cfDNA Analysis Research Articles

P-570 A cell-free DNA test in semen of nonobstructive azoospermic men to predict spermatogenesis

Abstract Study question Can a genetic assessment of cell-free DNA, isolated from seminal plasma of nonobstructive azoospermic (NOA) men, predict successful sperm extraction at testicular biopsy? Summary answer Seminal cell-free DNA (cfDNA) isolated from the ejaculates of NOA men with failed testicular sperm retrieval outcomes concurrently and consistently displayed mutations on three genes. What is known already The most puzzling form of azoospermia is the testicular type, where the chances of successful microdissection testicular sperm extraction (micro-TESE) are unpredictable. Therefore, developing a method that could identify or predict individuals with absent spermatogenesis would be most useful. While histopathology is considered the most reliable method for predicting successful micro-TESE, it is invasive and often inconclusive. Recently, cfDNA testing has been gaining momentum as a noninvasive method for prenatal screening and even with the potential for eventually replacing PGT-A. However, the prognostic value of using cfDNA to predict the presence/absence of spermatozoa in men undergoing micro-TESE is relatively unknown. Study design, size, duration Over a period of 8 months, we recruited azoospermic men with normal peripheral karyotypes and for whom an extensive semen analysis did not identify spermatozoa. For consenting patients (n = 12), we performed genetic assessments on cfDNA from their ejaculates (IRB 1006101085). These patients subsequently underwent micro-TESE, and their cfDNA mutation profiles were compared according to whether spermatozoa were successfully retrieved ((+)Sperm, n = 5), or not ((-)Sperm, n = 7). Participants/materials, setting, methods Using a commercially available kit, circulating cfDNA was eluted from the ejaculates of consenting NOA men, as well as a normozoospermic control (n = 2). The isolated cfDNA specimens subsequently underwent whole exome sequencing (WES) on an Illumina HiSeq at 2x150bp. Gene mutations were detected by CLC Genomic Server 9.0 and compared within, as well as between, the +Sperm and -Sperm cohorts in comparison to the control group. Main results and the role of chance All 12 men (35.5±4yrs) had normal peripheral karyotypes and tested negative for Y-microdeletions. They had no history of genetic diseases and were off any supplements, medications, and/or hormones before or during the study. Genetic assessment on cfDNA from the +Sperm cohort (n = 5; 38.3±2yrs) identified 8 genes (ADAMTS7, ATP9B, CBFA2T3, FAM20C, FCGBP, PEBP4, PHF2, RPS9) that were concurrently mutated among all NOA men from this group. Although ADAMTS7 plays a potential role in sperm-egg fusion, the remaining genes are unrelated to sperm production. Whole exome sequencing of cfDNA from the -Sperm cohort (n = 7; 33.5±4yrs) consistently detected mutations on 3 genes, including one involved in spermatogenesis (CFAP54), one essential for oxidative regulation of spermatozoa (GSTT4), and one that encodes testis antigens that are normally expressed during spermatogenesis (BAGE2). Moreover, we concurrently and consistently identified pathogenic mutations on an additional gene in the -Sperm cohort. This gene (MUC2) is involved in the epithelial coating of mucus membrane-containing organs, and its expression has been correlated with spermatogenesis. Interestingly, these genes were all unaffected in men with successful micro-TESE outcomes. Limitations, reasons for caution Although we detected candidate gene mutations that correlate with micro-TESE outcome, these are preliminary findings that need further validation in a larger study cohort. Furthermore, while cfDNA analysis has shown great promise, it is not specific enough on its own and should be corroborated by epigenetic testing. Wider implications of the findings Genetic screening of seminal cfDNA provides a noninvasive alternative to histopathology and can be used to predict the absence of spermatogenesis, sparing these men from unnecessary surgery and cost. This novel biomarker approach lays the groundwork for precision medicine in male infertility testing. Trial registration number n/a

OTHR-20. MINIMALLY-INVASIVE MOLECULAR DIAGNOSIS OF INFILTRATING BRAINSTEM GLIOMAS THROUGH CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING

Abstract BACKGROUND Accurate molecular diagnosis of infiltrating brainstem tumors, including diffuse midline gliomas and IDH mutant astrocytomas, is essential for prognostication and optimal therapy. Surgical biopsy of the brainstem carries risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally invasive “liquid biopsy” analyzing circulating tumor cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) could represent an alternative molecular diagnostic modality. METHODS We performed MSK-IMPACT, a New York State Department of Health authorized hybridization capture-based next-generation panel DNA sequencing clinical assay on 44 CSF samples from 37 unique patients with brainstem tumors. Ages at time of CSF collection ranged from 1–47 years (median: 29 years). For samples in which diagnostic alterations could not be detected, we performed a series of secondary analyses for known hotspot mutations such as H3F3A, IDH1/2, and BRAF, including manual review for mutant reads below the official clinical reporting threshold, as well as droplet digital PCR (ddPCR). RESULTS Among 44 samples included in this study, 10/44 (23%) samples had mutations detected by MSK-IMPACT using standard clinical calling criteria and 14 of the remaining 34 samples (41%) had supporting evidence of a diagnostically relevant mutation based on manual review. Testing by ddPCR was performed on 11 samples (based on DNA availability), uncovering 4 additional mutations. Overall positive diagnostic yield was 64% (28/44). In biopsied patients, CSF confirmed disease-defining mutations in 80% (16/20) of CSF samples, including H3F3A K28M (9/10; 90%) and IDH1/2 (5/6; 83%). Among 24 unbiopsied patients, a disease-defining mutation was identified in 60% (15/25) of CSF samples. CONCLUSIONS Analysis of CSF cfDNA in patients with brainstem tumors has a high diagnostic yield and obviates the need for tissue biopsy in a majority of patients. A tiered testing approach using next-generation sequencing and ddPCR assays helps maximize diagnostic information and sensitivity.

