Free DNA Research Articles

Evaluating circulating cell-free DNA and DNA integrity index as biomarkers in non-small cell lung cancer

BackgroundAnalysis of free DNA molecules shed from tumour cells in plasma of patients referred as circulating tumour DNA (ctDNA) with reference to physiological circulating cell-free DNA (cfDNA) is nowadays exploited as liquid biopsy and is considered a new emerging promising biomarker for diagnosis, selection of proper treatment, and prognosis of cancer. DNA integrity index (DII) is assessed by calculating the ratio between the concentration of long cfDNA strands released from tumour cells (ALU247) and the short strands released from normal cells (ALU115). The aim of the current study was to evaluate DII as a potential diagnostic and prognostic biomarker of NSCLC.MethodsOur study included 48 NSCLC patients diagnosed as primary NSCLC before starting treatment, 30 COPD patients diagnosed clinically, radiologically, and subjected to chest high-resolution computerized tomography, and 40 healthy controls. cfDNA concentration and DII were measured by quantitative real-time polymerase chain reaction (qPCR).ResultsALU115, ALU247, and DII were significantly higher in NSCLC compared to COPD patients (p < 0.0001) and controls (p < 0.0001) and in COPD patients compared to control subjects (p < 0.0001). DII positively correlated with the stage of tumour (p = 0.01), tumour metastasis (p = 0.004), and with adenocarcinoma compared to other histopathological types (p = 0.02). To evaluate clinical utility of DII in NSCLC, ROC curve analysis demonstrated an AUC of 0.91 at a cut-off value of 0.44 with total accuracy = 85.6%, sensitivity = 90%, specificity = 83%, PPV = 78.1%, and NPV = 92.1%.ConclusioncfDNA and DII represent a promising diagnostic and prognostic tool in NSCLC. This type of noninvasive liquid biopsy revealed its chance in the screening, early diagnosis, and monitoring of NSCLC.

Assessment of associations between neutrophil extracellular trap biomarkers in blood and thrombi in acute ischemic stroke patients

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients’ blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.Graphical

Advancements in breast cancer progression monitoring: Analysis of methylated circulating cell- free DNA.

e12548 Background: Breast cancer is a leading cause of mortality among women, with a significant number developing metastasis. Existing markers like CA-153 and CEA have limited sensitivity. Circulating cell-free DNA (ccfDNA) in plasma can be used for the noninvasive sampling of cancer cells obtained from patients with breast cancer. Aberrant circulating methylated GCM2 and TMEM240 have been reported in patients with breast cancer. The study aims to evaluate the potential of simultaneous detection of methylated GCM2 and TMEM240 in plasma for enhanced prediction of residual disease and tumor progression. Methods: In a prospective study conducted in Taipei, Taiwan, we enrolled a total of 396 patients between 2016 and 2023. The retrospective study involving plasma samples from Western populations included a total of 134 samples. Utilizing the Monte Carlo replicates module within the R software, is employed to automate the random partitioning of 70% of the entire patient dataset into a training set, while the remaining 30% is assigned to the validation set. The model's performance, including sensitivity, specificity, accuracy, false positives, and false negatives, will be thoroughly assessed to ensure its robustness. Results: The test demonstrated an accuracy of 91.5%, a sensitivity of 95.7%, and a specificity of 90.3% for prediction of residual disease and tumor progression. CA15-3 and CEA markers show limited sensitivity (53.3% and 33.3%, respectively). The tests also assess the tumor burden dynamically through monitoring circulating methylated GCM2 and TMEM240 in plasma of patients with breast cancer following their treatment. Conclusions: The simultaneous detection of circulating methylated GCM2 and TMEM240 in plasma presents a novel and accurate noninvasive tool for predicting tumor progression. Clinical trial information: N201708049, N202105091, N202202061.

Multi-feature next-generation liquid biopsy for diagnosis and prognosis in HPV-associated head and neck cancer.

