Liquid Biopsy Analysis Research Articles

Investigating the Retained Inhibitory Effect of Cobimetinib against p.P124L Mutated MEK1: A Combined Liquid Biopsy and in Silico Approach.

Simple SummaryThis work focuses on the peculiar contribution made by molecular dynamics simulation and in silico tools, in the choice of an effective second line therapy for a BRAF-mutated melanoma patient who developed resistance to the undergoing targeted therapy with BRAF/MEK inhibitors. Among the MEK inhibitors, we identified a drug alternative to trametinib, able to block the target even in the presence of a damaging mutation, and supported these findings, gathered by an in silico approach, with a liquid biopsy tracking of the response to treatment. The evolution of the disease, before and after the therapy change, was followed by analysis of the circulating tumor DNA and circulating melanoma cells.The systemic treatment of metastatic melanoma has radically changed, due to an improvement in the understanding of its genetic landscape and the advent of targeted therapy. However, the response to BRAF/MEK inhibitors is transitory, and big efforts were made to identify the mechanisms underlying the resistance. We exploited a combined approach, encompassing liquid biopsy analysis and molecular dynamics simulation, for tracking tumor evolution, and in parallel defining the best treatment option. The samples at different time points were collected from a BRAF-mutant melanoma patient who developed an early resistance to dabrafenib/trametinib. The analysis of the circulating tumor DNA (ctDNA) identified the MEK1 p.P124L mutation that confers resistance to trametinib. With an in silico modeling, we identified cobimetinib as an alternative MEK inhibitor, and consequently suggested a therapy switch to vemurafenib/cobimetinib. The patient response was followed by ctDNA tracking and circulating melanoma cell (CMC) count. The cobimetinib administration led to an important reduction in the BRAF p.V600E and MEK1 p.P124L allele fractions and in the CMC number, features suggestive of a putative response. In summary, this study emphasizes the usefulness of a liquid biopsy-based approach combined with in silico simulation, to track real-time tumor evolution while assessing the best treatment option.

Network analysis of hepatocellular carcinoma liquid biopsies augmented by single-cell sequencing data.

Liquid biopsy, the analysis of body fluids, represents a promising approach for disease diagnosis and prognosis with minimal intervention. Sequencing cell-free RNA derived from liquid biopsies has been very promising for the diagnosis of several diseases. Cancer research, in particular, has emerged as a prominent candidate since early diagnosis has been shown to be a critical determinant of disease prognosis. Although high-throughput analysis of liquid biopsies has uncovered many differentially expressed genes in the context of cancer, the functional connection between these genes is not investigated in depth. An important approach to remedy this issue is the construction of gene networks which describes the correlation patterns between different genes, thereby allowing to infer their functional organization. In this study, we aimed at characterizing extracellular transcriptome gene networks of hepatocellular carcinoma patients compared to healthy controls. Our analysis revealed a number of genes previously associated with hepatocellular carcinoma and uncovered their association network in the blood. Our study thus demonstrates the feasibility of performing gene co-expression network analysis from cell-free RNA data and its utility in studying hepatocellular carcinoma. Furthermore, we augmented cell-free RNA network analysis with single-cell RNA sequencing data which enables the contextualization of the identified network modules with cell-type specific transcriptomes from the liver.

Monitoring Somatic Genetic Alterations in Circulating Cell-Free DNA/RNA of Patients with "Oncogene-Addicted" Advanced Lung Adenocarcinoma: A Real-World Clinical Study.

Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases—641 plasma samples from 57 patients—collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.

Serial Analysis of Gene Mutations and Gene Expression during First-Line Chemotherapy against Metastatic Colorectal Cancer: Identification of Potentially Actionable Targets within the Multicenter Prospective Biomarker Study REVEAL.