OTHR-26. MINIMALLY-INVASIVE MOLECULAR DIAGNOSIS OF DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR (DLGNT) USING CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING IN PEDIATRIC AND YOUNG ADULT PATIENTS

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumor (DLGNT) presents with nodular leptomeningeal disease throughout the neuroaxis and is characterized by the presence of KIAA1549::BRAF fusion and chromosome arm 1p deletion. Concomitant gain of chromosome arm 1q is associated with poor prognosis. Diagnosis by meningeal biopsy is often non-diagnostic due to sampling error or yields insufficient material for molecular testing. We hypothesized that analysis of cell free DNA (cfDNA) from cerebrospinal fluid (CSF) could represent a superior diagnostic modality. METHODS We analyzed CSF samples from 9 suspected DLGNT patients, median age of 19.5 years with a 5:4 male:female ratio using MSK-IMPACT, a hybridization capture-based next-generation DNA sequencing panel, including matched germline. RESULTS Prior tissue biopsy was done in 7 patients; 6 were negative for the KIAA1549:BRAF fusion and 1 was positive. Of the 6 biopsies negative for the fusion, analysis of CSF cfDNA detected KIAA1549::BRAF fusion in 3/6 (50%) of patients, who were all subsequently started on MEK inhibitor therapy. The other 3 patients were negative for KIAA1549:BRAF fusion on both biopsy and CSF cfDNA. One patient received biopsy confirmation of KIAA1549::BRAF fusion after CSF cfDNA analysis detected the mutation. In addition, in one patient with prior tissue-confirmed KIAA1549::BRAF fusion, the fusion was not detected in CSF cfDNA during/after treatment with a MEK inhibitor, but additional somatic alterations and DNA copy number profiles were similar to those found in prior biopsies. Additional somatic alterations were identified in ATRX, BCOR, RARA, STAT5A, SPOP, PPM1D, MED12, KMT2C, RECQL andSETD2, including likely oncogenic secondary driver alterations that have not been previously reported in DLGNT. CONCLUSIONS Given the high molecular diagnostic yield and concordance with open biopsy, cfDNA CSF sequencing should be considered as a first line diagnostic maneuver for patients with suspected DLGNT, followed by surgical biopsy only if CSF testing is non-diagnostic.

BIOM-09. TOWARDS CLINICAL IMPLEMENTATION OF LIQUID BIOPSY ANALYSIS FOR PAEDIATRIC BRAIN TUMOUR PATIENTS – A PAN-EUROPEAN EFFORT FOR STANDARDIZATION

Abstract BACKGROUND Liquid biopsies (LBs) allow for analysis of tumour-derived molecules, such as cell-free DNA (cfDNA) and microRNAs (miRNAs), complementing cancer diagnostics, therapy monitoring, and minimal residual disease detection. While circulation-based LB has benefit for extracranial tumour entities, peripheral blood has not historically been a reliable source for diagnosis and disease-monitoring in brain tumours. For these tumours, cerebrospinal fluid (CSF) analysis is better suited for detection despite being a more invasive procedure. However, standardized protocols for sample collection and pre-analytical parameters for these LBs currently are limited. AIM We aim to establish an evidence-based gold standard protocol for cfDNA and miRNA analyses from CSF in paediatric brain tumour patients. In addition to higher compliance in trial-related CSF sampling, this initiative seeks to facilitate identification of clinically relevant indications and time-points for collection, thereby paving the way for well-informed, routine CSF molecular diagnostics. METHODS AND RESULTS We have initiated the Liquid Biopsy Working Group within the framework of the International Society of Paediatric Oncology Europe Brain Tumour Group (SIOPE-BTG). Available literature on LB in brain tumours was critically assessed regarding pre-analytical parameters, downstream analyses and LB assay performance. To directly evaluate confounding by pre-analytical factors, we experimentally compared the impact of CSF sampling sites, storage conditions, and different pre-processing protocols. cfDNA and miRNA yields, purity, and the fraction of tumour-derived material were selected as read-outs and correlated with tumour tissue information. To prepare for clinical translation, we consulted clinical paediatric brain tumour trial coordinators to identify major bottlenecks for CSF sampling in routine clinical practice. In addition, clinical practitioners and patient/ parent representatives have been consulted to comprehensively represent the perspectives and needs of all stakeholders. Together, results of this collaborative effort will be fundamental to the successful clinical integration of LBs to improve the future management of paediatric brain tumour patients.