6064 Background: Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+HNSCC) releases circulating tumor HPV DNA (ctHPVDNA) into the blood which is a highly accurate biomarker of disease status. Existing approaches based on droplet digital PCR (ddPCR) have limitations in sensitivity, genotype coverage, and do not annotate additional prognostic genomic features. In this study, we tested the diagnostic and prognostic performance of a custom HPV whole-genome next-generation sequencing (NGS) liquid biopsy, termed HPV-DeepSeek, at the time of diagnosis with HPV+HNSCC, compared to ctHPVDNA ddPCR, HPV serology, and clinical work-up. Methods: 304 participants (152 untreated HPV+HNSCC patients, 152 population-level controls) were prospectively enrolled in this single-center prospective cohort study. Plasma samples were analyzed using HPV-DeepSeek, a multiplexed serology assay, single-plex ddPCR, and a 5-genotype multiplexed ddPCR assay. We tested the hypothesis that HPV-DeepSeek would have the highest diagnostic accuracy. We secondarily examined the accuracy of HPV-DeepSeek for prognostic feature annotation including high-risk HPV16 single nucleotide polymorphisms (SNPs), integration events, and PIK3CA mutations. Results: Of 152 cases, 114 (75%) were AJCC stage 1 and 138/152 (91%) were oropharynx primary site cancers. The sensitivity (98.7%), specificity (98.7%), and diagnostic accuracy (0.974) of HPV-DeepSeek was superior (P&lt;0.001) to singleplex ddPCR (94.2%, 98.6%, 0.928), multiplex ddPCR (90.6%, 96.3%, 0.869), HPV serology (86.4%, 96.3%, 0.827), and first attempt clinical diagnostic biopsy (78% sensitive). Utilizing HPV-DeepSeek, 8 different genotypes were detected. 4 patients were found to have multi-genotype infections. 137/152 cases were classified as HPV16, with A1 the most common sub-lineage (90/137). 21 of 137 HPV+HNSCC HPV16 cases (15%) displayed high risk SNPs. Head-to-head comparison of HPV SNP genotyping by HPV-DeepSeek, with the gold-standard tissue-based HPV whole genome sequencing assay, demonstrated 89% concordance in tissue and 85% in blood. The internal concordance rate for HPV-DeepSeek between tissue and blood was 91%. 48 of 152 cases (32%) had at least one viral integration event detected and 36 of 152 cases (24%) had a PIK3CA mutation detected by HPV-DeepSeek. Fragmentomic analysis demonstrated a mean ctHPVDNA fragment size of 148bp for ctHPVDNA versus 167bp for human cell free (cf)DNA from HPV+HNSCC patients and 168bp for control patient cfDNA. Baseline ctHPVDNA quantity was strongly correlated with viral integration status, T stage and N stage (P&lt;0.001). Conclusions: HPV-DeepSeek has improved diagnostic accuracy relative to ddPCR, HPV serology and clinical work-up at the time of diagnosis and demonstrates detection of prognostic features including high-risk HPV16 SNPs, viral integration events, and PIK3CA mutations.

Trifluridine/Tipiracil Based Chemoradiation in locally Advanced Rectal Cancer: The Phase I/II TARC Trial

BackgroundOptimizing functional outcomes and securing long-term remissions are key goals in managing patients with locally advanced rectal cancer. In this proof-of-concept study, we set out to further optimize neoadjuvant therapy by integrating the radiosensitizer trifluridine/tipiracil and explore the potential of cell free tumor DNA (ctDNA) to monitor residual disease. MethodsAbout 10 patients were enrolled in the phase I dose finding part which followed a 3 + 3 dose escalation design. Tipiracil/trifluridine was administered concomitantly to radiotherapy. ctDNA monitoring was performed before and after chemoradiation with patient-individualized digital droplet PCRs. ResultsNo dose-limiting toxicities were observed at the maximum tolerated dose level of 2 × 35 mg/m² trifluridine/tipiracil. There were 9 grade 3 adverse events, of which 8 were hematologic with anemia and leukopenia. Chemoradiation yielded a pathological complete response in 1 out of 8 assessable patients, downstaging in nearly all patients, and 1 clinical complete response referred for watchful waiting. Three of 4 assessable patients with residual tumor cells at pathological assessment remained liquid biopsy positive after chemoradiation, but 1 turned negative. ConclusionIn this exploratory phase I trial, the novel combination of neoadjuvant trifluridine/tipiracil and radiotherapy proved to be feasible, tolerable, and effective. However, the application of liquid biopsy as a potential marker for therapeutic de-escalation in the neoadjuvant setting requires additional research and prospective validation.The trial was registered at ClinicalTrials.gov: NCT04177602.

317P Targeted next generation sequencing (NGS) of cell free (cf) DNA in biliary tract cancer (BTC)

317P Targeted next generation sequencing (NGS) of cell free (cf) DNA in biliary tract cancer (BTC)

Trends over time in liquid biopsy testing in patients with metastatic breast cancer (MBC) across the Mayo Clinic enterprise.

e13036 Background: In January 2023, the FDA approved elacestrant for ESR1-mutated, ER+/HER2- MBC based on a companion diagnostic cell free (cf)DNA test (Guardant Health). In this study, we analyze the trends of liquid biopsy utilization at Mayo Clinic (MC) over a 3 year period to understand the impact of this drug approval on testing patterns and identify emergent alterations in patients (pts) with MBC. Methods: We extracted clinically performed Guardant 360 and Guardant 360 CDx ordered at MC sites in Rochester (MN), Florida (FL), Arizona (AZ), and across 9 additional Mayo Clinic Health System (MCHS) sites. Pts with MBC were identified using the EMR and analyzing visit diagnosis codes and staging information. Shifts in testing patterns were analyzed over a 3 year period from January 1, 2021 - December 31, 2023. Data was collected on the number of tests, serial testing, frequency of pathogenic mutations, and emergent mutations in ESR1 and PIK3CA. Chi-square test was used to compare the categorical values. Results: Between 2021-2023, 2,741 pts with MBC were seen across all sites. A total of 408 cfDNA tests were performed in 370 unique patients with MBC. Most pts (90%) had 1 test performed. The proportion of pts with MBC tested was 85/1350 (6.3%) in 2021, 77/1495 (5.2%) in 2022, and 208/1558 (13.4%) in 2023. The increased proportion of pts tested in 2023 was statistically significant compared to prior years (p&lt;0.0001), while no difference was observed between 2021 and 2022 (p 0.19). Testing patterns varied by site, with FL ordering the highest number of tests, followed by MN and AZ. Of the 34 providers practicing during the entire period seeing more than 10 MBC pts, the number of providers ordering Guardant testing increased from 15/34 (2021) and 14/34 (2022) to 20/34 (2023), though not statistically significant (p 0.15). Of the 41% with EMR-documented staging available, testing was highest in the ER+/HER2- (81.3%) subtype, followed by TNBC (13.6%) and then HER2+ (5.2%). The most commonly identified mutations identified in the first testing were PIK3CA (41.1%), TP53 (40.5%), ESR1 (30.5%), and ATM (13.2%). D538G (8.0%), Y537N (4.4%), and Y537S (3.5%) were frequent emergent mutations seen in ESR1. Conclusions: We observed an increase in cfDNA testing at the start of 2023 which aligns with the FDA approval of elacestrant. We observed variation by site and individual providers that warrants further exploration to improve cfDNA testing in MBC pts at Mayo Clinic.

