER2-negative breast cancer were enrolled, and plasma samples were collected subsequent to first-line aromatase inhibitor (AI) treatment and before the initiation of second-line treatment with fulvestrant. The study employed the PredicineCARE ULTRA, an ultra-sensitive liquid biopsy assay with a proprietary NGS panel and exceeds 100,000x coverage, allowing for the ultra-sensitive identification of genomic alterations, surpassing the detection capabilities of standard ctDNA NGS assays with approximately 20,000x coverage. Results: Within the cohort of 46 patients, the ultra-sensitive assay detected 193 somatic mutations, 95 gene copy number variants (CNVs), and a gene fusion involving FGFR3. The most commonly mutated genes, with a prevalence of 20% or more, were TP53 (33%), PIK3CA (26%), ATM (24%), ESR1 (22%), and BRCA2 (20%). These findings were benchmarked against results from a down-sampling algorithm designed to emulate ctDNA assays with 20,000x coverage. The ultra-sensitive assay revealed 31.3% more mutations (193 vs. 147) and 30.1% more CNVs. It was especially proficient in detecting mutations with allele frequencies (MAF) below 0.1%, identifying 10 additional mutations not observed at the standard ctDNA assay sensitivity, with the lowest MAF detected being 0.03% compared to the 0.1% threshold of the standard assay. This heightened sensitivity was evident in genes associated with targeted therapies and drug resistance, identifying 30.7% more variations in PIK3CA and 11.1% more in ESR1, with the lowest MAFs of 0.08% and 0.06%, respectively. The FGFR3-BAIAP2L1 gene fusion was also uniquely detected by the ultra-sensitive assay, with a MAF of 0.09%. Conclusions: The ultra-sensitive ctDNA NGS assay demonstrated superior detection of low-frequency mutations in HR-positive, HER2-negative breast cancer, significantly outperforming standard liquid biopsy assays. This enhanced sensitivity may offer profound implications for identifying more diagnostically positive patients who may benefit from targeted therapy such as PIK3CA and ESR1 inhibitors. This ultra-sensitive ctDNA assay will also enable early detection of treatment resistance and refined disease monitoring, potentially guiding more effective personalized therapies.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values ​< ​0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

Differential genomic profiling of the progesterone receptor (PR) negative and estrogen receptor (ER) low metastatic breast cancer subtypes through circulating tumor DNA: A TNBC-like disease subtype?

1039 Background: ER positive/HER2 negative metastatic breast cancer (MBC) is typically regarded as a single entity, although significant clinical differences are often observed, especially in the ER low subgroup (ER&lt;10% regardless of PR status). This study aims to investigate genomic differences among HER2 negative subtypes (i.e., ERlow, PRpos [ER &gt;10%/PRpositve], PRneg [ER &gt;10%/PRnegative], triple negative [TNBC]) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS). Methods: The study analyzed a retrospective cohort of 879 patients (pts) with HER2 negative MBC with ctDNA testing using the Guardant360 NGS panel within a large multicenter academic consortium. ER, PR and HER2 status was defined based on the most updated biopsy. Associations across single nucleotide and copy number variations (SNVs and CNVs) and ER/PR status were tested by multinomial logistic regression in terms of Relative Risk Ratio (RRR), adjusting for significant clinical characteristics (i.e., lines of treatment, metastatic sites, histology). Oncogenic pathway was defined based on reference (1). Prognosis was analyzed through Cox regression for overall survival (OS) defined from time of ctDNA collection. Results: Among the 879 analyzed pts, PRpos was the most prevalent subtype (N:420, 48%), followed by PRneg (N:216, 24%), TNBC (N:209, 24%), and ERlow (N:34, 4%). Compared to PRpos, lobular MBC was less common in TNBC (RRR: 0.33, P=0.001), while lung involvement was more frequent in TNBC (RRR: 1.53, P=0.019), and liver involvement in PRneg (RRR: 1.44, P=0.035). In contrast, bone metastases were less common in TNBC and ERlow (RRR: 0.26, P&lt;0.001 and RRR: 0.45, P=0.03, respectively). After multivariable analysis, ERlow and TNBC were associated with a higher incidence of TP53 SNVs (RRR: 5.42, P&lt;0.001 and RRR: 3.08, P=0.007, respectively) and a lower incidence of ESR1 SNVs (RRR: 0.23, P=0.025 and not detected, respectively). TNBC exhibited significantly fewer PIK3CA SNVs (RRR: 0.41, P=0.005) and CCND1 CNVs (RRR: 0.20, P=0.009), along with a higher frequency of CCNE1 CNVs (RRR: 6.47, P&lt;0.001). PRneg had a lower incidence of ESR1 SNVs (RRR: 0.52, P=0.013) and more frequent FGFR1 CNVs (RRR: 2.61, P=0.004). Similar results were observed across oncogenic pathways. Compared to PRpos, OS analysis indicated poorer outcomes across all subgroups (PRneg HR: 1.29 P = 0.035, TNBC HR: 1.96 P&lt;0.001, ERlow HR: 2.32 P&lt;0.001). Conclusions: This study highlights several clinical and molecular similarities between TNBC and ERlow MBC, while suggesting specific endocrine resistance mechanisms in PRneg that are less reliant on ESR1 and more exposed to alternative mechanisms such as FGFR1 amplification. These findings underscore the importance of more precise disease subtyping to inform future drug development trials. 1. Sanchez-Vega F et al, Cell. 2018.