BIOM-02. DISSECTING CDK4/6 INHIBITOR RESISTANCE MECHANISMS IN RECURRENT PEDIATRIC BRAIN TUMORS WITH LONGITUDINAL CSF LIQUID BIOPSIES FROM THE SJDAWN CLINICAL TRIAL

Abstract BACKGROUND SJDAWN (NCT03434262) is a St. Jude Children’s Research Hospital phase 1 trial that assessed the safety and benefit of CDK4/6 inhibitor-based therapy in patients with relapsed CNS tumors. To capture tumor evolution dynamics and arising resistance mechanisms in response to treatment, 143 longitudinal CSF samples were collected from 34/68 (50%) patients for cell-free DNA (cfDNA) analysis. METHODS cfDNA was extracted from CSF liquid biopsies and analyzed using a novel methylation-based platform that integrates copy number, tumor classification, and deconvolution of methylation signatures into normal vs. disease fractions. Samples with measurable residual disease (MRD) were further characterized by deep whole-genome sequencing to discover mutation-driven resistance mechanisms. When available, pre- and post-SJDAWN tumor tissues were processed for single-nucleus multi-omics to track tumor evolution at single-cell resolution. RESULTS In the context of CDK4/6 inhibitor-based therapy, longitudinal cfDNA assessment revealed treatment-induced selection for subclones with focal amplification of positive cell cycle regulators CDK6, CCND2, and MYCN. Emergence of these resistance mechanisms notably preceded disease progression on MRI and CSF cytology by up to 6 months. Of the 68 patients enrolled on SJDAWN, 4 patients (3 Group 3/4, subtype III medulloblastomas and 1 anaplastic ependymoma) achieved >2-year progression-free survival. Interestingly, serial cfDNA profiling of 1 long-term survivor (Group 3/4, subtype III medulloblastoma) exhibited undetectable MRD status for >1.5 years prior to reemergence, suggesting tumor senescence by CDK4/6 inhibition may impede circulating tumor DNA (ctDNA) release kinetics. CSF liquid biopsies were further evaluated in comparison to matched tumor tissues, and changes in CSF-specific subclones were observed in response to treatment. CONCLUSIONS In addition to early MRD detection, liquid biopsies capture tumor evolution dynamics and can inform developing resistance mechanisms. Focal amplification of CDK6, CCND2, and MYCN facilitates CDK4/6 inhibitor resistance. In responders, treatment-induced senescence may inhibit ctDNA release kinetics.

The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection

An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.

Genomic alterations and transcriptional phenotypes in circulating tumor DNA and matched metastatic tumor.

Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), a cancer whose aggressive clinical course making it exceedingly challenging to obtain tumor biopsies. Here, a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC, we study cfDNA low pass whole genome (0.1X coverage) and exome (130X) sequencing in comparison with time-point matched tumor, characterized using exome and transcriptome sequencing. Direct comparison of cfDNA versus tumor biopsy reveals that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not found in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Genomic sequencing coverage of plasma DNA fragments around transcription start sites shows distinct treatment-related changes and captures the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors, allowing prediction of SCLC neuroendocrine phenotypes and treatment responses. These findings have important implications for non-invasive stratification and subtype-specific therapies for patients with SCLC, now treated as a single disease.

Prospective monitoring for immunotherapy by liquid biopsy and signal pathway alterations in head and neck cancers.

e18000 Background: Clinical biomarkers that predict response or prognosis to immune checkpoint inhibitors (ICIs) are unclear in head and neck squamous cell carcinoma (HNSCC). Comprehensive genome profiling (CGP) testing including liquid biopsy using cell-free DNA (cfDNA) has become clinically available; however, CGP testing is approved only once per patient under the Japanese health insurance system. Therefore, whether liquid biopsy is useful for detecting sequential genetic change and disease progression as a real-time monitoring tool is still unknown. This study aims to analyze the cfDNA in the plasm of recurrent or metastatic (R/M) HNSCC patients during ICI therapy. This study was prospectively conducted to examine the relationship between sequential genetic changes and treatment response. Methods: In this prospective study, we examined ten evaluable R/M HNSCC cases treated with ICI monotherapy (nivolumab or pembrolizumab) from October 2020 to December 2023. Blood samples were collected and analyzed using Guardant360 at differing three-time points (0, 4, and 24 weeks or at progressive disease, PD). Results: The ten cases consisted of 5 males and 5 females, with a median age of 71 years old (52-92 years old). Seven patients received pembrolizumab as a first line and three received nivolumab as the second line treatment. The best response to ICI was partial response (PR) in 3 cases , stable disease (SD) in 1 and PD in 6. Three cases discontinued the study at the second point because of the rapid disease progression. Microsatellite status is stable in all cases. Eight of the ten cases had at least one pathogenic mutation, one case had only variants of unknown significance, and one case that had target lesions at lung metastases had none of them through a series of liquid biopsies. The most frequent gene alterations observed were TP53 (60%), TERT promoter (20%), GNAS (20%), and EGFR amplification (20%). In particular, EGFR amplification had emerged at the second point in two PD cases and these patients showed the sensitivity to cetuximab-based therapy at the following treatment line. In all PD cases, the highest variant allele fraction (VAF) was increased at the second point, which might help predict the response to ICI therapy. In one case achieving PR, BRAF mutation had emerged at the second point. Then it was undetectable at the third point with PD, and GNAS mutation was newly detected. In another case, APC inactivation had disappeared at the second point with PR, even though GNAS mutation emerged, and this case maintained the response to ICI. These results suggest that switching the signals in the MAPK or Wnt pathway could impact the response to ICI therapy. Conclusions: The findings of this prospective study demonstrated that the highest VAF alterations and genetic mutations detected by cfDNA analysis could reflect and predict the response to ICI.

