Circulating Tumor DNA Research Articles

Prognostic biomarkers in melanoma: a 2023 update from clinical trials in different therapeutic scenarios.

Over the past decade, significant advancements in the field of melanoma have included the introduction of a new staging system and the development of immunotherapy and targeted therapies, leading to changes in substage classification and impacting patient prognosis. Despite these strides, early detection remains paramount. The quest for dependable prognostic biomarkers is ongoing, given melanoma's unpredictable nature, especially in identifying patients at risk of relapse. Reliable biomarkers are critical for informed treatment decisions. This review offers a comprehensive review of prognostic biomarkers in the context of clinical trials for immunotherapy and targeted therapy. It explores different clinical scenarios, including adjuvant, metastatic, and neo-adjuvant settings. Key findings suggest that tumor mutational burden, PD-L1 expression, IFN-γ signature, and immune-related factors are promising biomarkers associated with improved treatment responses. Identifying practical prognostic factors for melanoma therapy is challenging due to the tumor's heterogeneity. Promising biomarkers include tumor mutational burden (TMB), circulating tumor DNA, and those characterizing the tumor microenvironment, especially the immune component. Future research should prioritize large-scale, prospective studies to validate and standardize these biomarkers, emphasizing clinical relevance and real-world applicability. Easily accessible biomarkers have the potential to enhance the precision and effectiveness of melanoma management.

Ultrasensitive and Rapid Circulating Tumor DNA Liquid Biopsy Using Surface-Confined Gene Amplification on Dispersible Magnetic Nano-Electrodes.

Circulating tumor DNA (ctDNA) detection has been acknowledged as a promising liquid biopsy approach for cancer diagnosis, with various ctDNA assays used for early detection and treatment monitoring. Dispersible magnetic nanoparticle-based electrochemical detection methods have been proposed as promising candidates for ctDNA detection based on the detection performance and features of the platform material. This study proposes a nanoparticle surface-localized genetic amplification approach by integrating Fe3O4-Au core-shell nanoparticles into polymerase chain reactions (PCR). These highly dispersible and magnetically responsive superparamagnetic nanoparticles act as nano-electrodes that amplify and accumulate target ctDNA in situ on the nanoparticle surface upon PCR amplification. These nanoparticles are subsequently captured and subjected to repetitive electrochemical measurements to induce reconfiguration-mediated signal amplification for ultrasensitive (∼3 aM) and rapid (∼7 min) metastatic breast cancer ctDNA detection in vitro. The detection platform can also detect metastatic biomarkers from in vivo samples, highlighting the potential for clinical applications and further expansion to rapid and ultrasensitive multiplex detection of various cancers.

Circulating tumour DNA analysis and new uses of anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer

Currently, tumour tissue biopsy to determine RAS/BRAF gene alterations, assess microsatellite instability status, and determine HER‑2/neu gene amplification/hyperexpression is the gold standard of diagnosis and allows the selection of optimal molecularly targeted therapy when considering treatment strategies for patients with metastatic colorectal cancer. However, biopsy does not fully reflect the existing intratumoural heterogeneity and clonal evolution of tumour cells, which can often be the cause of therapeutic failures. In recent years, liquid biopsy has attracted increasing attention as an additional and potentially alternative non-invasive tool for molecular tumour profiling. Assessment of circulating tumour DNA allows changes in the genetic status of the tumour to be monitored and the «burden» of disease to be measured dynamically in real time. Advances in liquid biopsy technology have led to promising new strategies for the management of patients with metastatic colorectal cancer in late-line therapy. The standard drug arsenal in this group of patients is limited to either repeat administration of previously effective therapy or regorafenib and the combination of trifluridine/tipiracil with bevacizumab, which are characterized by limited clinical activity. However, thanks to the discovery of the NeoRAS wild-type phenomenon and the rechallenge strategy of anti-EGFR monoclonal antibodies based on the study of clonal selection and evolution of tumour cells, the administration of epidermal growth factor inhibitors in a molecularly selected by liquid biopsy population is accompanied by good tolerability and efficacy. Numerous clinical studies are ongoing to further understand the mechanisms of tumour resistance and to develop new evidence-based treatment approaches in order to realise the concept of personalised medicine.