Comparison of plasma and blood cell samples in metagenomic next-generation sequencing for identification of the causative pathogens of fever

BackgroundMetagenomic next-generation sequencing (mNGS) of plasma DNA has become an attractive diagnostic method for infectious diseases; however, the rate of false-positive results is high. This study aims to evaluate the diagnostic accuracy of mNGS in plasma versus blood cell samples for immunocompromised children with febrile diseases. MethodsThe results of conventional microbiological test (CMT) and mNGS using plasma and blood cells in 106 patients with 128 episodes of febrile diseases from the Department of Hematology/Oncology were analyzed and described. ResultsThe positivity rates for CMT and mNGS of plasma and blood cells were 35.9 %, 84.4 % and 46.9 %, respectively (P < 0.001). Notably, mNGS identified multiple pathogens in a single specimen in 68.5 % of plasma samples and 38.3 % of blood cell samples (P < 0.001). Furthermore, plasma and blood cell mNGS identified causative pathogens in 58 and 46 cases, accounting for 53.7 % and 76.7 % of the mNGS-positive cases for each sample type, respectively (P = 0.002). By integrating results from both plasma and blood cell samples, causative pathogens were identified in 77 cases (60.2 %), enhancing sensitivity to 87.5 % but reducing specificity to 15.0 %, compared to plasma (65.9 % sensitivity and 20.0 % specificity) and blood cell samples (52.3 % sensitivity and 80.0 % specificity). ConclusionsmNGS of plasma is sensitive but has a high false-positive rate, while mNGS of blood cells has low sensitivity but higher specificity.

Quantitative analysis of the effects of brushing, flossing, and mouthrinsing on supragingival and subgingival plaque microbiota: 12-week clinical trial

BackgroundTranslational microbiome research using next-generation DNA sequencing is challenging due to the semi-qualitative nature of relative abundance data. A novel method for quantitative analysis was applied in this 12-week clinical trial to understand the mechanical vs. chemotherapeutic actions of brushing, flossing, and mouthrinsing against the supragingival dental plaque microbiome. Enumeration of viable bacteria using vPCR was also applied on supragingival plaque for validation and on subgingival plaque to evaluate interventional effects below the gingival margin.MethodsSubjects with gingivitis were enrolled in a single center, examiner-blind, virtually supervised, parallel group controlled clinical trial. Subjects with gingivitis were randomized into brushing only (B); brushing and flossing (BF); brushing and rinsing with Listerine® Cool Mint® Antiseptic (BA); brushing and rinsing with Listerine® Cool Mint® Zero (BZ); or brushing, flossing, and rinsing with Listerine® Cool Mint® Zero (BFZ). All subjects brushed twice daily for 1 min with a sodium monofluorophosphate toothpaste and a soft-bristled toothbrush. Subjects who flossed used unflavored waxed dental floss once daily. Subjects assigned to mouthrinses rinsed twice daily. Plaque specimens were collected at the baseline visit and after 4 and 12 weeks of intervention. Bacterial cell number quantification was achieved by adding reference amounts of DNA controls to plaque samples prior to DNA extraction, followed by shallow shotgun metagenome sequencing.Results286 subjects completed the trial. The metagenomic data for supragingival plaque showed significant reductions in Shannon-Weaver diversity, species richness, and total and categorical bacterial abundances (commensal, gingivitis, and malodor) after 4 and 12 weeks for the BA, BZ, and BFZ groups compared to the B group, while no significant differences were observed between the B and BF groups. Supragingival plaque vPCR further validated these results, and subgingival plaque vPCR demonstrated significant efficacy for the BFZ intervention only.ConclusionsThis publication reports on a successful application of a quantitative method of microbiome analysis in a clinical trial demonstrating the sustained and superior efficacy of essential oil mouthrinses at controlling dental plaque compared to mechanical methods. The quantitative microbiological data in this trial also reinforce the safety and mechanism of action of EO mouthrinses against plaque microbial ecology and highlights the importance of elevating EO mouthrinsing as an integral part of an oral hygiene regimen.Trial registrationThe trial was registered on ClinicalTrials.gov on 31/10/2022. The registration number is NCT05600231.

Characterization of FOLH1 Expression in Renal Cell Carcinoma.