Mutational analysis of cfDNA to identify predictive biomarkers in previously untreated patients with metastatic pancreatic cancer receiving the GSK-3 inhibitor elraglusib (9-ING-41) in combination with gemcitabine/nab-paclitaxel in the 1801 phase 2 study.

e16306 Background: Glycogen Synthase Kinase-3beta has been well-credentialed as a therapeutic target in patients with advanced cancer including pancreatic cancer. Elraglusib (9-ING-41), a novel best-in-class GSK-3 inhibitor, is being developed in combination with gemcitabine/nab-paclitaxel (GnP) as first-line therapy for patients with metastatic pancreatic cancer (mPDAC). Using liquid biopsy samples from an ongoing randomized phase 2 trial (1801 Part 3B; NCT03678883), we examined whether gene mutations in cfDNA correlated with clinical outcomes in mPDAC patients treated with GnP or elraglusib/GnP. Methods: Pre-dose plasma samples were obtained from 37 (GnP arm) and 89 (elraglusib/GnP arm) mPDAC patients previously untreated for advanced disease. Mutational analysis of cfDNA extracted from plasma samples was performed by Tempus (Chicago, IL) using the xF+ platform. The Tempus xF+ assay is an NGS cfDNA liquid biopsy tumor profiling assay for identifying genomic alterations in 523 genes. Tempus xF+ reports were used for analysis of gene mutations. Results: In 126 pancreatic cancer patients, frequently mutated genes ( > 10%) consisted of KRAS (93/126, 74%), TP53 (79/126, 63%), CDKN2A (26/126, 21%), DNMT3A (25/126, 20%), NOTCH1/2 (25/126, 20%), and MLL3 (23/126, 18%). Gain-of-function mutations in KRAS, and loss-of-function mutations of TP53, and CDKN2A genes were associated with worse overall survival (OS) in patients treated with elraglusib/GnP (P < 0.05) but not in GnP-treated patients. Multivariate analysis of frequently mutated genes showed that patients with double (KRAS+TP53) and triple (KRAS+TP53+CDKN2A) mutations had significantly worse OS only in the elraglusib/GnP arm. The presence of DNMT3A, NOTCH1/2 or MLL3 gene mutations did not correlate with any clinical outcomes. Conclusions: Our preliminary analysis of gene mutations in liquid biopsy samples obtained from patients in the ongoing 1801 Part 3B phase 2 trial identified potential predictive biomarkers of clinical outcome in mPDAC patients treated with combination of elraglusib/GnP. Clinical trial information: NCT03678883 .

Serial analysis of ESR1 and PIK3CA mutations in cell-free DNA from metastatic breast cancer during 1st line endocrine therapy.

e13060 Background: Activating mutations ofESR1 and PIK3CA genes are known mechanisms of endocrine resistance. Recent analysis supported benefit of early intervention at presence of ESR1 mutation in blood (bESR1) before clinical progression. We aimed to investigate ESR1 and PIK3CA mutations detected in cell-free DNA (cfDNA) from patients with hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) and its impact on progression-free survival (PFS). Methods: Thirty patients undergoing 1st line endocrine therapy (ETx) were identified from a prospective cohort. Seven ESR1 hotspot mutations in the ligand binding domain were tested in cfDNA with droplet digital polymerase chain reaction (ddPCR) assay (Genopeaks Co., Ltd). Eleven PIK3CA hotspot mutations were tested using amplicon-targeted sequencing method (Genopeaks Co., Ltd) in both tumor and cfDNA. PFS analyses were performed using Kaplan-Meier methods and compared with the log-rank test. Results: We examined serial cfDNA samples from 26 patients collected every 3-6 months. ESR1 mutations were found in 61.5% (16/26) patients at any timepoint during 1st line endocrine therapy, with D538G being the most common followed by Y537C and 68.8% (11/16) being polyclonal. Among the 50.0% (8/16) bESR1 positive patients with clinical progression, 4 had bESR1 detected before clinical progression while 4 were diagnosed clinical progression concurrently, suggesting the necessity of closer intervals of cfDNA analyses. While presence of bESR1 did not affect PFS overall, 18.8% of patients with bESR1 detected within 6 months after 1st line ETx, displayed worse outcome (median PFS 5 vs 52 months). Also, patients with clearance of bESR1 mutation in subsequent cfDNA (81.3%, 13/16) were shown to have a better outcome (median PFS 52 vs 41 months). Among 14 patients with clinical progression, 8 had bESR1 mutation. Median PFS between patients with bESR1 positive vs negative was 14 vs 9 months. PIK3CA mutations were detected in 69.5% (16/23) of cfDNA at diagnosis of distant metastasis, among them 53.3% (8/15) had co-occurrence with bESR1 mutations. While presence of PIK3CA mutation in primary tumor tissue did not affect disease-free survival nor PFS, patients with blood PIK3CA mutation at diagnosis of metastasis displayed significantly worse outcome (p = 0.041). Conclusions: Substantial number of hormone receptor-positive, HER2-negative MBC patients were detected with ESR1 and PIK3CA mutation in plasma. Though it was a small-sized analysis to accurately assess statistical significance, we observed worse outcome in patients with 1) early detection (<6 months) of bESR1 and with 2) sustained bESR1 during palliative ETx. Patients with clinical progression without bESR1 mutation displayed shorter PFS than patients with bESR1. PIK3CA mutations in cfDNA were prognostic supporting the benefit of combined targeted therapy.