Simple SummaryThe emergence of resistant cells remains a major obstacle for chemotherapy treatment of metastatic colorectal cancers. Improvement of the therapeutic response requires a thorough understanding of the mechanisms of resistance as well as informative biomarkers. In the REVEAL study, we have systematically compared the mutational patterns and expression profiles of primary tumor specimens before and after first-line chemotherapy treatment in the metastatic situation. In addition, we analyzed liquid biopsies pre, during, and after treatment. Alterations in gene expression appeared as the major drivers of chemotherapy resistance. We identified a gene expression signature differentiating primary tumors and metastases and validated this signature in two independent patient cohorts. Moreover, we evaluated the expression of two signature genes, SFRP2 and SPP1, as prognostic and potentially druggable biomarkers.Most metastatic colorectal cancer (mCRC) patients succumb to refractory disease due to secondary chemotherapy resistance. To elucidate the molecular changes associated with secondary resistance, we recruited 64 patients with mCRC and hepatic metastases before standard first-line chemotherapy between 2014 and 2018. We subjected DNA from primary tumor specimens (P), hepatic metastasis specimens after treatment (M), and liquid biopsies (L) taken prior to (pre), during (intra), and after (post) treatment to next generation sequencing. We performed Nanostring expression analysis in P and M specimens. Comparative bioinformatics and statistical analysis revealed typical mutational patterns with frequent alterations in TP53, APC, and KRAS in P specimens (n = 48). P and pre-L (n = 42), as well as matched P and M (n = 30), displayed a similar mutation spectrum. In contrast, gene expression profiles classified P (n = 31) and M (n = 23), distinguishable by up-regulation of immune/cytokine receptor and autophagy programs. Switching of consensus molecular subtypes from P to M occurred in 58.3% of cases. M signature genes SFRP2 and SPP1 associated with inferior survival, as validated in an independent cohort. Molecular changes during first-line treatment were detectable by expression profiling rather than by mutational tumor and liquid biopsy analyses. SFRP2 and SPP1 may serve as biomarkers and/or actionable targets.

Cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n=3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.

Partial advances in the diagnosis and treatment of gastrointestinal cancer.

Partial advances in the diagnosis and treatment of gastrointestinal cancer.

Gastric cancer and genomics: review of literature

Gastric cancer (GC) is a major health concern in many countries. GC is a heterogeneous disease stratified by histopathological differences. However, these variations are not used to determine GC management. Next-generation sequencing (NGS) technologies have become widely used, and cancer genomic analysis has recently revealed the relationships between various malignant tumors and genomic information. In 2014, studies using whole-exome sequencing (WES) and whole-genome sequencing (WGS) for GC revealed the entire structure of GC genomics. Genomics with NGS has been used to identify new therapeutic targets for GC. Moreover, personalized medicine to provide specific therapy for targets based on multiplex gene panel testing of tumor tissues has become of clinical use. Recently, immune checkpoint inhibitors (ICIs) have been used for GC treatment; however, their response rates are limited. To predict the anti-tumor effects of ICIs for GC and to select patients suitable for ICI treatment, genomics also provides informative data not only of tumors but also of tumor microenvironments, such as tumor-infiltrating lymphocytes. In therapeutic strategies for unresectable or recurrent malignant tumors, the target is not only the primary lesion but also metastatic lesions, and metastatic lesions are often resistant to chemotherapy. Unlike colorectal carcinoma, there is a heterogeneous status of genetic variants between the primary and metastatic lesions in GC. Liquid biopsy analysis is also helpful for predicting the genomic status of both primary and metastatic lesions. Genomics has become an indispensable tool for GC treatment and is expected to be further developed in the future.

Efficacy of Ipilimumab vs FOLFOX in Combination With Nivolumab and Trastuzumab in Patients With Previously Untreated ERBB2-Positive Esophagogastric Adenocarcinoma