Prospective validation of ctHPVDNA for detection of minimal residual disease and prediction of recurrence in patients with HPV-associated head and neck cancer treated with surgery.

6010 Background: Prediction of minimal residual disease (MRD) following surgery, and thus the need for adjuvant therapy, is currently based on clinicopathologic risk factors which have poor individual prognostic capacity. We previously reported that MRD detection by ctHPVDNA droplet digital (dd)PCR as early as post-operative day (POD) 1 is predictive of recurrence in stage I-II HPV+HNSCC patients. Here, we applied a significantly more sensitive custom whole genome hybrid-capture-based next generation sequencing assay, termed HPV-DeepSeek, to validate the prognostic capacity of ctHPVDNA detection and explore the optimal timing of MRD testing in HPV+HNSCC patients. We tested the primary hypothesis that patients with MRD detection within 6 weeks of surgery would have inferior PFS at 2 years and the secondary hypothesis that patients with ctHPVDNA positivity detected at any point following treatment completion would have inferior PFS at 2 years. Methods: 98 patients with HPV+HNSCC were prospectively enrolled with a mean follow-up of 712 days. All patients underwent surgery as primary treatment and clinicopathologic adjusted adjuvant treatment. 10ml blood samples were collected before surgery, in the post-operative period (POD 1-42), and serially in follow-up. MRD was defined as a lack of ctHPVDNA clearance during the 6 weeks following surgery. Cell free DNA was extracted from plasma and run on HPV-DeepSeek, and on existing ddPCR assays for head to head comparisons. Results: 96/98 (98%) of patients had detectable ctHPVDNA pre-treatment. 30/98 patients (31%) were MRD positive. Patients who were MRD positive had significantly worse 2 years PFS compared to MRD negative patients (78% vs 98%, P=0.0009). Predictive performance improved by limiting time points to POD 7-42 (2 year PFS 60% vs 97%, P&lt;0.0001) as significantly fewer patients were MRD positive &gt;1 week after surgery, suggesting the use of an ultra-sensitive assay such as HPV-DeepSeek requires adjustment of MRD time points. Patients with detectable ctHPVDNA following completion of all treatment had significantly worse 2-year PFS compared to patients without detectable ctHPVDNA (0% vs 97%, P&lt;0.0001). 7/98 patients had cancer recurrence during follow-up that was detected by ctHPVDNA with a mean lead time of 207 days (35-518) to clinical diagnosis. Conclusions: ctHPVDNA detection by HPV-DeepSeek is a highly specific biomarker of MRD in HPV+HNSCC, accurately predicts disease progression and detects recurrence earlier than standard care clinical.

Classification of HER2 status across multiple cancers using epigenomic profiles from a novel liquid biopsy assay.