The clinical utility of plasma cell-free DNA (cfDNA) in NET-02: Randomised, phase II trial of liposomal irinotecan (nal-IRI)/5-fluorouracil (5-FU)/folinic acid or docetaxel as second-line (2L) therapy in patients (pts) with progressive poorly differentiated extra-pulmonary neuroendocrine carcinoma (PD-EP-NEC).

4130 Background: In NET-02, nal-IRI/5-FU, but not docetaxel, met the primary endpoint of 6-month (mo) progression-free survival (PFS) rate in pts with progressive PD-EP-NEC (1). The prognostic potential of cfDNA in progressive PD-EP-NEC has not been explored in a prospective randomised trial. Methods: NET-02 was a multi-centre, randomised (1:1), phase II trial of IV nal-IRI/5-FU/folinic acid, Q14 days (ARM A), or IV docetaxel, Q21 days (ARM B), as 2L therapy in pts with progressive PD-EP-NEC. Plasma samples were taken at baseline (T0), following 6 weeks of treatment (T1) and at progression (T2). Samples were analysed using a multi-omic next-generation sequencing (NGS) approach (T7-MBD-seq) (measures genome-wide methylation and low pass whole genome copy number alterations (CNA)). The tumour fraction (TF) of cfDNA was determined using ichorCNA and correlated with clinical outcomes using Cox proportional hazards model and Logistic regression. Results: Of 56 pts evaluable for efficacy, there were 54 (96%) T0 samples (48 passed quality control), 31 (55%) at T1 and 20 (36%) at T2. At T0, 36/48 (75%) samples had detectable TF by copy number (using 3% TF cut-off). For the entire cohort, T0 TF was negatively prognostic for overall survival (OS) (Hazard Ratio (HR) 1.17, 95% Confidence interval (CI) 1.00-1.37, P=.044), but not for 6 mo PFS rate (Odds ratio (OR) 1.25, 95% CI 0.91-1.73, P=.17), response rate (RR) (OR 1.11, 95% CI 0.67-1.76, P=.65) or PFS (HR 1.04, 95%CI 0.91-1.20, P=.53). In the entire cohort, median OS split by TF (N=16 each) was 10.2 mo (95% CI 4.1-not available (NA)) for low (TF≤8.6%), 6.9 mo (95% CI 3.4-14.8) for medium (8.6<TF≤24.3%) and 3.7 mo (95% CI 2.8-NA) for high (TF>24.3%). T0 TF was also negatively prognostic for 6 mo PFS rate and OS in ARM A (P=.02, P=.03), but not ARM B (P=.61, P=.48), but was not prognostic for RR or PFS (Table). TF correlated with the presence of liver metastases (Wilcoxon Rank Sum P=.009), but not with ECOG PS (P=.14), sex (P=.10), Ki-67 (P=.39) or age (Spearman rank correlation P=.45). Longitudinal copy number and methylation cfDNA analysis is on-going. Conclusions: These results suggest that it may be possible to stratify prognosis based on amount of baseline TF in patients with progressive PD-EP-NEC, and may also identify patients who would benefit most from the nal-IRI combination. 1. McNamara 2023, EClin Med. Clinical trial information: 03837977. [Table: see text]

Mutational Characteristics and Clinical Outcomes for Lung Adenocarcinoma With EGFR Germline Mutations