Given the emergence of PSMA-targeted diagnostic agents and therapeutics, we sought to investigate patterns of FOLH1 expression in RCC and their impacts on RCC outcomes. We conducted a pooled multi-institutional analysis of patients with RCC having undergone DNA and RNA next-generation sequencing. FOLH1-high/low expression was defined as the ≥75th/<25th percentile of RNA transcripts per million (TPM). Angiogenic, T-effector, and myeloid expression signatures were calculated using previously defined gene sets. Kaplan-Meier estimates were calculated from the time of tissue collection or therapy start. We included 1,724 patients in the analysis. FOLH1 expression was significantly higher in clear cell (71%) compared to non-clear cell RCC tumors (19.0 versus 3.3 TPM, p < 0.001) and varied by specimen site (45% primary kidney/55% metastasis, 13.6 versus 9.9 TPM, p < 0.001). FOLH1 expression was correlated with angiogenic gene expression (Spearman = 0.76, p < 0.001) and endothelial cell abundance (Spearman = 0.76, p < 0.001). While OS was similar in patients with FOLH1-high versus -low ccRCC, patients with FOLH1-high clear cell tumors experienced a longer time on cabozantinib treatment (9.7 versus 4.6 months, respectively, HR 0.57, 95% CI 0.35-0.93, p < 0.05). We observed differential patterns of FOLH1 expression based on histology and tumor site in RCC. FOLH1 was correlated with angiogenic gene expression, increased OS, and a longer duration of cabozantinib treatment.

Transcriptionally Active Human Papillomavirus Infection in a Minority of Esophageal Squamous Cell Carcinomas in North America.

The role of Human papillomavirus (HPV) infection in esophageal squamous cell carcinoma (ESCC) is a topic of ongoing debate. This study used two screening approaches to look for evidence of HPV infection in esophageal squamous cell carcinoma. We initially checked for HPV infection in a randomly selected group of 53 ESCC cases. We did not detect any tumors positive for high-risk HPV. However, during clinical practice, we identified an HPV-positive ESCC in the distal esophagus, which tested positive for HPV16. This index case was TP53 wild-type, as determined by next-generation DNA sequencing (NGS). Since TP53 mutations are rare in other HPV-driven cancers, we improved our screening method by limiting our screen to a subset of ESCC cases without TP53 mutations. A second screen of 95 ESCCs (from 93 patients) sequenced by NGS revealed an additional 7 ESCCs with TP53 wild-type status (7.3% of the total). Of the 7 cases, 2 cases were found to be high-risk HPV positive. Both patients also tested positive for circulating cell-free HPV DNA and had a complete response to neoadjuvant chemoradiation. The index patient had microscopic residual tumor following neoadjuvant therapy. The patient underwent adjuvant immunotherapy and remained disease free after 22 months of surveillance. This study affirms the transcriptionally active status of high-risk HPV in a minority of ESCC patients in North America.

Advancement of Next-Generation DNA Sequencing through Ionic Blockade and Transverse Tunneling Current Methods.

DNA sequencing is transforming the field of medical diagnostics and personalized medicine development by providing a pool of genetic information. Recent advancements have propelled solid-state material-based sequencing into the forefront as a promising next-generation sequencing (NGS) technology, offering amplification-free, cost-effective, and high-throughput DNA analysis. Consequently, a comprehensive framework for diverse sequencing methodologies and a cross-sectional understanding with meticulous documentation of the latest advancements is of timely need. This review explores a broad spectrum of progress and accomplishments in the field of DNA sequencing, focusing mainly on electrical detection methods. The review delves deep into both the theoretical and experimental demonstrations of the ionic blockade and transverse tunneling current methods across a broad range of device architectures, nanopore, nanogap, nanochannel, and hybrid/heterostructures. Additionally, various aspects of each architecture are explored along with their strengths and weaknesses, scrutinizing their potential applications for ultrafast DNA sequencing. Finally, an overview of existing challenges and future directions is provided to expedite the emergence of high-precision and ultrafast DNA sequencing with ionic and transverse current approaches.

117 Gene editing on the farm: Current status and future prospects

Abstract Livestock with enhanced disease resistance genes offer both health advantages to animals and economic opportunities for farmers aiming to meet the rising demand for affordable, high-quality protein. This study builds upon prior research that utilized gene editing to modify the porcine CD163 gene, demonstrating resistance to the RNA virus responsible for porcine reproductive and respiratory syndrome (PRRS). To amplify the impact of this disease resistance trait, it is crucial for it to be present in commercially significant breeding populations, facilitating the widespread production and distribution of resistant pigs. In pursuit of this objective, a groundbreaking gene editing program was implemented, leveraging CRISPR-Cas technology, next-generation DNA sequencing, in vitro and in vivo off-target analyses, whole-genome sequencing (WGS), and a simplified breeding strategy. This innovative approach aimed to introduce a single modified CD163 allele into four genetically elite and diverse porcine lines, rendering the cells and animals resistant to PRRS virus (PRRSV) infection. The resulting founder population is currently undergoing large-scale phenotype testing, multiplication through classical breeding methods, ongoing genetic refinement, and poised for commercialization pending regulatory approvals. This presentation will provide insights into the process and data associated with this scaled production, marking a significant stride towards the realization of commercially elite PRRS virus resistant pigs.