Updates on the Management of Colorectal Cancer in Older Adults

Colorectal cancer (CRC) poses a significant global health challenge. Notably, the risk of CRC escalates with age, with the majority of cases occurring in those over the age of 65. Despite recent progress in tailoring treatments for early and advanced CRC, there is a lack of prospective data to guide the management of older patients, who are frequently underrepresented in clinical trials. This article reviews the contemporary landscape of managing older individuals with CRC, highlighting recent advancements and persisting challenges. The role of comprehensive geriatric assessment is explored. Opportunities for treatment escalation/de-escalation, with consideration of the older adult’s fitness level. are reviewed in the neoadjuvant, surgical, adjuvant, and metastatic settings of colon and rectal cancers. Immunotherapy is shown to be an effective treatment option in older adults who have CRC with microsatellite instability. Promising new technologies such as circulating tumor DNA and recent phase III trials adding later-line systemic therapy options are discussed. Clinical recommendations based on the data available are summarized. We conclude that deliberate efforts to include older individuals in future colorectal cancer trials are essential to better guide the management of these patients in this rapidly evolving field.

Undetectable circulating tumor DNA confirms the inability of pseudomyxoma peritonei to systemic dissemination

The study of circulating tumor DNA (ctDNA) plays a pivotal role in advancing precision oncology, providing valuable information for individualized patient care and contributing to the ongoing effort to improve cancer diagnosis, treatment, and management. However, its applicability in pseudomyxoma peritonei (PMP) remains unexplored. In this multicenter retrospective study involving 21 PMP patients, we investigated ctDNA presence in peripheral blood using three distinct methodologies. Despite mucinous tumor tissues exhibiting KRAS and GNAS mutations, ctDNA for these mutations was undetectable in blood samples. In this pilot study, circulating tumor DNA was not detected in blood when the tumor harbored mutations of known significance. In the future, a study with a larger sample size is needed to confirm these findings and to determine whether ctDNA could identify patients at risk for early recurrence and/or systemic metastases.

Integrated approach to generate artificial samples with low tumor fraction for somatic variant calling benchmarking

BackgroundHigh-throughput sequencing (HTS) has become the gold standard approach for variant analysis in cancer research. However, somatic variants may occur at low fractions due to contamination from normal cells or tumor heterogeneity; this poses a significant challenge for standard HTS analysis pipelines. The problem is exacerbated in scenarios with minimal tumor DNA, such as circulating tumor DNA in plasma. Assessing sensitivity and detection of HTS approaches in such cases is paramount, but time-consuming and expensive: specialized experimental protocols and a sufficient quantity of samples are required for processing and analysis. To overcome these limitations, we propose a new computational approach specifically designed for the generation of artificial datasets suitable for this task, simulating ultra-deep targeted sequencing data with low-fraction variants and demonstrating their effectiveness in benchmarking low-fraction variant calling.ResultsOur approach enables the generation of artificial raw reads that mimic real data without relying on pre-existing data by using NEAT, a fine-grained read simulator that generates artificial datasets using models learned from multiple different datasets. Then, it incorporates low-fraction variants to simulate somatic mutations in samples with minimal tumor DNA content. To prove the suitability of the created artificial datasets for low-fraction variant calling benchmarking, we used them as ground truth to evaluate the performance of widely-used variant calling algorithms: they allowed us to define tuned parameter values of major variant callers, considerably improving their detection of very low-fraction variants.ConclusionsOur findings highlight both the pivotal role of our approach in creating adequate artificial datasets with low tumor fraction, facilitating rapid prototyping and benchmarking of algorithms for such dataset type, as well as the important need of advancing low-fraction variant calling techniques.

Human papillomavirus circulating tumor DNA assays as a mechanism for head and neck cancer equity in rural regions of the United States

The rates of human papillomavirus-positive oropharyngeal cancer (HPV-OPC) are rising worldwide and in the United States, particularly in rural regions including Appalachia. Rural areas face unique health challenges resulting in higher cancer incidence and mortality rates, and this includes HPV-OPC. The recent advent of highly sensitive liquid biopsies for the non-invasive detection of HPV-OPC recurrence (circulating tumor HPV DNA, HPV ctDNA) has been swiftly adopted as part of surveillance paradigms. Though knowledge gaps persist regarding its use and clinical trials are ongoing, the ease of collection and cost-effectiveness of HPV ctDNA make it more accessible for HPV-OPC survivors than usual surveillance methods of frequent exams and imaging. Herein, we discuss how implementing HPV ctDNA assays in rural regions of the United States provide one poignant example of how liquid biopsies can improve cancer care equity.