IntroductionGermline mutations driving lung cancer have been infrequently reported in the literature, with EGFR T790M being a known germline mutation identified in 1% of NSCLCs. Typically, a somatic EGFR mutation is acquired to develop lung adenocarcinoma. Osimertinib has become a standard-of-care treatment for EGFR T790M-positive lung cancer. MethodsWe perform a retrospective analysis through the Lung Cancer Moon Shot GEMINI database at the University of Texas MD Anderson Cancer Center. Of the patients that underwent cell-free DNA analysis, germline mutations were identified by those with high variant allelic fraction approximating 50%, followed by further confirmation on genetic testing. ResultsWe identified 22 patients with germline EGFR mutations, with the majority harboring an EGFR T790M mutation (95.5%) and an EGFR L858R somatic mutation (50%). Notably, most patients were female (86.4%), non-smokers (81.8%), white (86.4%), had a family history of lung cancer (59.1%), and stage IV at diagnosis (72.7%). A distinct radiographic pattern of small multifocal ground-glass pulmonary nodules was observed in the majority of our cohort (72.7%).Among the 18 with advanced-stage NSCLC, 12 patients (66.7%) were treated with first-line osimertinib, demonstrating a median progression-free survival (PFS) of 16.9 months (95% confidence interval [CI]: 6.3–not reached [NR]). Others were treated with first-line afatinib (11.1%) or chemotherapy (22.2%). Among the 17 patients treated with osimertinib (in first or second-line), median PFS was 20.4 months (95% CI: 6.3–NR) and median overall survival was 82.0 months (95% CI: 28.4–NR). ConclusionsBased on our institutional cohort, NSCLC driven by EGFR germline mutations occurs more frequently in non-smoking, white females with multi-focal pulmonary nodules radiographically. Osimertinib for advanced germline EGFR-mutated NSCLC renders similar PFS compared to somatic T790M EGFR-mutated NSCLC.

Serial Cell-Free DNA Sequencing in ROS1 Fusion-Positive Lung Cancers During Treatment With Entrectinib.

Patients with metastatic ROS1 fusion-positive non-small cell lung cancer (NSCLC) are effectively treated with entrectinib, a multikinase inhibitor. Whether serial targeted gene panel sequencing of cell-free DNA (cfDNA) can identify response and progression along with mechanisms of acquired resistance to entrectinib is underexplored. In patients with ROS1 fusion-positive NSCLC, coclinical trial plasma samples were collected before treatment, after two cycles, and after progression on entrectinib (global phase II clinical trial, ClinicalTrials.gov identifier: NCT02568267). Samples underwent cfDNA analysis using MSK-ACCESS. Variant allele frequencies of detectable alterations were correlated with objective response per RECIST v1.1 criteria. Twelve patients were included, with best response as partial response (n = 9, 75%), stable disease (n = 2, 17%), and progressive disease (PD; n = 1, 8%). A ROS1 fusion was variably detected in cfDNA; however, patients without a ROS1 fusion in cfDNA had no other somatic alterations detected, indicative of possible low cfDNA shedding. Clearance of the enrolling ROS1 fusion or concurrent non-ROS1 alterations (TP53, CDH1, NF1, or ARID1A mutations) was observed in response to entrectinib therapy. Radiologic PD was accompanied by redemonstration of a ROS1 fusion or non-ROS1 alterations. On-target resistance was rare; only one patient acquired ROS1 G2032R at the time of progression. Several patients acquired new off-target likely oncogenic alterations, including a truncating alteration in NF1. Serial cfDNA monitoring may complement radiographic assessments as determinants of response and resistance to entrectinib in ROS1 fusion-positive lung cancers in addition to detecting putative resistance mechanisms on progression.

Sequential Analysis of cfDNA Reveals Clonal Evolution in Patients with Neuroblastoma Receiving ALK-Targeted Therapy.

The study of cell-free DNA (cfDNA) enables sequential analysis of tumor cell-specific genetic alterations in patients with neuroblastoma. Eighteen patients with relapsing neuroblastoma having received lorlatinib, a third-generation ALK inhibitor, were identified (SACHA national registry and/or in the institution). cfDNA was analyzed at relapse for nine patients and sequentially for five patients (blood/bone marrow plasma) by performing whole-genome sequencing library construction followed by ALK-targeted ddPCR of the hotspot mutations [F1174L, R1275Q, and I1170N; variant allele fraction (VAF) detection limit 0.1%] and whole-exome sequencing (WES) to evaluate disease burden and clonal evolution, following comparison with tumor/germline WES. Overall response rate to lorlatinib was 33% (CI, 13%-59%), with response observed in 6/10 cases without versus 0/8 cases with MYCN amplification (MNA). ALK VAFs correlated with the overall clinical disease status, with a VAF < 0.1% in clinical remission, versus higher VAFs (>30%) at progression. Importantly, sequential ALK ddPCR detected relapse earlier than clinical imaging. cfDNA WES revealed new SNVs, not seen in the primary tumor, in all instances of disease progression after lorlatinib treatment, indicating clonal evolution, including alterations in genes linked to tumor aggressivity (TP53) or novel targets (EGFR). Gene pathway analysis revealed an enrichment for genes targeting cell differentiation in emerging clones, and cell adhesion in persistent clones. Evidence of clonal hematopoiesis could be observed in follow-up samples. We demonstrate the clinical utility of combining ALK cfDNA ddPCR for disease monitoring and cfDNA WES for the study of clonal evolution and resistance mechanisms in patients with neuroblastoma receiving ALK-targeted therapy.