3047 Background: The efficacy of trastuzumab deruxtecan in multiple cancers led to a recent FDA granting of priority review for the treatment of adults with HER2-positive solid tumors. This underscores the critical need for reliable assessment of HER2 expression across cancers. The current standard using IHC/ISH for HER2 scoring is fraught with challenges including scoring discordance and tumor heterogeneity. Here we describe the ability to classify HER2 status from 1 ml of plasma, using epigenomic signatures from a novel multi-analyte liquid biopsy (LBx) platform, offering a minimally invasive approach for patient (pt) selection across cancers. Methods: We selected 179 samples from 172 pts with advanced breast (BC), gastro-esophageal (GEA) and ovarian (OV) cancers who had associated HER2 status scored from tissue-based IHC/ISH according to ASCO/CAP guidelines (table). Samples were taken at baseline or at progression and those with detectable cell free DNA, as assessed by iChorCNA, were profiled for genome-wide epigenomic signals across histone modifications associated with active enhancers, promoters and DNA methylation. A regularized regression model developed to classify HER2 status in BC cell lines and was refined and validated for HER2 status prediction for each pt cohort (HER2+ = 3+, 2+/ISH+; HER2- = 2+/ISH-, 1+, 0). The BC cell-line derived HER2 classifier was refined to incorporate GEA and OV cancer specific features before being applied to those samples. Performance was assessed via AUC in a leave-one-out cross-validation schema. We also evaluated HER2 status at progression in a subset of pts with benchmarked HER2 IHC to assess dynamic changes in receptor status by LBx. Results: The epigenomic HER2 classifier was applied to all samples with ctDNA detectable by ichorCNA (90 of 179; 50%). HER2 classification of BC pts by epigenomic liquid biopsy was concordant with standard tissue-based IHC for 64/72 (89%) BC samples (AUC 0.9, table). HER2 classifier predictions for all longitudinally collected samples were concordant with IHC-based HER2 status, including the one patient whose status switched from HER2+ to HER2- at progression. Accurate classification of 11/14 (79%, GEA) and 4/4 (100%, OV) pts was achieved using the indication-refined HER2 classifier. Conclusions: We demonstrate proof of concept for a HER2 classification approach using comprehensive epigenomic signals from 1 ml of plasma that could be applied across multiple cancers. With further development, our genome-wide profiling approach could alleviate clinical constraints associated with multiple tissue-based HER2 scoring assays (IHC/ISH) and enable longitudinal monitoring of HER2 status on therapy. [Table: see text]

Integration of circulating tumor DNA and metabolic parameters for outcome prediction in unresectable locally advanced non-small cell lung cancer.

8080 Background: An effective biomarker to direct precise consolidation treatment after CRT is still lacking. Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. Hereby, we applied CAPP-Seq ctDNA analysis to plasma samples collected before and after definitive chemoradiotherapy (CRT) or radiotherapy (RT) in unresectable LA-NSCLC. Methods: A total of 62 unresectable LA-NSCLC patients were prospectively enrolled, providing 62 baseline and 49 post-definitive CRT/RT plasma samples. All patients underwent a PET/CT scan at baseline and 33 patients received a mid-treatment PET/CT scan upon reaching a RT dose of 40Gy/20f during therapy. These 33 patients were randomly assigned to either receive or not receive adaptive dose-escalated RT. A group of patients who received immune checkpoint inhibitor (ICI) consolidation after CRT/RT was compared with patients who were ctDNA-/high △TMTV post-treatment. Results: ctDNA was detected at baseline in 44 (71.0%) patients. Pre-treatment ctDNA concentration was significantly correlated with TMTV (p = 0.004) and TLG (p = 0.010). Baseline ctDNA detection and concentration were not able to differentiate patients with varying treatment responses or predict survival. However, patients with undetectable ctDNA and low TMTV exhibited significantly better PFS (p = 0.024). One month after completing CRT or RT, ctDNA was detected in 25 (47.2%) patients. While the concentration of circulating free DNA (cfDNA) remained relatively stable (p = 0.652), both the mean ctDNA Variant Allele Frequency (VAF) (p = 0.002) and ctDNA concentration (p = 0.043) showed a significant decrease. Patients with undetectable ctDNA post-treatment exhibited significantly longer PFS and OS. A lower ∆TMTV was significantly associated with longer PFS and OS. Compared to the non-response group, the response group exhibited significantly lower change in △SUVmax (p = 0.024), with a trend towards lower change in TMTV, although not significant (p = 0.064). Compared with the matched 30 patient receiving ICI consolidation, those with negative ctDNA and high △TMTV after CRT demonstrated significantly better PFS (p = 0.042) and OS (p = 0.039). Conclusions: Baseline ctDNA combined with TMTV can enhance the predictive ability for survival. Post-CRT ctDNA and △TMTV possess strong prognostic capabilities. Patients who are ctDNA- and with high △TMTV may be exempt from ICI consolidation therapy.

A phase II trial of olaparib and durvalumab in patients with recurrent IDH-mutated gliomas.