In metastatic esophagogastric adenocarcinoma (EGA), the addition of programmed cell death 1 (PD-1) inhibitors to chemotherapy has improved outcomes in selected patient populations. To investigate the efficacy of trastuzumab and PD-1 inhibitors with cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors or FOLFOX in first-line treatment of advanced ERBB2-positive EGA. This phase 2 multicenter, outpatient, randomized clinical trial with 2 experimental arms compared with historical control individually was conducted between March 2018 and May 2020 across 21 German sites. The reported results are based on a median follow-up of 14.3 months. Patients with previously untreated, metastatic ERBB2-positive (local immunohistochemistry score of 3+ or 2+/in situ hybridization amplification positive) EGA, adequate organ function, and eligibility for immunotherapy were included. Data analysis was performed from June to September 2021. Patients were randomized to trastuzumab and nivolumab (1 mg/kg × 4/240 mg for up to 12 months) in combination with mFOLFOX6 (FOLFOX arm) or ipilimumab (3 mg/kg × 4 for up to 12 weeks) (ipilimumab arm). The primary end point was survival improvement with a targeted increase of the 12-month overall survival rate from 55% (trastuzumab/chemotherapy-ToGA regimen) to 70% in each arm. A total of 97 patients were enrolled, and 88 were randomized (18 women, 70 men; median [range] age, 61 [41-80] years). Baseline Eastern Cooperative Oncology Group performance status was 0 in 54 patients (61%) and 1 in 34 patients (39%); 66 patients (75%) had EGA localized in the esophagogastric junction and 22 in the stomach (25%). Central post hoc biomarker analysis (84 patients) showed PD-1 ligand 1 (PD-L1) combined positive score of 1 or greater in 59 patients (72%) and 5 or greater in 46 patients (56%) and confirmed ERBB2 positivity in 76 patients. The observed overall survival rate at 12 months was 70% (95% CI, 54%-81%) with FOLFOX and 57% (95% CI, 41%-71%) with ipilimumab. Treatment-related grade 3 or greater adverse events (AEs) and serious AEs occurred in 29 and 15 patients in the FOLFOX arm and in 20 and 17 patients in the ipilimumab arm, respectively, with a higher incidence of autoimmune-related AEs in the ipilimumab arm and neuropathy in the FOLFOX arm. Liquid biopsy analyses showed strong correlation of early cell-free DNA increase with shorter progression-free and overall survival and emergence of truncating and epitope-loss ERBB2 resistance sequence variations with trastuzumab treatment. In this randomized clinical trial, trastuzumab, nivolumab, and FOLFOX showed favorable efficacy compared with historical data and trastuzumab, nivolumab, and ipilimumab in ERBB2-positive EGA. The ipilimumab arm yielded similar OS compared with the ToGA regimen. ClinicalTrials.gov Identifier: NCT03409848.

Mind the target: human epidermal growth factor receptor 2 in colorectal cancer.

In this article, we briefly summarise the current knowledge about human epidermal growth factor receptor 2 (HER2) alterations in colorectal cancer (CRC) and provide an overview of the latest published evidence especially regarding standardisation of detection methods/diagnostic criteria, prognostication, prediction and targeted treatments. Over the last 18 months, the results of many studies have been presented confirming the therapeutic potential of established anti-HER2 agents either as a monotherapy or in combination, as well as new anti-HER2 agents like antibody-drug-conjugates and tyrosine kinase inhibitors. Also, we have seen confirmation of the utility of liquid biopsy and ctDNA analyses as tool for HER2 detection and patient selection. Despite concerning only 5% of metastatic CRC, HER2 represents a valuable target for emerging anti-HER2 therapies that might significantly improve the outcome of these patients. Standardising HER2 detection methods/diagnostic criteria, and producing high-quality, randomised evidence are the next challenges to meet the standards of regulatory authorities and ultimately have anti-HER2 agents available for use in routine practice.

Liquid Biopsy Landscape in Patients with Primary Upper Tract Urothelial Carcinoma.

Simple SummaryThe standard of care for patients diagnosed with upper tract urothelial carcinoma (UTUC) is surgery. However, the current standard tools for the diagnosis and preoperative risk stratification of primary UTUC leave much to be desired. Using a simple blood sample, we can detect rare events, such as circulating tumor cells (CTCs) and large extracellular vesicles (LEVs), which may reveal biomarkers indicative of disease and provide insight into progression. This study demonstrates a wide variety of CTCs and LEVs that are identifiable in the blood of patients with UTUC at higher counts compared to normal donors. The data presented indicate that a blood-based liquid biopsy can detect UTUC and help guide clinical decisions.Urothelial carcinomas (UCs) are a broad and heterogeneous group of malignancies, with the prevalence of upper tract urothelial carcinoma (UTUC) being rare, accounting for only 5–10% of total malignancies. There is a need for additional toolsets to assist the current clinical paradigm of care for patients with UTUC. As a non-invasive tool for the discovery of cancer-related biomarkers, the liquid biopsy has the potential to represent the complex process of tumorigenesis and metastasis. Herein, we show the efficacy of the liquid biopsy as a source of biomarkers for detecting UTUC. Using the third-generation high-definition single-cell assay (HDSCA3.0) workflow, we investigate liquid biopsy samples collected from patients with UTUC and normal donors (NDs) to provide critical information regarding the molecular and morphological characteristics of circulating rare events. We document several important findings from the liquid biopsy analysis of patients diagnosed with UTUC prior to surgery: (1) Large extracellular vesicles (LEVs) and circulating tumor cells (CTCs) are detectable in the peripheral blood. (2) The rare-event profile is highly heterogeneous. (3) Clinical data elements correlate with liquid biopsy analytes. Overall, this study provides evidence for the efficacy of the liquid biopsy in understanding the biology of UTUC with the future intent of informing clinical decision making, ultimately improving patient outcomes.