Interaction of sound-audition traits between eared insects and arthropodophagous bats: using a DNA approach to assess diet

Abstract Arthropod–bat interactions are often considered as a base model for studying factors underlying predator–prey coevolutionary processes. Bats developed ultrasonic echolocation to hunt, and in response some arthropods developed defense mechanisms such as ultrasonic hearing, allowing them to elude bat predators. The present study analyzes the feeding patterns of bats, focusing on sonic-auditory sensory mechanisms in predator–prey interactions. Next-generation DNA sequence data from fecal samples were used to analyze the diet of 17 bat species from Mexico. Arthropod prey taxa were classified according to their auditory traits, and echolocation data were recompiled from literature review. We: (i) classified arthropod families according to their hearing ability; (ii) estimated arthropod taxon richness and proportion in the diet of each bat species; and (iii) used multidimensional scaling, principal component analysis, and regression to analyze prey consumption patterns in relation to their auditory traits and in relation to echolocation characteristics of bats. Finally, we analyzed the relationship between foraging time and auditory characteristics of prey. Families with hearing organs correspond to the orders Lepidoptera and Orthoptera. We registered 20 families of Lepidoptera and 5 of Orthoptera—7 and 3 with hearing organs, respectively. Of these orders, families lacking ears were recorded in the diet of a few bat species. Our results support the allotonic frequency hypothesis predicting a difference in emission frequency intervals between predator and prey. However, we found that the consumption of earless moths is less frequent and is related to diurnal and twilight activity—hence, their consumption is limited to bat species foraging early. Results indicate bats feed on arthropod prey successfully despite the ultrasonic hearing ability of the prey. These results may be due to counteradaptations that allow maintenance of an asymmetric “arms race” between bats and eared insects that favors the predator.

Abstract PO3-23-09: ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer

Abstract Mutations in ESR1 (the gene encoding the estrogen receptor, ERα) are major drivers of resistance to antiestrogen therapy in ER+ breast cancer. However, it remains unclear whether ESR1 mutations also drive resistance to CDK4/6 inhibitors (CDK4/6i). We analyzed de-identified next generation sequencing (NGS) data of tumor tissue DNA (n=1,820) and circulating tumor (ct) DNA (n=2,138) from patients with ER+/HER2- metastatic breast cancer. NGS of tumor tissue DNA and ctDNA previously had been performed using the Tempus xT tumor assay (595-648 genes) and Tempus xF liquid biopsy (105-523 genes), respectively. Patients were stratified into treated (xT:1,070; xF:1,885) or untreated (xT:750; xF:253) with CDK4/6i prior to biopsy, and untreated patients were restricted to those biopsied within 30 days of metastatic diagnosis. ESR1 was the only gene for which mutations were significantly enriched in patients who received CDK4/6i vs. those who did not in both xT (32% vs. 4.8%, q&amp;lt; 0.001) and xF (29% vs. 9.5%, q&amp;lt; 0.001) cohorts. To determine whether ESR1 mutations drive resistance to CDK4/6i, we used MCF-7 cells carrying ESR1 wild-type (WT), Y537S, or D538G ‘hotspot’ knock-in mutations to assess cell proliferation upon treatment with the CDK4/6i palbociclib. Cells harboring the Y537S and D538G mutations displayed a 3-fold increase in the IC50 of palbociclib single agent compared to ESR1WT cells. When treated with palbociclib + fulvestrant or palbociclib + estrogen-free medium (mimicking estrogen suppression in patients treated with aromatase inhibitors), cells harboring the Y537S and D538G mutations displayed an 11.2 to 46.9-fold increase in the IC50 of palbociclib. Treatment with palbociclib arrested growth of MCF-7 ESR1WT xenografts established in ovariectomized nude mice whereas MCF-7 ESR1Y537S and MCF-7 ESR1D538G tumors continued to grow. These results suggest that ESR1 mutations enable cell proliferation and tumor growth in the presence of CDK4/6i and the combination of CDK4/6i + antiestrogens. Next, we performed RNA-seq to investigate the mechanisms underlying ESR1 mutation-mediated resistance to CDK4/6i. Gene set enrichment analysis (GSEA) of the RNA-seq data showed significant upregulation of cell cycle-related Hallmark gene signatures such as E2F targets, G2/M checkpoints, and mitotic spindle in ESR1Y537S vs. ESR1WT and ESR1D538G vs. ESR1WT MCF-7 cells upon treatment with palbociclib. Upregulation of these gene signatures in ESR1-mutant vs. WT cells was also observed upon treatment with palbociclib + fulvestrant and palbociclib in estrogen-free media, suggesting that ESR1 mutations hamper the inhibitory effects of CDK4/6i alone or in combination with antiestrogens on cell cycle progression. Additionally, RNA-seq analysis showed that cells expressing either the Y537S or D538G mutation had elevated basal gene signatures (METABRIC_basal, TCGA_basal, and Huper_basal) compared to ESR1WT cells, consistent with previous studies suggesting that ESR1 mutations promote a luminal-to-basal subtype switch in ER+ breast cancers. We are currently investigating whether this lineage reprogramming underlies resistance to CDK4/6i in ESR1-mutant cells. In addition, we will validate our findings by analyzing the tumor tissue transcriptome from the patients included in this study. Conclusions: ESR1 mutations are enriched following treatment with CDK4/6i in a cohort of 3,958 patients with ER+/HER2- metastatic breast cancer. Mutations in ESR1 in ER+ breast cancer cells directly promote resistance to CDK4/6i alone or CDK4/6i + antiestrogens in vitro and in vivo. Citation Format: M Rosario Chica-Parrado, Chang-Ching Lin, Ellen Jaeger, Michelle Harris, Lei Guo, Emmanuel Bikorimana, Fabiana Napolitano, Calvin Chao, Ariella Hanker, Carlos Arteaga. ESR1 mutations drive resistance to CDK4/6 inhibitors in ER+ Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-09.