2013 Background: Isocitrate dehydrogenase mutations (IDHmt) define astrocytomas and oligodendrogliomas. IDHmt results in accumulation of R-2-hydroxyglutarate (2HG), leading to epigenetic dysregulation and defective homologous recombination repair, providing a rationale for poly (adenosine 5’-diphophate-ribose) polymerase (PARP) inhibitors. PARP inhibition upregulates PD-L1 so the combination with immune checkpoint inhibition is potentially synergistic. Methods: Patients (pts) with recurrent high-grade IDHmt gliomas were enrolled in this phase II open-label study (NCT03991832). Eligibility included progressive disease with up to 2 prior lines of systemic therapies and ECOG 0–1. Pts received olaparib 300 mg twice daily continuously and durvalumab 1500 mg IV every 4 weeks. Simon’s optimal two-stage design was used. The primary objective was overall response rate (ORR) and disease control rate (DCR) by RANO criteria. Secondary objectives included overall survival (OS), progression free survival (PFS) and safety.Exploratory biomarkers of response and resistance were assessed with serial blood samples using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as well as multiplex-immunohistochemistry. Results: In the 29 pts enrolled between January 2020–February 2023, median age was 40.5 (range 23–66) and 41% were female. Initial tumor grade was 2 in 9, 3 in 8 and 4 in 12 pts, respectively. Median time to enrollment from tumour diagnosis was 5.9 years. All had prior resection and median number of prior systemic therapies was 2. One patient clinically deteriorated before starting treatment. ORR was 10%, 95% CI 2.2–27%, with responses in 3 pts. One pt with grade 4 astrocytoma had a complete response and remains on treatment after 33 months. The other 2 responders with grade 4 astrocytoma had response durations of 4.1 and 9.8 months. DCR was 28% (95% CI 12.7–47.2%) with 5 additional pts demonstrating stable disease. With a median follow-up of 33 months, mOS was 9.5 months (95% CI 4.3–19.3) and mPFS was 1.9 months (95% CI 1.8–3.0). There was no treatment-related grade 3–4 toxicities. Any grade toxicities included fatigue (48%), nausea (17%), diarrhea (10%), and cytopenias (3%). Using serial cfMeDIP-seq, cell free DNA methylomes comparing responders versus progressors using the top differentially methylated regions can predict treatment response with high accuracy (AUC 0.833). Post progression, differentially methylated genes converge on TGF-β and signal transduction pathways, suggesting possible mechanisms of resistance. Multi-modal analysis of long-term responders will additionally be presented. Conclusions: Combination treatment with olaparib and durvalumab for pts with IDHmt glioma is well tolerated but has limited efficacy in unselected pts. cfMeDIP-seq can reliably predict tumour progression providing a blood-based biomarker of treatment response. Clinical trial information: NCT03991832 .

Utility of liquid biopsy for predicting cancer type and informing treatment of carcinoma with unknown primary.

e13606 Background: The advent of next-generation sequencing has greatly facilitated physicians in recommending tailored treatments for cancer patients. Despite these advancements, the dependence on the primary tumor site remains crucial for accurate diagnoses, posing a substantial obstacle in cases of carcinomas of unknown primary (CUPs) and hindering optimal patient care. To address this challenge, we propose the implementation of a machine learning model capable of identifying the tissue of origin (TOO) across 16 cancer types. Current standards gravitate to the use of tumor tissue for identifying primary tumor site. Leveraging the advantages of cell-free DNA (cfDNA), our approach provides a non-invasive and highly sensitive alternative to traditional biopsy or radiography methods. Methods: Cell-free DNA was extracted from patients diagnosed with carcinoma of known origin. The model underwent training using a dataset comprising 2,793 cfDNA samples, while the validation set consisted of 1,863 samples. The training and validation cohorts exhibited comparable demographics in terms of sex, age, and cancer types. Employing low-coverage whole genome sequencing (WGS), samples were analyzed to capture cfDNA features, including DNA fragment size distribution, copy number variation, nucleosome coverage pattern, mutational signature, and hotspot regions associated with cancer-related mutations. The final prediction model's accuracy was evaluated based on top 1 and top 2 predictions in accordance with the true tumor site of origin. Results: Overall accuracy of TOO predictions in the training cohort is 80.20% (2240/2793) for top 1 site and 90.26% (2521/2793) for top 2 sites. Overall accuracy of TOO predictions in the validation cohort is 81.80% (1524/1863) for top 1 site and 90.07% (1678/1863) for top 2 sites. Accuracy of tumor site prediction in the validation cohort is highest for lung cancer (Top 1 = 95.9%; Top 2 = 98.0%) tumor sites and lowest for biliary system and kidney tumor types (Top 1 = 54.3% &amp; 57.4%; Top 2 = 71.6% &amp; 63.2%). In the clinical setting, TOO estimation of 12/16 patients with CUP were identified to be in concordance with majority pathological consensus. Of note, two patients with CUP were diagnosed as lung cancer patients through TOO estimation. Molecular biomarkers of lung adenocarcinoma were also identified through targeted sequencing which corroborated the diagnosis, and chemoradiotherapy was recommended to the patient. Conclusions: We developed an accurate predictor of TOO by leveraging machine learning algorithm and incorporating cfDNA mutational, fragmentomic, and epigenetic features. Our results demonstrate high concordance between model-predicted and actual tumor sites across all examined cancer types. This model shows promise in offering valuable insights for the identification of cancers with an unknown primary origin using non-invasive cell free DNA samples.

Molecular profiling in ABC: Is complementary testing with tissue and plasma necessary and clinically relevant?