The clinical utility of cfRNA for disease detection and surveillance: A proof of concept study in non-small cell lung cancer.

BackgroundCT scans are used in routine clinical practice for the diagnosis and treatment surveillance of non‐small cell lung cancer (NSCLC). However, more sensitive methods are desirable. Liquid biopsy analyses of RNA and DNA can offer more sensitive diagnostic approaches. Cell‐free RNA (cfRNA) has been described in several malignancies, but its clinical utility has not previously been explored.MethodsWe evaluated the clinical utility of cfRNA for early detection and surveillance of tumor disease in a proof‐of‐concept study. Using real‐time‐droplet digital polymerase chain reaction we characterized a candidate transcript (MORF4L2) in plasma samples from 41 advanced stage, 38 early stage NSCLC and 39 healthy samples. We compared its diagnostic performance with tumor markers and evaluated its utility for disease monitoring.Results MORF4L2 cfRNA was more abundant in patients than in healthy donors (p < 0.0001). Using the Youden index approach (cutoff value of 537 copies/ml was established) with a sensitivity of 0.73 (95% CI: 0.61–0.82) and a specificity of 0.87 (95% CI: 0.73–0.96). Positive and negative predictive values of 0.92 (95% CI: 0.83–0.95) and 0.59 (95% CI: 0.47–0.83) were achieved. Combination of cfRNA and Cyfra21‐1 improved its predictive value from 89.5% to 94.7%. Low baseline MORF4L2 levels were associated with better overall survival (HR:0.25, 95% CI: 0.09–0.7, p = 0.009) and progression‐free survival for patients treated with tyrosine kinase inhibitors (p = 0.011) and chemotherapy (p = 0.019). MORF4L2 profile between baseline and follow‐up mirrored radiological response and tumor dynamics better than tumor markers. cfRNA transcripts allowed monitoring tumor dynamics in patients without tumor‐reported genetic alterations.ConclusionOur data support clinical utility of cfRNA for detection and surveillance of NSCLC. Further studies with larger cohorts are required.

Abstract LB113: Genomic classification to refine prognosis in clear cell renal cell carcinoma