#3031 Cell free DNA methylation analysis reveals cerebral cell death during hemodialysis

Abstract Background and Aims Patients treated with hemodialysis demonstrate a significant cognitive decline, likely explained by cerebrovascular disease and hypoperfusion. Despite the significant morbidity and mortality associated, no method exists to detect subclinical cerebral injury. This prevents early diagnosis and precludes mitigation strategies. Following cell death, cell free DNA (cfDNA) is released into the peripheral blood. Since DNA methylation is cell-type specific, cfDNA methylation analysis can reveal the cellular origin of cfDNA and cell-type specific changes in cell death. Here, we evaluate if subclinical cerebral ischemia during hemodialysis is detectable through cfDNA methylome analyses. Method Chronic hemodialysis patients with a high cardiovascular risk profile were studied during a single hemodialysis session. Brain tissue oxygenation was measured continuously by near-infrared spectroscopy using two sensors on the forehead. Plasma samples were taken immediately before and after hemodialysis. cfDNA was extracted from the blood plasma and converted into whole genome bisulfite sequencing libraries using tailored protocols. Next, in-solution target enrichment was applied to analyze 4,989 genomic loci, selected for showing specific methylation in one of 15 tissues or cell types. After sequencing, reads were aligned to the hg38 reference genome using BisMark, and deconvoluted using EMeth-Laplace to disclose the relative contribution of the 15 selected tissues or cell types. Results 41 hemodialysis patients participated in the study, with a mean age of 75 ± 8 years (46% male). The median ultrafiltration volume was 2.3 L (IQR 1.6-3.0 L). After targeted bisulfite sequencing and QC-based filtering, 35 patients remained for cfDNA methylation analysis. In 30 patients, cfDNA concentrations were increased following dialysis. Deconvolution analysis revealed a relative increase in granulocyte-derived cfDNA from 36.7% to 55% before versus after dialysis. Consequently, for most other tissues and cell types we observed a relative decrease in their cfDNA contribution. Interestingly, a subset of patients (n = 13) showed an increase in neuron-derived cfDNA from 0% to 0.26%, while no neuron-derived cfDNA was detectable in the remaining 22 patients. Near-infrared spectroscopy showed a significantly reduced cerebral oxygenation in these 13 patients (P &amp;lt; 0.0001) at 2 separate time windows during hemodialysis treatment (at 20-60 and 207-251 minutes into the session) (Fig.). Conclusion In patients with a high cardiovascular risk profile, cfDNA in peripheral blood increased after hemodialysis. cfDNA methylome analyses suggested this to be due to an increased contribution of granulocytes, indicating elevated levels of NETosis. In addition, we observed more neuron-derived cfDNA after dialysis in about 1 in 3 patients. This was most apparent in patients with a reduced brain tissue oxygenation during hemodialysis. Together, these findings suggest that hypoperfusion during hemodialysis can evoke neuronal cell death, detectable through methylome analyses of cfDNA isolated from a minimally invasively obtainable bodily fluid. Follow-up studies are ongoing to assess if this translates to progressive cognitive impairment.

The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples.

Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.

Liquid Biopsy Profiling with Multiple Tests in Patients with Metastatic Breast Cancer

The chief goal of the Blood Profiling Atlas in Cancer (BloodPAC) consortium is to promote collaborative efforts that support the development and implementation of liquid biopsy tests. Here, we report the results of a pilot study conducted by three BloodPAC members that aimed to demonstrate a multisite liquid biopsy testing framework using longitudinal blood specimens from 38 patients with metastatic breast cancer. Three laboratories receiving identical samples from two clinical sites each applied a different targeted sequencing platform to analyze mutations in cell-free DNA (cfDNA). The resulting mutational profiles reflected common breast cancer alterations, including clinically actionable mutations for 40% of hormone- receptor-positive patients. In 12 genes with shared target regions across sequencing panels, perfect inter-assay concordance was also observed for mutations detected above the lowest common assay limit of detection. Whole-genome copy number profiling of cfDNA and circulating tumor cells (CTCs) further revealed marked heterogeneity in copy number alterations and cfDNA tumor fractions across patients. Additionally, comparison of tumor fraction and CTC abundance demonstrated the complementary nature of cfDNA and CTC analyses. Overall, the framework described in this study may serve as a resource for future trials aiming to identify multimodal liquid biopsy biomarkers to guide clinical care.

Ampholytic ion-exchange magnetic beads: a promising tool for selecting short fragments in circulating cell-free DNA analysis.

For liquid biopsy of cancer, the extraction of circulating cell-free DNA (cfDNA) from plasma is required. We evaluated the efficacy of use of magnetic submicron particles coated with abundant small zwitterions (MSP-ZEWBs) for extracting short fragments of cfDNA. We developed and optimized an MSP-ZEWB-based cfDNA extraction method using ampholytic ion-exchange materials and compared its results with those using a control kit. We measured the cfDNA concentration by quantitative polymerase-chain-reaction and using the Qubit method and analyzed cfDNA fragmentation patterns using a bioanalyzer. The fragment size of cfDNA isolated from glycine hydrochloric acid at a pH of 2.2 exhibited a better alignment with the DNA marker. The highest DNA intensity was observed at the final concentration of 0.8% polyethylene glycol 8000. The intensity of cfDNA decreased significantly when isolated from plasma with DNA marker using MSP-ZEWBs with an adsorption buffer containing guanidine hydrochloride or isothiocyanoguanidine. All fragments were successfully extracted using MSP-ZEWBs from both plasma and phosphate-buffered saline. Notably, the intensity of short cfDNA fragments isolated using MSP-ZEWBs remained consistent for recovery of long DNA fragments. indicating a potential selective of small fragments. The extraction of plasma cfDNA with MSP-ZEWBs requires no protein denaturation, shows resistance to cells remaining in plasma, and demonstrates higher overall efficiency and better reproducibility than other extraction methods. Use of MSP-ZEWBs may greatly enhance liquid biopsy of cancers through the analysis of plasma cfDNA in clinical practice.

