Cell-Free DNA Profiling for Prenatal Screening and Oncology-Directed Liquid Biopsy.

Background: It has been recognized that intratumoral heterogeneity together with therapeutic intervention-associated multiple genomic alterations added complexity for precision therapy in late-stage colorectal cancer (CRC). The clinical utility/validity of ctDNA assays have been systematically analyzed and proved under clinical settings in select cancer types with demonstrated specificity and sensitivity. Notably, cfRNA sequencing confirmed and enhanced detection sensitivity for variants with low allele frequency. In this study, plasma-based ctDNA mutation and cfRNA gene expression in patients with advanced CRC were simultaneously tested using NGS liquid biopsy panels. Methods: ctDNA and cfRNA were extracted from plasma of 25 treatment-naive patients with advanced CRC (stage III/IV), then were tested using the 600-gene PredicineATLAS ctDNA NGS assay and 180-gene PredicinePLUS cfRNA NGS assay, respectively. Various genomic alterations including single-nucleotide variation (SNV), Indel, copy number variation (CNV) and genome rearrangement are detected, along with tumor mutation burdens (TMB) evaluation/measurement. RNA-based variants and gene expression are also analyzed. Results: At ctDNA level, 96% (24/25) ctDNA samples were detected with at least 1 genomic alteration and total 265 SNV/Indels and 27 CNVs were identified across samples, including 180 genes with reported SNVs/indels and 23 genes with CNV. 20% (5/25) of the patients with RAS somatic mutations, 32% (8/25) with TP53 mutations, 8% (2/25) with APC mutations, and 8% (2/25) with PIK3CA mutations. TPM3-NTRK1 fusion was also detected in one patient. A subset of these variants detected at ctDNA level were also confirmed by cfRNA sequencing. Conclusions: Our results demonstrated the clinical utility/feasibility of simultaneous multiparametric profiling of ctDNA and cfRNA for comprehensive molecular insight/characterization in advanced-stage cancer. cfRNA-derived variant detection confirms the variants originally detected in ctDNA test and further validates the high-specificity results for report interpretation. Further investigation of exploring concordance between temporally matched tissue and plasma samples is ongoing. The ctDNA and cfRNA-based combined liquid biopsy solution provides holistic molecular overview for detecting driver mutations, monitoring efficacy/disease burden, and identifying subclonal alterations that may related to drug resistance and tumor heterogeneity. Citation Format: Zheng Feng, Danyi Wang, Dennis Merkle, Jianjun Yu, Shidong Jia, Juergen Scheuenpflug. Simultaneous multiparametric profiling of cell-free RNA (cfRNA) and cell-free tumor DNA (ctDNA) in advanced-stage colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A22.

Cell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy–based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers.What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsiesWhat is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies

Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. However, a standard blood draw yields an average of only 10 ng of cfDNA, of which DNA derived from the tumor is a small minority. Therefore, we are faced with a dilemma when utilizing the limited sample to obtain maximum information. Genetic sequencing provides information on actionable somatic mutations but detection of a few loci in a minority of the sample is challenging. Modified cytosine profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, they can be used to distinguish tissue-of-origin of the tumor. However, methods such as bisulfite sequencing, EM-seq and TAPS sacrifice genetic information (namely C->T mutations, which are the most common mutation in cancer) to measure modified cytosine. Genetic and modified cytosine data together have been shown to be more powerful for the detection of early cancer than either alone. Here we present a technology, which sequences at base resolution the complete genetic sequence integrated with modified cytosine from low nanogram amounts of cfDNA. It consists of (i) a single pre-sequencing workflow, which creates a copy of the original DNA and performs enzymatic base conversions which discriminate genetic and epigenetic states and (ii) post-sequencing data processing which resolves the resultant sequencing data to an information-rich 16-state code and derives genetic variants integrated with modified cytosine levels, within easy-to-use software. It is, in principle, compatible with any sequencing methodology and is shown here optimized for the Illumina fleet. In cfDNA we show accuracy of modC measurement is higher than that of bisulfite sequencing and EM-seq; and the accuracy of genetic sequencing is higher than that of Illumina alone. Accuracy of measurement is extremely important for liquid biopsy where tumor DNA is in the minority and observed in a small number of reads. We demonstrate the impact of varying base calling error rate on limit of detection for rare alleles in cfDNA samples. Further we show derivation of cfDNA fragment characteristics and that this is produced in combination with genetic and modC information. This further increases the signal available from cfDNA in only one workflow. We suggest this method will help advance the field of liquid biopsy towards its promise. Citation Format: Fabio Puddu, Casper K. Lumby, Nick Harding, David J. Morley, Jamie Scotcher, Robert Crawford, Jens Füllgrabe, Walraj S. Gosal, Shirong Yu, Daniel Brudzewsky, Jane Haywood, Andrada Tomoni, Philippa Burns, Joanna D. Holbrook, Paidi Creed. Refining liquid biopsy: generating more information from cell free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6602.

Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. While early work in liquid biopsy focused on the identification of actionable somatic variations at specific loci, the past decade has seen an expansion into non-genetic features, notably methylation. 5-methylcytosine (5-mC) profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, recent research has suggested that 5-hydroxymethylcytosine (5-hmC) profiles in cfDNA can be a marker for early cancer. However, a standard blood draw yields an average of only 10ng of cfDNA, presenting the dilemma of how to use limited sample to obtain maximum information. Existing methods for measuring methylation using next-generation sequencing cannot distinguish between 5-mC and 5-hmC and are limited in their ability to detect mutations, with approaches that measure both 5-mC and 5-hmC requiring two separate workflows. Hence, to measure complete genetics, 5-mC and 5-hmC using existing technologies requires three separate workflows, each of which require separate DNA input. We will present a technology which sequences at single base resolution the complete genetic sequence of input DNA fragments integrated with the modification status (unmodified, 5-mC or 5-hmC) for each CpG from low nanogram input quantities of DNA. A hairpin construct is used to create a copy of the original DNA strand. Enzymatic conversion followed by next-generation sequencing enables coupled decoding of bases across the original and copy strand, uniquely reporting one of A, C, G, T, 5-mC, or 5-hmC for each position in the input DNA fragment. Using this technology, we generated whole genome 6-letter data (measuring A, C, G, T, 5-mC, and 5-hmC at single-base resolution across the genome) on cell-free DNA extracted from plasma of healthy volunteers and patients with colorectal cancer at various stages of progression. We demonstrate how the technology can be used to compare 5-mC and 5-hmC methylomic profiles, genomics, and fragmentomics, across different stages of colorectal cancer. In particular, we show how disease progression can be marked by changes in 5-mC and 5-hmC across several loci. We propose that the ability to measure 5-mC and 5-hmC at high accuracy and single base resolution, alongside genomic and fragmentomic profiles, from a limited quantity of DNA will enable greater insight into the ctDNA in plasma and help advance the field of liquid biopsy towards fulfilling its promise. Citation Format: Tom J. Charlesworth, Fabio Puddu, Robert Crawford, Annelie Johansson, Ermira Lleshi, Aurelie Modat, Jamie Scotcher, Michael Wilson, Nicholas Harding, Jean Teyssandier, Jens Fullgrabe, Walraj Gosal, Paidi Creed. More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2299.

Simple SummaryLiquid biopsies seek to isolate tumor derived genetic material that circulates in blood plasma or cerebrospinal fluid. The less-invasive character of liquid biopsies combined with the option for serial analyses bears enormous potential for treatment monitoring or surveillance. We aimed to establish robust sampling protocols and pre-analytical workflows to allow for site independent multi-layer liquid biopsy testing. For an optimal usage of precious material, we explored sample stabilization in various conservation tubes and describe a protocol for the parallel isolation of cell-free DNA and RNA. Quantification and quality control steps were optimized for minimal sample use with both high sensitivity and reproducibility. We provide detailed step-by-step information on how to i) choose the best-suited protocol and ii) implement this in the liquid biopsy workflow. We believe that our study has potential to increase comparability of liquid biopsy approaches to bring these one step closer to routine clinical application.Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

The link between cell-free DNA, inflammation and the initiation of spontaneous labor at term