Abstract Renal cell carcinomas (RCC) are characterized by their heterogenous clinical outcomes, and due to their indeterminate behavior and the absence of routine biomarkers, it is difficult to identify patients who are at high-risk for relapse after curative nephrectomy. To identify genomic biomarkers for clear cell RCC (ccRCC) risk-stratification we interrogated somatic mutation status of 12 RCC-relevant genes using next-generation sequencing (NGS) in tumor-normal pairs from 943 patients with matched follow up data from the Cancer Genomics of the Kidney (CAGEKID) study. We examined associations between genomically-defined patient groups, explained below, and disease-free as well as RCC-specific survival independently in two cohorts of patients (N=469 for cohort 1; 474 for cohort 2). We used the Kaplan-Meier method with log-rank tests to compare survival functions, and Cox proportional hazards models to stratify for patient stage and age to estimate association of each group with survival. RCC-specific survival was assessed with a competing-risks method to include deaths from other causes. Within these cohorts, 76.4% of patients harbored somatic mutations in VHL, the most common driver gene in ccRCC. The most commonly mutated genes within VHL-mutated tumors were PBRM1 (39.7%), SETD2 (19%), BAP1 (14.3%), and KDM5C (8.3%). Less frequently mutated genes included ATM, COL11A1, DMD, TP53, and TRRAP (~3-5%).Among VHL-driven tumors, we identified a new genomic classifier on the basis of the number of mutations in additional RCC driver genes in the panel examined. Patients were classified based on the presence of mutations only in VHL (VHL+0), those with mutations in VHL and one other driver gene (VHL+1), two other driver genes (VHL+2), and 3 or more other driver genes (VHL≥3). We observed within both cohorts that both the risk of disease recurrence as well as RCC-specific death were associated with an increased number of mutations within this classification. When stratified for patient stage and age, the hazard-ratio for 5-year disease-free survival for VHL≥3 patients was 6.69 (p=0.000212), 4.31 for VHL+2 (p=0.000862), and 2.43 for VHL+1 (p=0.035662), compared to patients with only mutations in VHL. These observations were replicated in the second patient cohort, with hazards ratios of 4.55, 2.49, and 1.40, for VHL≥3, VHL+2, and VHL+1 classified patients respectively, indicating that risk of disease recurrence increases with the number of driver mutations. Notably, tumor mutational burden (TMB) was not significantly different between the aforementioned groups, demonstrating that our classifier is independent of TMB. We created a model based on a set of 12 RCC-relevant genes, which can predict risk of relapse for the ~80% of patients with ccRCC that are VHL-driven. This classification can be defined based on a small panel of genes, making it easily applicable to the clinic, in the context of tumor or liquid biopsy analysis. Citation Format: Kate I. Glennon, Naveen S. Vasudev, Ghislaine Scelo, Michelle Wilson, Louis Letourneau, Robert Eveleigh, Nazanin Nourbehesht, Madeleine Arseneault, Antoine Paccard, Lars Egevad, Juris Viksna, Edgars Celms, Sharon M. Jackson, Behnoush Abedi-Ardekani, Anne Y. Warren, Peter J. Selby, Sebastian Trainor, Michael Kimuli, Naeem Soomro, Adebanji Adeyoju, Poulam Patel, Magdalena B. Wozniak, Ivana Holcatova, Antonin Brisuda, Vladimir Janout, Estelle Chanudet, David Zaridze, Anush Moukeria, Oxana Shangina, Lenka Foretova, Marie Navratilova, Dana Mates, Viorel Jinga, Ljiljana Bogdanovic, Bozidar Kovacevic, Anne Cambon-Thomsen, Guillaume Bourque, Alvis Brazma, Jörg Tost, Paul Brennan, Mark Lathrop, Yasser Riazalhosseini, Rosamonde E. Banks. Genomic classification to refine prognosis in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB113.

Abstract 541: Evaluation of low-pass whole genome sequencing for the diagnosis and monitoring of patients with pediatric solid tumors

Abstract Liquid biopsy assays based on sequence analysis of cell-free circulating tumor DNA (cfDNA) have the potential to transform patient care. Recent advances, however, have primarily focused on adult patients with solid tumors from whom access to sufficient volumes of blood, urine or other bodily fluids is not a barrier to testing. For pediatric cancer patients, noninvasive procedures are of paramount importance for early diagnosis as well as effective monitoring of disease, but clinical diagnostic tests need to be optimized for low volumes of blood. Furthermore, most childhood solid tumors have a limited number of driver mutations that can be interrogated by targeted next generation sequencing (NGS) panels. We evaluated analysis of liquid biopsy using Low-Pass Whole Genome Sequencing (LP-WGS) of plasma cfDNA as a potential approach for diagnosing and monitoring pediatric solid tumors. We profiled samples from 42 patients with a variety of solid tumors including sarcoma (n = 28), germ-cell (n = 4), and renal tumors ( n = 10). cfDNA was extracted with the MagMax cell-free total nucleic acid extraction kit from 4-6ml peripheral blood collected in EDTA tubes at the time of diagnosis (if available), during and after chemotherapy, and when available, at relapse. Whole-genome sequencing libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA and paired-end sequenced on an Illumina NextSeq 500 at an average of 1x depth of coverage. Data was analyzed using iChor software. The LP-WGS data from the cfDNA samples was compared with copy number alteration (CNA) profiles of the primary tumors generated from clinical chromosomal microarray analysis (CMA) (CytoScanHD or OncoScan). The CNA profiles detected in plasma using LP-WGS were very similar to those of the primary tumors observed by CMA. Of 42 patients analyzed to date, 19 had non-informative profiles either at diagnosis or relapse. The loss of 9p and 17p, including CDKN2A and TP53 respectively, were the most common pathogenic CNAs detected in the abnormal cases. One patient’s germ cell tumor profiled with our OncoKids NGS panel had a confirmed KRAS mutation and 12p amplification that were also detected by LP-WGS in the liquid biopsy. Marked genomic instability characteristic of osteosarcoma was readily detectable by LP-WGS and an indication of response to therapy was mirrored by the absence of CNAs in the liquid biopsy specimen after treatment. Combined, these data suggest that liquid biopsy with LP-WGS may be used to monitor pediatric patients with solid tumors, from diagnosis through treatment and recurrence. Citation Format: Eirini Christodoulou, Dejerianne Ostrow, Anya Zdanowicz, Venkata Yellapantula, Cameron O'Connell, Moiz Bootwalla, Pan Yachen, Jennifer Cotter, James Amatruda, Leo Mascarenhas, Fariba Navid, Gai Xiaowu, Jaclyn Biegel. Evaluation of low-pass whole genome sequencing for the diagnosis and monitoring of patients with pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 541.