A quadratic isothermal amplification fluorescent biosensor without intermediate purification for ultrasensitive detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) is an auspicious tumor biomarker released into the bloodstream by tumor cells, offering abundant information concerning cancer genes. It plays a crucial role in the early diagnosis of cancer. However, due to extremely low levels in body fluids, achieving a simple, sensitive, and highly specific detection of ctDNA remains challenging. Here, we constructed a purification-free fluorescence biosensor based on quadratic amplification of ctDNA by combining nicking enzyme mediated amplification (NEMA) and catalytic hairpin assembly (CHA) reactions. After double isothermal amplification, this biosensor achieved an impressive signal amplification of nearly 107-fold, enabling it to detect ctDNA with ultra-sensitivity. And the detection limit of this biosensor is as low as 2 aM. In addition, we explored the influence of human serum on the performance of the biosensor and found that it showed favorable sensitivity in the presence of serum. This biosensor eliminates the need for an intermediate purification step, resulting in enhanced sensitivity and convenience. Thus, our purification-free fluorescent biosensor exhibits ultra-high sensitivity when compared to other biosensors and has the potential to serve as an effective diagnostic tool for early detection of cancer.

Evaluation of Molecular Residual Disease by a Fixed Panel in Resectable Colorectal Cancer.

Molecular residual disease (MRD) is a promising biomarker in colorectal cancer (CRC) for prognosis and guiding treatment, while the whole-exome sequencing (WES) based tumor-informed assay is standard for evaluating MRD based on circulating tumor DNA (ctDNA). In this study, we assessed the feasibility of a fixed-panel for evaluating MRD in CRC. 75 patients with resectable stage I-III CRC were enrolled. Tumor tissues obtained by surgery, and pre-operative and post-operative day 7 blood samples were collected. The ctDNA was evaluated using the tumor-agnostic and tumor-informed fixed assays, as well as the WES-based and panel-based personalized assays in randomly selected patients. The tumor-informed fixed assay had a higher pre-operative positive rate than the tumor-agnostic assay (73.3% vs 57.3%). The pre-op ctDNA status failed to predict disease-free survival (DFS) in either of the fixed assays, while the tumor-informed fixed assay-determined post-op ctDNA positivity was significantly associated with worse DFS (HR, 20.74, 95%CI 7.19-59.83; p<0.001), which was an independent predictor by multivariable analysis (HR, 28.57, 95%CI 7.10-114.9; p<0.001). Sub-cohort analysis indicated the WES-based personalized assay had the highest pre-operative positive rate (95.1%). The two personalized assays and the tumor-informed fixed assay demonstrated same results in post-op landmark (HR, 26.34, 95%CI, 6.01-115.57; p<0.001), outperforming the tumor-agnostic fixed panel (HR, 3.04, 95%CI, 0.94-9.89; p=0.052). Our study confirmed the prognostic value of the ctDNA positivity at post-op day 7 by the tumor-informed fixed panel. The tumor-informed fixed panel may be a cost-effective method to evaluate MRD, which warrants further studies in future.

Quality and Accessibility of Liquid Biopsy Information

This cross-sectional study evaluates the information on a circulating tumor DNA test available to the public on popular internet resources.

Liquid biomarkers in prostate cancer: recent advancements and future directions.