Lung cancer is the leading cause of cancer-associated deaths in North America, with the vast majority being non-small cell lung cancer (NSCLC) with a five-year survival rate of only 24%. Non-invasive discovery of biomarkers associated with early-diagnosis of NSCLC can enable precision oncology efforts using liquid biopsy-based multiomic profiling of plasma cell-free DNA. Although tissue biopsies are currently the gold standard for tumor profiling, this method presents many limitations since these are invasive, risky, and sometimes hard to obtain as well as only giving a limited tumor profile. Blood-based, biomarker directed liquid biopsies provide a less-invasive, more robust approach to interrogate both tumor- and non-tumor-derived signals. Liquid biopsies can delineate complex tumoral genomic and proteomic profiles throughout tumor evolution and are increasingly used for cancer diagnostics that enables a precision oncology approach. We intend to examine 30 stage III-IV NSCLC pre-surgical patients and collect plasma samples. Cell-free DNA (cfDNA) will be extracted from plasma, and next-generation sequencing (NGS) performed as well as comparative traditional diagnostics. Through the analysis of tumor-specific alterations used as biomarkers, including single nucleotide variants (SNVs), insertions, deletions, copy number variations (CNVs), and methylation alterations we intend to identify tumor-derived DNA—ctDNA, among the total pool of cfDNA. Analysis of cancer genotyping for diagnostic purposes will also be compared to accuracy of traditional, imaging and tissue-based techniques. Using liquid biopsies offer opportunities to improve the surveillance of cancer patients during treatment and would supplement current diagnosis and tumor profiling strategies previously not readily available in Trinidad and Tobago. There are a vast number of advantages to use molecular biomarkers like liquid biopsies in diagnosis and tumor profiling as well as to monitor cancer patients, providing early information regarding disease evolution and treatment efficacy, and reorient treatment strategies rapidly, thereby improving clinical oncology outcomes. With the development of both experimental protocols and computational methods dedicated to liquid biopsy, the implementation of multi-omics strategies into the clinical workflow, especially in an under-performing clinical diagnostic system, will benefit the clinical management of cancers including decision-making guidance and patient outcome improvement in the underserved communities of Trinidad and Tobago. Citation Format: Samuel M. West, Nicole Ramlachan. Using novel multiomic plasma profiling from liquid biopsies to identify potential signatures for disease diagnostics in late-stage non-small cell lung cancer (NSCLC) in Trinidad and Tobago [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C028.

Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Inflammatory breast cancer (IBC) currently accounts for 2% to 6% of all breast cancer cases in the United States and up to 20% of all breast cancer cases globally. IBC exhibits distinctively aggressive clinical features compared to all breast cancers, and accounts for a disproportionally high mortality rate—15% of breast cancer-related deaths in U.S. In addition, the survival rates between stage-matched IBC and non-IBC differ drastically. Therefore, we are in need of better understanding the molecular abnormalities driving IBC aggressive phenotype. We performed a study to evaluate the genomic alterations in cell free DNA (cfDNA) from plasma from 13 IBC patients, including 9 with triple-negative disease. In 6 patients, mutation analyses were also studied in tumor samples (tumor tissue or tumor cells from pleural effusions). Mutation analysis was performed by next-generation sequencing (NGS) using a unique molecular identifiers (UMI) assay and a panel of 93 breast cancer-related genes (Qiagen). The data were analyzed using the Qiagen's GeneGlobe portal and Biomedical Genomics Workbench and interpretation was performed with Qiagen's QCI. Somatic mutations detected in cfDNA samples were seen in: TP53 (7/13), RB1 (2/13), GEN1 (2/13) and EP300 (2/13); additional somatic mutations were found in PIK3CA (1/13), ERBB2 (1/13), PALB2 (1/13) and MUC16 (1/13). In 4 patients with plasma and tumor samples taken at the same time of the disease progression, complete concordance was not found in the somatic mutations detected in cfDNA and tumor cells DNA. Interestingly, in 12 of 13 IBC patients some variants were observed with high variant allele frequencies (VAF), ~50% or ~100% at a total coverage depth ≥230 reads, in the following genes: BRCA2 (1/13), RAD51D (2/13), PALB2 (1/13), RAD51C (1/13), AR (1/13) and MUTYH (1/13), which were classified as pathogenic or likely pathogenic, and BARD1 (3/13), SYNE1 (2/13), KMT2C (2/13), BRIP1 (1/13), XRCC3 (1/13), RET (1/13), APC (1/13), RAD50 (1/13) and MUC16 (1/13), classified as variants of uncertain significance. Moreover, 11 of these patients had a family history of different cancers including breast, colon, prostate, stomach, bladder, cervical cancers, and melanoma and myeloma. In the 6 patients where cfDNA and tumor samples were available, mutations with high VAF were found in both samples (100% concordance), suggesting that variants with high VAF are germline variants. These results suggest that the described germline variants could increase the risk of IBC and somatic mutation information obtained from cfDNA is complementary to that obtained from tissue samples. Studies on more samples are in progress. Citation Format: Jianming Pei, Jacqueline Talarchek, Jennifer S. Winn, Katherine Alpaugh, Massimo Cristofanilli, Sandra V. Fernandez. Genomic profiling of cell free DNA (cfDNA) from patients with inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4593.