e12566 Background: This retrospective analysis explores the concordance between molecular analyses of tissue and plasma amongst Advanced Breast Cancer (ABC) patients and investigates the utility of simultaneous profiling for enhanced clinical insights. Methods: A total of 187 (177 Cancertrack paired and 10 Guardant 360) patient samples with availability of both tissue and ctDNA specimens, were subjected to comparative molecular profiling. Median age was 52 years (Range 23 – 82 years), 174 (93%) patients had advanced breast cancer and 177 (95%) patients had received prior treatments. We assigned tier 1 status to predictive biomarkers with FDA approved or guideline recommended therapies in breast cancer. Tier 2 status was assigned to alterations for which therapies were approved in different tumor type. Tier 3 had either clinical evidence in small studies or had clinical trial inclusion criteria. Results: Paired tissue-ctDNA profiling showed 139/187 (74%) samples had at least one concordant molecular alteration. Simultaneous tissue and ctDNA profiling led to the opportunity to offer Tier 1 recommended therapies in 113/187 (60%) patients. Amongst these, 71/113 (63%) exhibited concordance in tissue and circulating free DNA (cfDNA). ctDNA missed Tier 1 alterations in 14 patients which were detected by tissue biopsy. On the contrary tissue biopsy failed to detect Tier 1 alterations in 18 patients which were identified by ctDNA only. When Tier 2 and 3 alterations were considered, ctDNA identified additional alterations compared to tissue in 29 patients (37 variants), whereas tissue identified additional alterations in 20 patients (24 variants). Complementary tissue and ctDNA profiling showed alterations with potential to impact clinical management (Tier1/2/3) in 82% patients. When including only Tier 1 and 2 alterations, simultaneous profiling approach gives an opportunity to offer personalized therapy in 68% patients. The Tier1 ctDNA only alterations were most frequent in ESR1 gene. In fact, a total of a total of 29 ESR1 mutations were detected in these patients, of which 10 mutations were detected in both tissue and liquid. In 3 patients’ tissues were positive for ESR1 mutations but the corresponding ctDNA was negative. Similarly, ctDNA detected 16 ESR1 variants which were negative in the tissue sample. The most common ESR1 variants were D538G and Y537S. Conclusions: The simultaneous profiling of tissue and ctDNA enhanced overall clinical utility of molecular analysis in advanced breast cancer by identifying predictive biomarkers. The integration of data from both sources allowed for comprehensive understanding of the molecular heterogeneity in breast cancer. It underscores the potential of simultaneous profiling to guide treatment decisions and improve patient outcomes. By detecting acquired resistance ESR1 mutations more effectively, ctDNA serves as a valuable tool for monitoring as well.

Use of early circulating tumour DNA dynamics in patients with stage III melanoma receiving neoadjuvant combination immunotherapy.

9577 Background: Neoadjuvant therapy in resectable stage III cutaneous melanoma improves relapse-free survival compared to adjuvant therapy alone and is now a standard of care.While pathological response can help predict recurrence risk and the need for further treatment, more biomarkers are required. We investigated circulating tumour DNA (ctDNA) as a predictive biomarker for recurrence in stage IIIB/C melanoma patients following neoadjuvant immunotherapy and surgery. Methods: We retrospectively analysed plasma samples collected at baseline and six weeks post-surgery from 30 patients enrolled in the OpACIN-neo and PRADO clinical trials, who received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent pre-amplification followed by tumour-informed mutation detection analysis using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system. BRAF mutations were identified in 60% of patients through tumour tissue sequencing. Following neoadjuvant therapy, 7/30 (23%) achieved complete pathological response (pCR), 5/30 (17%) near pCR, 4/30 (13%) partial response, and 14/30 (47%) non-response (pNR). Results: Baseline ctDNA was detectable in 15/30 (50%) patients using the pre-amplification ddPCR method. At median follow-up of 47 months, 9/30 (30%) patients recurred, with a median time to recurrence of 8 months (range 6 to 29 months). Post-surgery, ctDNA was detectable in 4/9 patients who recurred; all four were pathological non-responders and recurred with distant, unresectable disease. Among the 15 ctDNA-positive patients at baseline, 13/15 (87%) patients achieved ctDNA clearance while 2/15 (13%) remained persistently ctDNA-positive; with a subsequent relapse rate of 0% and 100% respectively. ctDNA detection post-surgery predicted patients at higher risk of disease recurrence [relapse-free survival (RFS) hazard ratio (HR) 7.83, 95%CI 0.82-74.48; p=0.0002] and poorer melanoma-specific survival (MSS) [HR 6.35, 95% CI 0.36-110.70; p=0.0337]. Conclusions: Post-surgery ctDNA positivity can signal imminent recurrence, thus offering a window for personalised adjuvant therapy modification.

Molecular profiling of serial liquid biopsy specimens utilizing cell free DNA (cfDNA) and circulating tumor cells (CTCs) in TBCRC041: A phase II study of alisertib in endocrine resistant metastatic breast cancer (MBC).