Traditional diagnostic approaches of prostate cancer like PSA are limited by high false-positive rates and insufficient capture of tumour heterogeneity, necessitating the development of more precise tools. This review examines the latest advancements in liquid biomarkers for prostate cancer, focusing on their potential to refine diagnostic accuracy and monitor disease progression. Liquid biomarkers have gained prominence because of their minimally invasive nature and ability to reflect the molecular characteristics of prostate cancer. Circulating tumour cells provide insight into tumour cell dissemination and are indicative of aggressive disease phenotypes, with single-cell analyses revealing genomic instability and treatment resistance. Circulating tumour DNA offers real-time tumour genomic information, aiding in treatment decision-making in advanced prostate cancer, where it has been associated with clinical progression. MicroRNAs act as oncogenes or tumour suppressors and exhibit diagnostic and prognostic potential; however, their clinical utility is constrained by the lack of consistent validation. Extracellular vesicles contain tumour-derived biomolecules, with specific proteins demonstrating prognostic relevance. Applications of these markers to urinary testing have been demonstrated. Liquid biomarkers show potential in refining prostate cancer management. Future research should aim to integrate these biomarkers into a cohesive framework in line with precision medicine principles.

Circulating tumor DNA- and cancer tissue-based next-generation sequencing reveals comparable consistency in targeted gene mutations for advanced or metastatic non-small cell lung cancer.

Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy. This study aimed to investigate the consistency of next-generation sequencing (NGS) results between circulating tumor DNA (ctDNA)-based and tissue-based in non-small cell lung cancer (NSCLC) and identify the patient characteristics that favor ctDNA testing. Patients who diagnosed with NSCLC and received both ctDNA- and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center, Sichuan University West China Hospital between December 2017 and August 2022 were enrolled. A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS. The unweighted Cohen's kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6. Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS. A total of 85 patients were enrolled, of which 22.4% (19/85) had stage III disease and 56.5% had stage IV disease. Forty-four patients (51.8%) showed consistent gene mutation types between ctDNA-based and tissue-based NGS, while one patient (1.2%) tested negative in both approaches. Advanced diseases and metastases to other organs would be fit for the ctDNA-based NGS, and the generalized linear model showed that T stage, M stage, and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS. ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS, and it could be considered in advanced or metastatic NSCLC.

Loss of copy numbers of retrotransposons (HERVK) on chromosome 7p11.2 impacts EGFR (Epidermal Growth Factor Receptor)-induced phenotypes for platinum sensitivity and long-term survival in ovarian cancer-A study from the OVCAD consortium.

We analyzed variations in the epidermal growth factor receptor (EGFR) gene and 5'-upstream region to identify potential molecular predictors of treatment response in primary epithelial ovarian cancer. Tumor tissues collected during debulking surgery from the prospective multicenter OVCAD study were investigated. Copy number variations in the human endogenous retrovirus sequence human endogenous retrovirus K9 (HERVK9) and EGFR Exons 7 and 9, as well as repeat length and loss of heterozygosity of polymorphic CA-SSR I and relative EGFR mRNA expression were determined quantitatively. At least one EGFR variation was observed in 94% of the patients. Among the 30 combinations of variations discovered, enhanced platinum sensitivity (n = 151) was found dominantly with HERVK9 haploidy and Exon 7 tetraploidy, overrepresented among patients with survival ≥120 months (24/29, p = .0212). EGFR overexpression (≥80 percentile) was significantly less likely in the responders (17% vs. 32%, p = .044). Multivariate Cox regression analysis, including age, FIGO stage, and grade, indicated that the patients' subgroup was prognostically significant for CA-SSR I repeat length <18 CA for both alleles (HR 0.276, 95% confidence interval 0.109-0.655, p = .001). Although EGFR variations occur in ovarian cancer, the mRNA levels remain low compared to other EGFR-mutated cancers. Notably, the inherited length of the CA-SSR I repeat, HERVK9 haploidy, and Exon 7 tetraploidy conferred three times higher odds ratio to survive for more than 10 years under therapy. This may add value in guiding therapies if determined during follow-up in circulating tumor cells or circulating tumor DNA and offers HERVK9 as a potential therapeutic target.