ACOG approves new trisomy screen for high‐risk pregnancies

Follow the trace of death: methylation analysis of cell-free DNA for clinical applications in non-cancerous diseases.

To describe recent advances in the application of advanced genomic technologies towards the identification of biomarkers of prognosis and treatment response in breast cancer. Advances in high-throughput genomic profiling such as massively parallel sequencing have enabled researchers to catalogue the spectrum of somatic alterations in breast cancers. These tools also hold promise for precision medicine through accurate patient prognostication, stratification, and the dynamic monitoring of treatment response. For example, recent efforts have defined robust molecular subgroups of breast cancer and novel subtype-specific oncogenes. In addition, previously unappreciated activating mutations in human epidermal growth factor receptor 2 have been reported, suggesting new therapeutic opportunities. Genomic profiling of cell-free tumor DNA and circulating tumor cells has been used to monitor disease burden and the emergence of resistance, and such 'liquid biopsy' approaches may facilitate the early, noninvasive detection of aggressive disease. Finally, single-cell genomics is coming of age and will contribute to an understanding of breast cancer evolutionary dynamics. Here, we highlight recent studies that employ high-throughput genomic technologies in an effort to elucidate breast cancer biology, discover new therapeutic targets, improve prognostication and stratification, and discuss the implications for precision cancer medicine.