Culture time to optimize embryo cell-free DNA analysis for frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy

To investigate the ideal time in culture to optimize embryo cell-free deoxyribonucleic acid (cfDNA) analysis in frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy (PGT-A). Cell-free DNA is released into the spent blastocyst media (spent media) by the embryo. However, the optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing noninvasive PGT-A remains to be elucidated. In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March2022. Private fertility clinics. Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), whereas day-6 frozen-thawed blastocysts were cultured for 8 hours (Day-6 Short). The spent media and whole blastocysts were then collected for further analysis. Spent media and whole blastocysts were amplified using whole genome amplification and sequenced using next-generation sequencing. Informativity and concordance rates between cfDNA in spent media and whole blastocyst DNA were compared according to the different times in culture. When comparing time in culture, informativity rates for spent media were significantly higher for Day-5 Long and Day-6 Short (>91%) compared with the Day-5 Short group (<60%). A similar trend was observed for cases with and without a previous PGT-A. Regarding blastocyst expansion grade, informativity rates were lower on Day-5 Short compared with Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner-grade expansion grades 3, 4, and 5-6. In addition, for a similar time in culture, the grade of expansion did not have an impact on the informativity rates. For concordance rates, no significant differences were observed among the 3 groups. In all cases, concordance rates were 90.5% for Day-5 Short, 93.6% for Day-5 Long, and 92.3% for Day-6 Short. No impact of the expansion grade was observed on concordance rates. Noninvasive PGT-A in frozen-thawed blastocysts yields very high concordance rates with whole blastocysts, possibly limiting the need for invasive PGT-A and making it available for a wider range of patients.

Abstract PO5-16-05: Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, comprising only between 1-5% of all breast cancer cases in the United States. Despite its low incidence, IBC accounts for 10% of breast cancer-related deaths and is often diagnosed with metastasis, resulting in shorter survival compared to non-IBC cases. The 5-year relative survival rate for all stages of IBC is 39%, dropping to 19% for patients with distant metastasis. Liquid biopsy-based biomarkers have emerged as potential analytes for predicting prognosis and monitoring response to therapies in IBC patients. We explored the prognostic role of liquid biopsy encompassing both CTCs and cfDNA in the blood of patients with stage III/IV IBC and their clinical outcomes. Methods: CTC and cfDNA analysis was performed on 90 blood samples from 47 patients with stage III and IV IBC. Samples were collected before (pre-treatment, n=43) initiation and after (post-treatment, n=47) completion of neoadjuvant chemotherapy. CTCs were enriched and enumerated using the CellSearch® platform (Menarini-Silicon Biosystems). CfDNA was extracted from plasma using MagMax™ Cell-Free Total Nucleic Acid Isolation Kit (Applied Biosystems™) and quantified using the Qubit™ dsDNA High Sensitivity kit (Invitrogen™). Differences in cfDNA concentrations were used to plot a receiver operating characteristic (ROC) curve which was then used to determine Area Under the Curve (AUC) and a cfDNA cut-off point. ROC cut-off was calculated based on Youden Index J. Survival analysis was performed by the Kaplan-Meier estimator and Cox regression models. P-values were measured using the Student t-test. Results: Prior to neoadjuvant treatment, CTC was detected in 51% of patient samples, and the median cfDNA concentration was 5.87 ng/mL (range 0.95-24.11 ng/mL). After completion of neoadjuvant treatment, CTC was detected in 25% of patient samples, and the median cfDNA concentration was 11.19 ng/mL (range 3.41-88.13 ng/mL). The cut-off points determined for pre-treatment and post-treatment cfDNA concentration was &amp;gt;4.2 ng/mL and &amp;gt;9.5 ng/mL respectively. At pre-treatment, CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL were independently associated with disease relapse (P=0.007, 0.016, respectively). The combination of CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL was significantly associated with disease relapse (P=0.0004), decreased relapse free survival (RFS, P=0.005) and overall survival (OS, P=0.007). At the post-neoadjuvant treatment time point, the combination of CTC detection and cfDNA concentration &amp;gt;9.5 ng/mL was significantly associated with relapse regardless of pCR status (P=0.02). Conclusion: The combined presence of CTCs and elevated cfDNA concentrations in patients with stage III/IV IBC after completion of neoadjuvant chemotherapy was associated with disease relapse regardless of pCR status. A combination of elevated cfDNA and CTC detection signatures after completion of neoadjuvant chemotherapy may be useful for risk stratification and adjuvant systemic therapy planning. Citation Format: Sridevi Addanki, Salyna Meas, Vanessa Sarli, Abdulkader Almosa, Wendy Woodward, Dhruvajyoti Roy, Anthony Lucci. Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory 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 PO5-16-05.