1037 Background: TBCRC041, a randomized phase 2 trial of alisertib (A) ± fulvestrant (F) in women with HR+/HER2- MBC demonstrated promising clinical activity for alisertib (1). Interrogation of baseline (PreC1) and end of cycle 1 (EOC1) cfDNA and CTCs may identify biomarkers associated with response to A±F. Methods: Plasma cfDNA was sequenced using the Guardant INFINITY platform, which includes genomic and methylated tumor fraction (mTF) analysis. Pathogenic variants were determined using publicly available databases and Mayo Clinic annotation pipelines. CTCs were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells and assessed for ER/HER2 staining (RareCyte, Seattle). PFS was defined as the time from randomization to progression or death. EOC1-PFS differs from PFS as time starts at C2D1. Stratified log-rank tests and conditional Cox modeling were used to assess whether PFS or EOC1-PFS differed by mutations in ESR1 or PIK3CA, pre-CTC ≥ 5, or mTF&gt;1%. Results: Of the 91 eligible pts, 84 progressed, and 73 were deceased. In the 86 pts with PreC1-cfDNA results, variants were identified in ESR1 (42; 48.8%), PIK3CA (36; 41.9%), AKT1 (7; 8.1%), PTEN(7; 8.1%), BRCA1 (4; 4.7%), and BRCA2 (7; 8.1%). Pts with a PIK3CAmut had significantly decreased PFS (HR 1.8; 95%CI: 1.1 -2.9, p=0.014) but not by ESR1mut status (p=0.583). PreC1 and EOC1-mTF were determined for 78 and 76 pts, respectively. PreC1-mTF was 0-1% in 21; 1.1-10.0% in 27; and 10.1-100% in 30. mTF levels remained between 0-1% in 17 pts fell to 0-1% in 11 pts, and was 0-1% in 5 pts without a preC1-mTF determination. PFS did not differ with respect to PreC1-mTF (p=0.210), but EOC1-PFS was worse in pts with mTF &gt;1% (HR 3.0; 95%CI: 1.6 -5.2, p&lt;0.001). PreC1 and EOC1-CTC were determined for 78 and 76 pts. 36 (46.2%) pts had a PreC1 CTC count ≥ 5. EOC1-CTC levels remained at (n=17) or increased to (n=1) ≥ 5 and was ≥ 5 in 2 pts without a preC1-CTC determination. EOC1-PFS was not significantly different for EOC1-CTC ≥ 5 (p=0.065), but was significantly decreased in pts with PreC1-CTC≥5 (HR=1.8; 95%CI: 1.1-3.0; p=0.018). Broader cfDNA NGS panel and CTC-derived ER/HER2 staining results will be presented at the meeting. Conclusions: Among patients receiving A±F, PFS varied based on the PIK3CAmutation status. cfDNA and CTC assessment PreC1 and EOC1 provided complementary information. Further validation in larger datasets is needed. 1. JAMA Oncol 2023;9:815. Clinical trial information: NCT02860000 .

Analysis of cell free DNA to predict outcome to bevacizumab therapy in colorectal cancer patients

To predict outcome to combination bevacizumab (BVZ) therapy, we employed cell-free DNA (cfDNA) to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients. Low-coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples, collected from 74 mCRC patients from the AC-ANGIOPREDICT Phase II trial (NCT01822444), and analysed for CIN and NFs. A validation cohort of plasma samples from the University Medical Center Mannheim (UMM) was similarly profiled. 61 AC-ANGIOPREDICT plasma samples collected before and following BVZ treatment were selected for targeted methylation sequencing. Using cfDNA CIN profiles, AC-ANGIOPREDICT samples were subtyped with 92.3% accuracy into low and high CIN clusters, with good concordance observed between matched plasma and tumor. Improved survival was observed in CIN-high patients. Plasma-based CIN clustering was validated in the UMM cohort. Methylation profiling identified differences in CIN-low vs. CIN high (AUC = 0.87). Moreover, significant methylation score decreases following BVZ was associated with improved outcome (p = 0.013). Analysis of CIN, NFs and methylation profiles from cfDNA in plasma samples facilitates stratification into CIN clusters which inform patient response to treatment.

CRISPR/Cas13a-based supersensitive circulating tumor DNA assay for detecting EGFR mutations in plasma

Despite recent technological advancements in cell tumor DNA (ctDNA) mutation detection, challenges persist in identifying low-frequency mutations due to inadequate sensitivity and coverage of current procedures. Herein, we introduce a super-sensitivity and specificity technique for detecting ctDNA mutations, named HiCASE. The method utilizes PCR-based CRISPR, coupled with the restriction enzyme. In this work, HiCASE focuses on testing a series of EGFR mutations to provide enhanced detection technology for non–small cell lung cancer (NSCLC), enabling a detection sensitivity of 0.01% with 40 ng cell free DNA standard. When applied to a panel of 140 plasma samples from 120 NSCLC patients, HiCASE exhibits 88.1% clinical sensitivity and 100% specificity with 40 μL of plasma, higher than ddPCR and Super-ARMS assay. In addition, HiCASE can also clearly distinguish T790M/C797S mutations in different positions at a 1% variant allele frequency, offering valuable guidance for drug utilization. Indeed, the established HiCASE assay shows potential for clinical applications.

Chemotherapy related changes in cfDNA levels in squamous non-small cell lung cancer: correlation with symptom scores and radiological responses.