Prospects of liquid biopsy in the prognosis and clinical management of gastrointestinal cancers

Gastrointestinal (GI) cancers account for one-fourth of the global cancer incidence and are incriminated to cause one-third of cancer-related deaths. GI cancer includes esophageal, gastric, liver, pancreatic, and colorectal cancers, mostly diagnosed at advanced stages due to a lack of accurate markers for early stages. The invasiveness of diagnostic methods like colonoscopy for solid biopsy reduces patient compliance as it cannot be frequently used to screen patients. Therefore, minimally invasive approaches like liquid biopsy may be explored for screening and early identification of gastrointestinal cancers. Liquid biopsy involves the qualitative and quantitative determination of certain cancer-specific biomarkers in body fluids such as blood, serum, saliva, and urine to predict disease progression, therapeutic tolerance, toxicities, and recurrence by evaluating minimal residual disease and its correlation with other clinical features. In this review, we deliberate upon various tumor-specific cellular and molecular entities such as circulating tumor cells (CTCs), tumor-educated platelets (TEPs), circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), exosomes, and exosome-derived biomolecules and cite recent advances pertaining to their use in predicting disease progression, therapy response, or risk of relapse. We also discuss the technical challenges associated with translating liquid biopsy into clinical settings for various clinical applications in gastrointestinal cancers.

Dynamic changes in tumor profiling reveal intra- and inter-tumoral heterogeneity focused on an uncharacterized HER2 mutation: a case report of a young breast cancer patient

Despite multiple recent advances in systemic therapy for metastatic breast cancer, cases which display suboptimal response to guideline-driven treatment are frequently seen in the clinic. Effective options for such patients are limited, particularly in later line of therapy, and selection of optimal treatment options is essentially empirical and based largely on considerations of previous regimens received. Comprehensive cancer profiling includes detection of genetic alterations in tissue and circulating tumor DNA (ctDNA), immunohistochemistry (IHC) from re-biopsied metastatic disease, circulating tumor cells (CTCs), gene expression analysis and pharmacogenomics. The advent of this methodology and application to metastatic breast cancer, facilitates a more scientifically informed approach to identification of optimal systemic therapy approaches independent of the restrictions implied by clinical guidelines. Here we describe a case of metastatic breast cancer where consecutive comprehensive tumor profiling reveals ongoing tumor evolution, guiding the identification of novel effective therapeutic strategies.

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

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

Abstract PO2-14-04: Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor

Abstract Background The presence of residual tumor at surgery (non-pathological complete response or non-pCR) occurs in about half of TNBCs treated with neoadjuvant chemotherapy (NAC) signals chemoresistance. Although the addition of further adjuvant chemotherapy (Capecitabine/Xeloda) significantly improves RFS in these patients, the majority of patients nevertheless have a good prognosis, and thus may not require adjuvant Capecitabine. One of the best prognostic biomarkers in these patients is Residual Cancer Burden (RCB), which relies on the pathological examination of the residual tumor bed and lymph nodes obtained at surgery. Circulating tumor DNA (ctDNA) is a plasma-based biomarker, which has been shown to accurately detect minimal residual disease in breast cancer. We have previously shown that detection of ctDNA after NAC but before surgery in patients with non-pCR signals very poor prognosis, while non-detectable ctDNA is associated with an excellent prognosis. We now compare ctDNA to RCB score in TNBC patients with non-pCR. Methods In the multicenter TRICIA study, 44 TNBC patients with non-pCR after NAC in 5 hospital centers had consented to serial plasma collection prior to and after surgery. RCB score was calculated from pathology records. Whole exome sequencing (WES) was performed on DNA extracted from primary tumor biopsies or residual tumors at surgery to identify tumor-specific mutations (5 variants/patient). Digital droplet PCR (ddPCR) assays were developed for these mutations and tests were run in an academic hospital setting. Patients with at least one detectable mutation were considered ctDNA positive for a given time point. The detection of ctDNA was correlated with relapse-free survival (RFS). Results: The median follow up was 40 months. 19 of the 44 patients relapsed within 2 years after surgery. Detection of ctDNA at the end of NAC but before surgery (T1) was correlated with very poor RFS (H.R. = 0.15, p&amp;lt; 0.0001 (95% CI = 0.067-0.35)). 14/15 patients (93%) without detectable ctDNA did not recur, while 76% of patients with detectable ctDNA relapsed. The RCB score distribution was as follows: 7 RCB1, 21 RCB2 and 16 RCB3. According to RCB score, 1/7 RCB1, 11/21 RCB2 and 11/16 RCB3 patients relapsed. All but one of the relapsed patients had detectable ctDNA in at least one timepoint. All 11 RCB3 patients that relapsed had detectable ctDNA while none of the 5 RCB3 patients without relapse had detectable ctDNA. The one RCB1 patient with detectable ctDNA also relapsed. In cox regression analysis, the lack of ctDNA detection was significantly associated with excellent RFS in RCB2 (HR=0.16, p=0.003, (95% CI=0.05-.53)) and RCB3 groups (HR=0.10, p=0.0005, 95% CI= 0.03-.36). Conclusion ctDNA testing using ddPCR in an academic hospital-based setting at the post-NAC time point identifies an excellent prognostic group in TNBC patients with non-pCR and can discriminate very good from very poor prognosis in both RCB2 and RCB3 score subgroups. The detection of ctDNA may be superior to RCB scores in assessing prognosis in patients with non-pCR. Citation Format: Mark Basik, Talia Roseshter, Anna Klemantovich, Luca Cavallone, Adriana Aguilar-Mahecha, Josiane Lafleur, Cathy Lan, Oluwadara Elebute, Sarah Jenna, Jean-Francois Boileau, Manuela Pelmus. Circulating tumor DNA after neoadjuvant chemotherapy is a better prognostic test than Residual Cancer Burden in patients with triple negative breast cancer and residual tumor [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 PO2-14-04.