Abstract PO4-15-06: Genomic landscape characterization after exposure to cyclin dependent kinase 4/6 inhibitors: a retrospective multi-institutional consortium analysis

Abstract Background: Limited data are available to determine the best therapeutic strategy for hormone-receptor positive (HR+) HER2 negative (HER2-) advanced breast cancer (ABC) after progression on cyclin dependent kinase 4/6 inhibitors (CDK4/6i). The aim of this study was to characterize the genomic and prognostic profile of patients (pts) that experienced disease progression after first-line therapy with a CDK4/6i. Methods: The study retrospectively analyzed a multi-institutional cohort of 75 patients (pts) with HR+/HER2- ABC after experiencing progression on first-line endocrine therapy (ET) with CDK4/6i and characterized by circulating tumor DNA (ctDNA) next-generation sequencing (Guardant 360). Oncogenic pathways (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, p53, cell cycle, notch, and PI3K) were defined based on previous research (Sanchez-Vega et al. Cell, 2018). Associations across single nucleotide variations (SNVs), copy number variations (CNVs), and pathway classification were tested by uni- and multivariable logistic regression with respect to a CDK4/6i naïve group comprising 247 patients with HR+/HER2- ABC (control). Prognosis was analyzed through Cox regression for overall survival (OS). Result: Our study cohort included 75 ABC pts who experienced progression after first line CDK4/6i plus ET: 43 pts (57.3%) were treated with ET while 31 (41.3%) with a non-ET- second line therapy. The most common histology was invasive ductal carcinoma (70.6%), 37.3% of pts were negative for progesterone expression and 26.7% had de novo metastatic disease. The PI3K (33.3%), p53 (28%) and ER (25.3%) pathways were the most mutated in the CDK4/6i-treated cohort. Comparing our population with a CDK4/6i naïve cohort (N=247), the multivariate analysis showed a higher prevalence in the study cohort of SNVs mutations in: i) ESR1 gene [Odds ratio (OR) 2.93; p=0.005]; ii) cell-cycle pathway (OR 4.24; p=0.033); iii) ER pathway (OR 2.04; p= 0.034). Moreover, in the study cohort a numerical but not significant increase of RB1 SNVs was also observed. Multivariable analyses of the 43 pts that received ET second line therapy showed a negative prognostic impact with TP53 SNVs in both progression-free survival (PFS) [Hazard ratio (HR) = 3.34; 95% CI: 1.15-9.66, p=0.026] and OS (HR = 3.85; 95% CI: 1.52-9.77, p=0.005). The prognostic role of p53 pathway mutations was also confirmed for both PFS (HR = 4.71, 95% CI: 1.64-13.50; p=0.004) and OS (HR = 3.25; 95% CI: 1.27-8.3; p=0.014). The potential interaction between the prognostic oncogenic pathways and the outcome was investigated according to the treatment strategy (ET vs non-ET). A consistent impact was observed across the above prognostic pathways both for PFS and OS. A numerical difference was observed in terms of PFS in pts with p53 pathway mutations that underwent non-ET second line. Conclusions: The genomic landscape of progressive disease after CDK4/6i exposure is significantly different from the genomic alterations detectable in treatment-naïve pts, which could potential impact future treatment strategies for patients with disease progression after adjuvant CDK4/6i exposure. Our data suggest that the choice of second-line treatment could potentially be guided by the identification of actionable mutation by ctDNA, although further prospective studies are needed to validate the clinical utility of this approach. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew Davis, Lorenzo Gerratana, Arielle Medford, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Ami N. Shah, Jeannine Donahue, Laura Munoz Arcos, Charles S. Dai, Jennifer Keenan, Amir Behdad, William Gradishar, Emilio Bria, Cynthia Ma, Aditya Bardia, Massimo Cristofanilli. Genomic landscape characterization after exposure to cyclin dependent kinase 4/6 inhibitors: a retrospective multi-institutional consortium analysis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-06.