There is limited data on prognostic value of baseline plasma cell free DNA (cfDNA) in advanced squamous non-small cell lung cancer (sq-NSCLC). This prospective observational study aimed to assess change in plasma cfDNA levels in locally-advanced/metastatic sq-NSCLC with chemotherapy and its correlation with symptom-scores and radiological-responses. Chemotherapy-naive patients with stages-IIIB/IIIC/IV sq-NSCLC (n = 59), smokers with chronic obstructive pulmonary disease [COPD, COPD-controls (CC); n = 27] and healthy-controls (n = 25) were enrolled. Respiratory symptom burden (RSB) and total symptom burden (TSB) were calculated from mean visual-analog-scores (VAS) of dyspnoea, cough, chest pain, hemoptysis RSB, anorexia and fatigue (all six for TSB). cfDNA was isolated from peripheral blood. All patients received platinum-doublet chemotherapy. RSB/TSB/cfDNA assessment and contrast-enhanced computed tomography (CECT)-thorax scans were done at baseline and post-chemotherapy. At baseline, 13/59 (22%) sq-NSCLC, 3/27 (11%) CC and none (0%) healthy-controls had detectable cfDNA. All three CC were heavy smokers with no evidence of malignancy and undetectable cfDNA levels on repeat testing. In sq-NSCLC group, majority were males (95%), current-smokers (88%), heavy-smokers (70%), had metastatic disease (59%) with median age of 65 years. Eastern Co-operative Oncology Group (ECOG) performance status (PS) was 0-1 (56%) and 2 (42%). Median RSB- and TSB-scores were 9 [interquartile range (IQR) = 5-14] and 16 (IQR = 9-23), respectively. Of the 59 patients, 54 received ≥ 1 cycle while 27 underwent post-C4 evaluation with detectable cfDNA levels in 18/27 (66.7%). No baseline characteristic correlated with cfDNA detectability. Median overall survival (OS) and progression-free survival (PFS) were 262 days and 167 days, respectively. ECOG PS ≥ 2, RSB-score > 9 and TSB-score > 16 were all associated with worse OS and PFS as was cfDNA detectability [median OS = 97 days vs. 298 days and median PFS = 97 days vs. 197 days; P = 0.025; hazard ratio (HR) = 2.17]. Baseline cfDNA detectability is independently associated with poor OS and PFS in patients with advanced sq-NSCLC on chemotherapy.

Da-yuan-yin decoction alleviates ulcerative colitis by inhibiting complement activation, LPS-TLR4/NF-κB signaling pathway and NET formation

Ethnopharmacological relevanceDa-yuan-yin decoction (DYY) is a classical traditional Chinese medicine prescription for ulcerative colitis (UC). Aim of studyThis study explored the protective effects and mechanisms of DYY on UC. Materials and methodsThe mice were fed 2.5% dextran sulfate sodium (DSS) for 7 days to establish UC. On the second day, DYY (0.4 g/kg, 0.8 g/kg, 1.6 g/kg) was orally administered daily for 7 consecutive days. The colon tissues and serum were measured by histopathological examination and biochemical analysis. ResultsDYY significantly reduced the disease activity index (DAI) and severity of colon shortening and alleviated pathological changes in the colon tissue. DYY restored the protein expression of intestinal tight junction (TJ) protein (ZO-1, occludin and claudin-3). DYY remarkably decreased the level of lipopolysaccharide (LPS), Lactic acid (LA), circulating free DNA (cfDNA), complement (C3, C3a, C3c, C3aR1, C5a and C5aR1) and regulated the levels of inflammatory cytokines in serum. DYY significantly inhibited the expressions of nuclear factor kappa-B p65 (NF-κB p65) and Toll-like receptor 4 (TLR4), citrullinated histone H3 (CitH3) and myeloperoxidase (MPO), reactive oxygen species (ROS) peptidylarginine deiminase 4 (PAD4) and CD 11b, the mRNA levels of PADI4, MPO and ELANE in colon tissues. ConclusionsDYY significantly attenuated DSS-induced UC, which was related with regulating the inflammatory response by the inhibition of complement activation, the LPS-TLR4/NF-κB signaling pathway and neutrophil extracellular traps (NETs) formation. DYY is a potential therapeutic agent for UC.

GR.5 Establishing the utility of multi-platform liquid biopsy by integrating the CSF methylome and proteome in CNS malignancy

Background: Liquid biopsy represents a major development in cancer research, with significant translational potential. Similarly, the integration of multiple molecular platforms has yielded novel insights into disease biology and heterogeneity. We hypothesise that applying contemporary multi-omic approaches to liquid biopsies will improve the power of current models. Methods: We have compiled a cohort of 51 patients with glioblastoma, brain metastasis, and primary CNS lymphoma who underwent CSF sampling as part of clinical care. Cell free methylated DNA and shotgun proteomic profiling was obtained from the CSF of each patient and used to build tumour-specific classifiers. Integrated classifiers were compared with single platform classifiers using multiple approaches. Results: In this study, we show that the DNA methylation and protein profiles of cerebrospinal fluid can be combined to fully discriminate lymphomas from their major diagnostic counterparts with perfect AUCs of 1 (95% confidence interval 1-1) and 100% specificity. Each integrated lymphoma classifier significantly outperforms single-platform classifiers, suggesting synergistic biology is obtained using multiple molecular platforms. Conclusions: We present the most specific and accurate CNS lymphoma classifier to date by integrating the methylome and proteome of CSF. This has important implications for the future of cancer diagnostics and generates immediate utility for patients with CNS lymphoma.