Abstract PO4-15-04: Differential genomic profiling of progesterone receptor (PR) negative, estrogen receptor (ER) positive HER2-negative metastatic breast cancer (MBC) through circulating tumor DNA

Abstract Background: MBC is usually classified based on ER and HER2 status. Although the absence of PR expression is commonly associated with a poorer response to endocrine therapy (ET) and an unfavorable prognosis, data regarding the underlining molecular features that occur in this subgroup are still unclear. The aim of this study was to investigate genomic differences between ER/PRpos HER2neg (PRpos) and ERpos PR/HER2neg (PRneg) MBC through circulating tumor DNA (ctDNA) profiling in a large multicenter consortium. Methods: This retrospective study analyzed a cohort of 1089 patients at Weill Cornell Medicine, Northwestern University, Massachusetts General Hospital, and Washington University in St. Louis with HER2neg MBC and ctDNA testing by Guardant360. Oncogenic pathway status (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, and PI3K) was defined based on previous research (Sanchez-Vega et al. Cell, 2018). Associations across single nucleotide variations (SNVs), copy number variations (CNVs), pathway classification, and ER/PR status were tested by multinomial logistic regression and corrected for significant clinical characteristics (i.e., lines of treatment, metastatic sites). Prognosis was analyzed through Cox regression for overall survival (OS) defined from time of ctDNA collection. Results: Among the 1089 analyzed patients, PRpos was the most represented subtype (N:580, 53%), followed by PRneg (N:300, 28%) and Triple Negative (TNBC) (N:209, 19%). In the hormone receptor positive subgroup, bone (N:663, 75%), liver (N:323, 37%) and lymph nodes (N:314, 36%) were the main sites of distant involvement. The TNBC group was exposed to significantly fewer lines of therapy (RRR:0.37, P= 0.005 for ≥5 lines). As compared to PRneg, TNBC had less frequent and PRpos had more frequent bone involvement (RRR:0.44, P&amp;lt; 0.001 and RRR:1.80, P&amp;lt; 0.001, respectively) and less frequent liver involvement (RRR:0.64, P=0.019 and RRR:0.71, P=0.020, respectively for TNBC and PRpos). After multivariable analysis, a significant difference with respect to PRneg was observed in SNVs for the PI3K and P53 pathways for TNBC (RRR 0.49 P = 0.013 and RRR 4.06 P &amp;lt; 0.001, respectively) but not for PRpos. On a single gene level, a lower incidence in CDH1 SNVs (RRR 0.09 P = 0.036) and a higher incidence of ESR1 SNVs (RRR 0.09 P = 0.036) was present in PRpos compared to PRneg. TNBC, on the other hand, was characterized by a lower prevalence of KRAS SNVs (RRR 0.09 P = 0.036) and PIK3CA SNVs (RRR 0.25 P &amp;lt; 0.001) and a higher prevalence of TP53 SNVs (RRR 0.09 P &amp;lt; 0.001). No ESR1mutations were detected in TNBC. Median OS was 15 months (mo) for TNBC, 21 mo for PRneg and 31 mo for PRpos (P &amp;lt; 0.001). The differential prognostic impact of ctDNA features on OS was then analyzed across the PRpos and PRneg subpopulations. ESR1 SNVs was prognostic for both subgroups in univariable analysis (HR 2.19 P &amp;lt; 0.001 and HR 1.96 P = 0.001 for PRpos and PRneg, respectively). In multivariable analysis corrected for lines of therapy, ESR1 SNVs retained its significance only in the PRpos subgroup (HR 1.41 P = 0.021) together with NF1 SNVs (HR 2.04 P = 0.034). On the other hand, TP53 SNVs had a significant impact on OS in the PRneg subgroup (HR 2.02 P = 0.014). Conclusions: Our study confirmed the prognostic impact of PR status in ER positive, HER2 negative MBC. Moreover, it suggested a differential profile in terms of ctDNA-detectable genomic features and their impact on survival, including well-established markers such as ESR1 SNVs, with a potential relevance for disease subtyping and future algorithms for drug development trials. Citation Format: Lorenzo Gerratana, Andrew Davis, Arielle Medford, Carolina Reduzzi, Katherine Clifton, Emily Podany, Whitney Hensing, Marko Velimirovic, Ami N. Shah, Lorenzo Foffano, Laura Munoz Arcos, Eleonora Nicolò, Charles S. Dai, Jennifer Keenan, Amir Behdad, Seth Wander, William Gradishar, Cynthia Ma, Fabio Puglisi, Aditya Bardia, Massimo Cristofanilli. Differential genomic profiling of progesterone receptor (PR) negative, estrogen receptor (ER) positive HER2-negative metastatic breast cancer (MBC) through circulating tumor DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-04.