Abstract PO5-15-02: Integration of synonymous mutations and variants of unknown significance for basal and longitudinal characterization of metastatic breast cancer by circulating tumor DNA

Abstract Background: Despite how commonly synonymous mutations (SYN) and unknown significance (VUS) are detected in tissue and circulating tumor DNA (ctDNA) next-generation sequencing (NGS), the potential for these somatic changes to impact personalized approaches in metastatic breast cancer (MBC) is unclear. Emerging evidence suggests that SYN mutations may play a role in cancer biology through changes in mRNA stability, splicing, and gene expression. The aim of this study was to characterize SYN and VUS in a large multicenter consortium and to analyze their interplay with pathogenic variants (PATH). Methods: The study retrospectively analyzed a multi-institutional cohort comprising 1189 patients with MBC characterized for ctDNA using NGS (Guardant360™, Guardant Health) before treatment start (BL) and at disease progression (PD). Pathway classification was defined based on prior research (Sanchez-Vega F et al, Cell. 2018). Single Nucleotide Variations (SNVs) were annotated for oncogenicity (OncoKB) and protein domain (UniProtKB). Associations between clinical characteristics and pathway classifications for SYN and VUS were explored by multinomial logistic regression; survival was tested through Cox regression in terms of overall survival (OS). Results: Hormone-receptor positive (HR+)/HER2 negative (luminal-like) was the most represented subtype (68.7%) followed by HER2-positive (14.6%), and triple-negative (TNBC) (16.7%). PATH were mainly detected in the TP53 DNA binding domain, ESR1 ligand binding domain, helical and catalytic domains of PIK3CA, SYN in the ligand binding domain of ESR1, MET Sema, KIT protein kinase domain, and FGFR1 Ig-like C2-type 1. In contrast, VUS were mainly found in the ESR1 ligand binding domain, MET Sema, ATM PI3K/PI4K catalytic domain, DDR2 Protein kinase and PIK3CA C2 PI3K-type. Analyzing pathways, after multivariable multinomial logistic regression, RAS SYN, PI3K and ESR PATH were significantly associated with HER2+ MBC (respectively RRR=4.14, P=0.046; RRR = 0.474, P &amp;lt; 0.001 and RRR = 0.306, P &amp;lt; 0.001). TNBC was associated with PATH PI3K (RRR = 0.366, P &amp;lt; 0.001), PATH P53 (RRR = 4.71, P &amp;lt; 0.001) and PATH RAS (RRR = 0.362, P = 0.026, 95% CI 0.147 - 0.888). In luminal-like MBC, a significant impact on OS was observed for SYN P53 (HR = 2.53, P = 0.008) and PATH ER (HR = 1.54, P &amp;lt; 0.001), PATH P53 (HR = 1.56, P &amp;lt; 0.001), PATH cell-cycle (HR = 1.64, P = 0.0299), and PATH RAS (HR = 1.62, P = 0.010). An impact on OS was observed in TNBC for SYN cell-cycle (HR = 5.41, P = 0.006), VUS ESR (HR = 2.87, P = 0.018), PATH P53 (HR = 1.84, P = 0.012) and PATH cell-cycle (HR = 5.07, P &amp;lt; 0.001). VUS MYC (HR = 38.5, P = 0.001) and PATH PI3K3 (HR = 2.32, P = 0.022) were significant among HER2 positive. Differences in SYN, VUS and PATH alterations were then compared between baseline and progressive disease. While a significant increase in mutant allele frequency (MAF) was observed for PATH (P=0.0010) and VUS (P=0.0152) at PD, no differences were highlighted for SYN (P=0.8362). Conclusions: In our cohort we characterized the MBC for PATH, VUS and SYN utilizing the ctDNA. In this study, considering the complex genomic landscape of MBC, we demonstrated that both SYN and VUS mutations had an impact on the survival outcome. Therefore, further analysis to validate the prognostic and predictive role of SYN and VUS, with or without PATH mutations, are needed to better characterize and monitoring of MBC through ctDNA. Moreover, functional studies are needed to better understand their role in the nuanced biology of MBC. Citation Format: Elisabetta Molteni, Carolina Reduzzi, Andrew Davis, Lorenzo Foffano, Arielle Medford, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Ami N. Shah, Laura Munoz Arcos, Charles S. Dai, Jennifer C. Keenan, Elyssa Denault, William Gradishar, Giuseppe Damante, Amir Behdad, Lorenzo Gerratana, Fabio Puglisi, Cynthia Ma, Aditya Bardia, Massimo Cristofanilli. Integration of synonymous mutations and variants of unknown significance for basal and longitudinal characterization of metastatic breast cancer by circulating tumor DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-15-02.