Background: Targeted therapies are increasingly under evaluation in clinical trials for the treatment of biliary tract cancer (BTC), and cell-free tumor DNA assays are an emerging technology for the detection of actionable alterations. Methods: ctDNA analysis was performed in patients with BTC using the CLIA-certified Guardant360 liquid biopsy assay as part of routine clinical care. The assay screens up to 73 genes for single-nucleotide variants (SNVs), 23 for insertions or deletions (INDELs), 18 for copy number variations (CNVs), and 6 for fusions. Results: 855 samples were analyzed from 751 unique BTC patients. Median age was 64 years (range, 29-89), 53% were female, and 631 (84%) had at least one alteration in their first sample. In the 751 patients, the most common genes altered were TP53 (39%), KRAS (15%), PIK3CA (13%), ARID1A (13%), EGFR (11%), FGFR2 (11%), ERBB2 (11%), NF1 (10%), IDH1 (10%), APC (9%), BRAF (9%), MYC (8%), MET (7%), CCNE1 (7%), and FGFR1 (7%). Alterations in DNA damage response genes were also seen, including BRCA2 (6%), BRCA1 (5%), ATM (4%), and MLH1 (1%). The median number of alterations per sample was 3 (range, 0-40). In a clinically annotated dataset from the Massachusetts General Hospital Cancer Center, 112 samples were obtained from 61 unique patients. Among these, 52 (85%) had intrahepatic cholangiocarcinoma (ICC), 3 (5%) had extrahepatic cholangiocarcinoma, and 6 (10%) had gallbladder cancer. 20/61 (33%) patients were treated with targeted therapy. Among 20 patients with both ctDNA and tumor sequencing results, the targetable mutation was found in both ctDNA and tumor tissue in 7/20 (35%), solely in ctDNA in 1/20 (5%), and solely in tissue in 12/20 (60%) patients. The patient with the alteration detected solely in ctDNA had insufficient tissue for analysis; she was found to have an FGFR2 F276C mutation and benefited for 8.5 months from an FGFR inhibitor on a clinical trial. Of the 12 patients whose targetable mutation was not detected in plasma, 10 (83%) had an FGFR2 fusion, 1 had an INDEL in FGFR2 (Leu262_Ala266delinsAsp), and 1 had an FGFR3 splice site mutation. 15/20 (75%) patients treated with targeted therapy were followed with serial ctDNA analysis, and a potential resistance mechanism was identified in 5/15 (33%). All 5 patients had FGFR2-fusion positive ICC and developed mutations in the FGFR2 kinase domain as a potential resistance mechanism to FGFR inhibition, including the FGFR2 V564F gatekeeper mutation in 4/5 patients. Conclusions: In patients with BTC, ctDNA-based genomic profiling can detect alterations in IDH, FGFR, PIK3CA, ERBB2, BRAF, and MET. The technology is still evolving for the detection of FGFR2 fusions, which is hindered in capture-based methods in plasma by the large number of unique breakpoints and fusion partners. ctDNA analysis may also provide a noninvasive mechanism to study resistance to targeted therapy and clonal dynamics in BTC. Citation Format: Lipika Goyal, Robin Kate Kelley, Lesli Kiedrowski, Daniel Catenacci, Kabir Mody, Rachna Shroff, Aparna Parikh, Stephanie Reyes, Jordan R. Maurer, Ryan B. Corcoran, Paul Fanta, Mike Cusnir, Becky Nagy, Richard Lanman, Michael Morse, Benjamin Tan, Milind Javle, Andrew Zhu. Blood-based genomic profiling of cell-free tumor DNA (ctDNA) in patients with biliary tract cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A183.

Colorectal cancer (CRC) is a worldwide health burden, with nearly 1.2 million new cases expected each year. Significant efforts are currently being employed to find patients who will benefit from chemotherapy. In fact, drug resistance is a limiting factor for the efficacy of chemotherapy in CRC treatment. In the present work, we aimed at identifying molecular markers in plasma cell-free DNA that could be associated with good/poor response and chemoresistance, comparing good responders (i.e., benefitting from chemotherapy, not having side effects like toxicity, no relapses, and having a complete response with no residual cancer) from non-/poor responders (patients with chemoresistance and lacking any response, or developing toxicity). Paired plasma samples from 10 colon cancer patients collected before and after adjuvant 5-fluorouracil (5-FU)-based therapy were profiled by a whole-exome sequencing (WES) approach in order to identify mutations associated with therapy response. In concomitance, DNA from tumor tissue from the same patients was also profiled by WES for mutation burden concordance. Analyses were performed on colon cancer patients treated with the same 5-FU-based therapy. The first sampling was performed at the time of the diagnosis (i.e., active disease), and the second after 6-9 months depending on patients’ conditions (i.e., covering the tumor resection, administration of adjuvant chemotherapy, etc.). After follow-up, 3 patients were clinically classified as “good responders” and 7 as “non-/poor responders.” All prepared libraries passed the quality control checkpoints and were sequenced. The number of total reads exceeds 2,57 × 109. Mean read length is about 100 nt. Alignment to human reference genome (hg38) is > 99.8%. The bioinformatics analyses are currently ongoing to profile the two different groups over time. Detailed results of the study will be presented during the meeting. The significance of the project lies in the improvement of the diagnosis and therapy efficacy in colon cancer patients. Supported by grant AZV MZ 17-30920A. Citation Format: Veronika Vymetalkova, Barbara Pardini, Marketa Urbanova, Klara Cervena, Pavel Vodicka, Alessio Naccarati. Cell-free tumor DNA profiling of colon cancer patients: Searching for mechanisms of chemoresistance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A35.

Noninvasive inferring expressed genes and in vivo monitoring of the physiology and pathology of pregnancy using cell-free DNA