Abstract PO4-14-10: Circulating Tumor DNA as a Biomarker for ADCs in Metastatic Breast Cancer

Abstract Background: Breast cancer is a complex disease characterized by heterogeneity, and the analysis of circulating tumor DNA (ctDNA) holds promise for capturing this heterogeneity. Recently, ctDNA testing has been employed in clinical practice to guide targeted therapies for metastatic breast cancer, particularly in cases of hormone receptor-positive disease with mutations in PIK3CA and ESR1. This approach becomes necessary when traditional tumor biopsies are inadequate for next-generation sequencing (NGS) testing or fail to capture the full extent of the cancer's heterogeneity. Moreover, the utilization of antibody-drug conjugates (ADC) in addressing tumor heterogeneity continues to expand. However, there has been no biomarker, outside of HER2 protein expression, to predict ADC response. In this project, we test the hypothesis that ctDNA can be utilized as a biomarker to predict response to novel therapies such as ADCs. Methods: We analyzed a subset of patients from the Dallas Metastatic Breast Cancer Study comprised of patients with metastatic breast cancer (n= 109) who underwent ctDNA testing using the Tempus xF liquid biopsy ctDNA sequencing panel. This panel detects 105-genes that are known oncogenic drivers and resistance mutations. The data was collected from a single academic medical center between the initial year of ctDNA collection in 2019 and 2023. Results: Among these patients, 85 (77.9%) had hormone receptor-positive disease, 13 (11.9%) had triple-negative breast cancer, and 11 (10.0%) had HER2-positive disease (including 3 (2.75%) patients with triple-positive disease). Only 22 of these patients had tissue biopsies that underwent NGS testing. Analysis of all patients revealed that the most common gene alteration was PIK3CA, which was identified in 70 patients. Specifically, the most frequently observed alteration was PIK3CA p.545K, found in 25 patients. Table 1 further details the frequency of each gene in this subset of patients. Among the patients, 34 out of 109 (31%) received an ADC, while only 8 out of 109 (7%) received immunotherapy. Patients with CDKN2A achieved the longest progression-free survival (PFS) on an ADC, with a median PFS of 7 months, whereas patients carrying mutations in TP53, PIK3R1, PIK3CA, PTEN, NF1, BRAF, RHOA, FGFR4, KRAS, TERT, GATA3, HNF1A, FB1, STOP had median PFSs of 2 months or less. Table 2 provides additional information on the response of patients with specific mutations to ADCs. Notably, only one patient received sequential ctDNA testing, and in that case, there was a gain of TP53 p.N239S following sacituzumab govitecan administration compared to the baseline ctDNA test, followed by a gain of PIK3CA p.P449_L455del after trastuzumab deruxtecan administration. Conclusions: As we observe changes in subtypes following treatment progression, researchers are actively seeking biomarkers to better characterize real-time changes in tumor biology that can predict response and resistance to treatments. Further studies are needed to identify additional genes present in ctDNA that can provide mechanistic insights into why certain patients respond favorably to ADCs while others do not. We are currently enrolling patients for a prospective study that aims to assess baseline ctDNA levels and monitor changes in variant allele frequency following treatment with ADCs and immunotherapy. Table 2 Median PFS on ADC by Gene Citation Format: Hannah Chang, Isabela Anawate, Alyssa Low, Shao-Po Huang, Julia Maues, Christine Hodgdon, Isaac Chan. Circulating Tumor DNA as a Biomarker for ADCs in Metastatic 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 PO4-14-10.