Diagnostic value of DNA or RNA-based metagenomic next-generation sequencing in lower respiratory tract infections

ObjectivesWe aimed to evaluate and compare the diagnostic performance of RNA-mNGS and DNA-mNGS workflow in bacterial pneumonia, fungal pneumonia and tuberculosis. MethodsA total of 134 cases suspected pneumonia undergoing both DNA and RNA based mNGS of bronchoalveolar lavage fluid (BALF) and also traditional etiological examination were evaluated retrospectively.Sensitivity, specificity, PPV, NPV and accuracy rate of DNA and RNA based mNGS were estimated. ResultsIn the diagnosis performance of bacterial pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS(82.3 % vs. 61.9 %, P < 0.01). There was no significant difference of sensitivity between the two process(71.4 % vs. 85.7 %, P = 0.375).In the diagnosis performance of fungal pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS (72.3 % vs. 27.3 %,p < 0.001). There was no significant difference of sensitivity between the two process(96.5 % vs. 98.8 %,p = 0.125).In the diagnosis performance of tuberculosis in LRTIs,the sensitivity of DNA-mNGS was higher than that of RNA-mNGS (91.7 % vs. 33.3 %,p = 0.016),the specificity was similar in the two process (100 %). ConclusionsRNA-mNGS may reduced the misdiagnosis rate of bacterial and fungal pathogens in LRTIs.Compared to RNA-mNGS, DNA-mNGS may could improve the diagnostic rate of tuberculosis.

Genomic Profiling of Rare Undifferentiated Sarcomatoid Subtypes of Pancreatic Carcinomas: In Search of Therapeutic Targets

PURPOSE The highly aggressive undifferentiated sarcomatoid carcinoma (USC) subtype of pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized because of its rarity. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy, but the prevalence of established predictive biomarkers of response is largely unknown. The objective of this study was to leverage comprehensive genomic profiling of USC PDAC tumors to determine the prevalence of biomarkers associated with potential response to targeted therapies. METHODS USC tumors (n = 20) underwent central pathology review by a board-certified gastrointestinal pathologist to confirm the diagnosis. These samples were compared with non-USC PDAC tumors (N = 5,562). Retrospective analysis of DNA and RNA next-generation sequencing data was performed. RESULTS USC PDACs were more frequently PD-L1+ by immunohistochemistry than non-USC PDAC (63% v 16%, respectively, P &lt; .001). Furthermore, USC PDAC had an increase in neutrophils (8.99% v 5.55%, P = .005) and dendritic cells (1.08% v 0.00%, q = 0.022) and an increased expression of PDCD1LG2 (4.6% v 1.3%, q = 0.001), PDCD1 (2.0% v 0.8%, q = 0.060), and HAVCR2 (45.9% v 21.7%, q = 0.107) than non-USC PDAC. Similar to non-USC PDAC, KRAS was the most commonly mutated gene (86% v 90%, respectively, P = 1). CONCLUSION To our knowledge, this work represents the largest molecular analysis of USC tumors to date and showed an increased expression of immune checkpoint genes in USC tumors. These findings provide evidence for further investigation into immune checkpoint inhibitors in USC tumors.

Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis. Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets. Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected). Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%. This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Utility of microbial cell free DNA next-generation sequencing for diagnosis and management of infectious diseases

Microbial cell-free DNA (mcfDNA) sequencing is a promising tool to identify infectious pathogens when traditional methods fail to identify the causative agent. We performed a retrospective observational cohort study to evaluate clinical outcomes among pediatric and adult patients who underwent mcfDNA testing. 127 mcfDNA tests were reviewed from 112 patients. Baseline characteristics included 61 (54.5 %) adults, 52 (40.9 %) tests were from female patients, and 67 (52.8 %) tests were obtained from patients designated as immunocompromised. Of all tests obtained, 59 (46.4 %) were deemed clinically relevant. 41 (32.3 %) of tests also led to a change in antimicrobial management for the corresponding patient. No statistically significant association was ascertained between patient-specific factors and clinically relevant test results. Testing in certain clinical scenarios or high-risk settings may be useful, however further studies are needed to assess the cost-benefit of this approach.

Classifying Glioma via Liquid Biopsy: Progress toward an Unmet Clinical Need.

The diagnosis and classification of glioma by liquid biopsy represent a critical unmet need in neuro-oncology. A recent study demonstrates targeted next-generation sequencing of cell-free DNA from cerebrospinal fluid as an evolving option for liquid biopsy in patients with glioma. See related article by Iser et al., p. 2974.