Abstract PO1-20-02: MiRaDor: A proof-of-concept study of treatment efficacy by monitoring Minimal Residual Disease (MRD) using circulating tumor DNA (ctDNA) in hormone receptor-positive/HER2-negative (HR+/HER2-) early breast cancer (EBC)

Abstract Background: Detection of MRD through ctDNA has been consistently associated with high risk of relapse. Longitudinal evaluation of ctDNA to monitor MRD is a minimally invasive tool that may predict disease recurrence and treatment response. MiRaDor aims to improve the clinical utility of ctDNA and understand its applicability in high-risk, HR+/HER2- EBC patients (pts). Trial Design: MiRaDor (NCT05708235) is a multicenter, open-label, non-comparative, phase II trial. Selection criteria include pts: a) with HR+/HER2- EBC at high risk of relapse, b) on adjuvant treatment with endocrine therapy (ET) for at least 2 years with 3 additional years of planned ET, c) no prior treatment with cyclin-dependent kinases 4/6 inhibitors or fulvestrant, and d) had surgery for their primary BC in the last 5 years. The trial has two phases, ctDNA surveillance (n=1260) and treatment (n=40-60). The surveillance phase will analyze ctDNA, with the FoundationOne® Tracker, every 3 months for the 1st year and every 6 months thereafter until ctDNA detection, end of the adjuvant ET, or completion of treatment phase accrual. Pts with positive ctDNA without radiological disease progression (PD) will be allocated to 1 of the treatment arms (n=10): A) standard ET; B) giredestrant; C) giredestrant + abemaciclib; or D) giredestrant + inavolisib (if PIK3CA mutation). Treatment will continue until evidence of PD, physician’s and/or patient’s decision, or until the end of study treatment (abemaciclib/inavolisib for up to 2 years, 5 years for ET). Tumor assessments will be periodically performed to rule out radiological PD. The primary objective is to evaluate the rate of pts with at least 90% decrease or clearance in ctDNA after 3 months of treatment. Key secondary objectives include the proportion of pts with 90% decrease in baseline ctDNA at 6, 9, and 12 months, 70%, and 50% decrease in ctDNA at 3, 6, 9, and 12 months, and treatment safety and tolerability. A cohort expansion up to 20 pts in 1 or 2 arms will occur if at 3 months a 90% ctDNA decrease is observed in at least 30% of pts and if, after 3 additional months, a 90% ctDNA decrease is maintained in at least 20% of pts. Citation Format: Antonio Llombart-Cussac, José Manuel Pérez-García, María Gion, Daniel Alcalá-López, Janet Fazal-Salom, Alexandros Lazaris, Mario Mancino, Eileen Shimizu, Susana Victorino, Rui Rui Zhang, Zuzana Machackova, Frauke Schimmoller, Matthew Wongchenko, Joseph Gligorov, Peter Schmid, Javier Cortés. MiRaDor: A proof-of-concept study of treatment efficacy by monitoring Minimal Residual Disease (MRD) using circulating tumor DNA (ctDNA) in hormone receptor-positive/HER2-negative (HR+/HER2-) early breast cancer (EBC) [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 PO1-20-02.