Abstract P19: Comprehensive Benchmark of Somatic Mutation Callers on Cell-Free DNA Next-Generation Sequencing Data in Cancer Liquid Biopsy

Abstract Circulating tumour DNA (ctDNA) has emerged as a rich source of biomarkers in precision oncology. However, ctDNA sequence data is often noisier than that derived from standard tissue biopsies, posing significant challenges for accurate cancer mutation detection. Besides, there is a lack of standardised and unbiased benchmark datasets to evaluate and compare performance of variant callers on ctDNA. To address this issue, we present a novel benchmarking approach and comprehensive benchmark of cancer somatic mutation callers on cell-free DNA (cfDNA) Next Generation Sequencing (NGS) data. To create a large-scale benchmark dataset, we developed a new methodology using real colorectal cancer patients plasma samples exhibiting either ultra-high and an ultra-low levels of ctDNA. This enabled the generation of dilution series of deep Whole Genome Sequencing (WGS, 150x) and the creation of high-quality consensus ground truth calls. We evaluated the performance of 9 SNV callers and 7 short INDEL callers frequently used in academic research, including 2 callers that were specifically designed for cfDNA. We further validated our findings at higher depth of coverage using deep Whole Exome Sequencing (WES, &amp;gt;2,000x) data. We further investigated each caller's features to develop practical guidelines for users depending on their application setting: unguided analysis focusing on overall accuracy vs. guided analysis focusing on sensitivity. Citation Format: Hanaé Carrié, Ngak Leng Sim, Pui Mun Wong, Anna Gan, Yi Ting Lau, Polly Poon, Saranya Thangaraju, Iain Tan, Yoon Sim Yap, Limsoon Wong, Anders Skanderup. Comprehensive Benchmark of Somatic Mutation Callers on Cell-Free DNA Next-Generation Sequencing Data in Cancer Liquid Biopsy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P19.

Safety and efficacy of anlotinib combined with taxane and lobaplatin in neoadjuvant treatment of clinical stage II/III triple-negative breast cancer in China (the neoALTAL trial): a single-arm, phase 2 trial

Anlotinib is a new type of tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors 1/2/3, platelet-derived growth factor receptors α/β, and fibroblast growth factor receptors 1-4 and c-Kit, with a broad spectrum of inhibitory effects on tumor angiogenesis and growth. It has been proven effective in HER2-negative metastatic breast cancer, but its efficacy in early-stage triple-negative breast cancer (TNBC) is unknown. This phase 2 study aims to evaluate the efficacy and safety of adding anlotinib to neoadjuvant chemotherapy in patients with TNBC. Patients with clinical stage II/III TNBC were treated with 5 cycles of anlotinib (12mg, d1-14, q3w) plus 6 cycles of taxanes (docetaxel 75 mg/m2 ,d1, q3w or nab-paclitaxel 125 mg/m2, d1 and d8, q3w) and lobaplatin (30 mg/m2, d1, q3w), followed by surgery. The primary endpoint was pathological complete response (pCR; ypT0/is ypN0) and the secondary endpoints include breast pCR (bpCR), axillary pCR (apCR), residual cancer burden (RCB), objective response rate (ORR), survival, and safety. Exploratory endpoints were efficacy biomarkers based on Fudan University Shanghai Cancer Center Immunohistochemical (FUSCC IHC) classification for TNBC and next-generation sequencing (NGS) of DNA from tumor tissue and blood samples of patients with 425-gene panel. This trial is registered with www.chictr.org.cn (ChiCTR2100043027). From Jan 2021 to Aug 2022, 48patients were assessed and 45 were enrolled. All patients received at least one dose of study treatment and underwent surgery. The median age was 48.5 years (SD: 8.7), 71% were nodal involved, and 20% had stage III. In the intention-to-treat population, 26 out of 45patients achieved pCR (57.8%; 90% CI, 44.5%-70.3%), and 39 achieved residual cancer burden class 0-I (86.7%; 95% CI, 73.2%-94.9%). The bpCR and apCR rate were 64.4% (29/45) and 71.9% (23/32), respectively. No recurrence or metastasis occurred during the short-term follow-up. Based on the FUSCC IHC-based subtypes, the pCR rates were 68.8% (11/16) for immunomodulatory subtype, 58.3% (7/12) for basal-like immune-suppressed subtype and 33.3% (4/12) for luminal androgen receptor subtype, respectively. NGS revealed that the pCR were 77% (10/13) and 50% (14/28) in MYC-amplified and wild-type patients, respectively, and 78% (7/9) and 53% (17/32) in gBRCA1/2-mutated and wild-type patients, respectively. The median follow-up time of the study was 14.9 months (95% CI: 13.5-16.3 months). There was no disease progression or death during neoadjuvant therapy. No deaths occurred during postoperative follow-up. In the safety population (N=45), Grade 3 or 4 treatment emergent adverse events occurred in 29patients (64%), and the most common events were neutropenia (38%), leukopenia (27%), thrombocytopenia (25%), anemia (13%), and hypertension (13%), respectively. The addition of anlotinib to neoadjuvant chemotherapy showed manageable toxicity and encouraging antitumor activity for patients with clinical stage II/III TNBC. Chongqing Talents Project, Chongqing Key Project of Technology Innovation and Application Development and Chongqing Outstanding Youth Natural Science Foundation.