Abstract 75: Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™

Abstract Liquid biopsies are a minimally invasive sampling approach to overcome the heterogeneity of tumors and represent a valuable source of circulating tumor DNA (ctDNA) for oncological biomarker analysis. ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. Digital PCR emerged as a powerful technology for the next-generation analysis of liquid biopsies. Stilla Technologies' 6-color naica® system is an ultrasensitive, easy-to-use digital PCR platform capable of simultaneously and precisely quantifying high numbers of biomarkers in a single reaction. Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated in NSCLC. A handful of anti-EGFR Tyrosine Kinase Inhibitor therapies are approved by the FDA; however, most patients develop resistance over time to these treatments. Both monitoring primary EGFR mutations to predict treatment response and precociously detecting resistance mutations to adapt treatments promptly through ctDNA analysis stand to improve the effectiveness of NSCLC patient management. Stilla has developed a highly multiplexed 6-color Crystal Digital PCR EGFR kit detecting more than 90% of EGFR mutations described in NSCLC from ctDNA. The assay detects 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, including both activating and resistant mutations. In this work, we evaluated the sensitivity, precision and specificity of the EGFR 6-color Crystal Digital PCR assay highly specific and sensitive for the detection of EGFR mutations, with a Limit of Detection in a high background of wild-type DNA ranging from 0.30 to 0.46 cp/μL (with observed MAFs ranging from 0.06 to 0.09%). Moreover, a high concordance was observed between those EGFR results obtained for NSCLC ctDNA samples using 6-color Crystal Digital PCR and other technologies. With a rapid time to results and straightforward ctDNA analysis workflow, Crystal Digital PCR on the naica system promises ultrasensitive, highly multiplexed 6-color mutation detection, maximizing the information obtained from precious samples. Citation Format: Cécile Jovelet, Myrtille Remy, Noémie Pata-Merci, Damien Vasseur, Ludovic Lacroix, Claudia Labrador-Rached, Allison C. Mallory. Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 75.

Liquid First Is "Solid" in Naïve Non-Small Cell Lung Cancer Patients: Faster Turnaround Time With High Concordance to Solid Next-Generation Sequencing.

PurposeMolecular profiling is crucial in naïve non-small cell lung cancer (NSCLC). While tissue-based analysis is challenged by turnaround time and scarcity of tissue, there is increasing demand for liquid biopsy. We aimed to analyze the use of upfront liquid biopsy as a molecular profiling approach.MethodsThis retrospective multicenter, non-interventional study compared findings and turnaround times of liquid vs. standard-of-care (SOC) tissue-biopsy molecular profiling. The study included naïve advanced NSCLC patients with available liquid biopsy (Guardant360 CDx).ResultsA total of 42 consecutive patients (60% men; median age, 69.5 [39–87] years; 86% stage IV NSCLC) were identified between September 2017 and December 2020. Liquid-biopsy analysis provided results for all 42 patients, whereas the tissue-based analysis failed in 5 (12%) patients due to insufficient tumor samples. In 17 patients, 18 actionable driver mutations were identified. Eleven mutations were detected by both approaches (i.e., concordance of 61%), 4 only by liquid biopsy and 3 only by tissue biopsy. The median time from the molecular request to receiving the molecular solid report on the last biomarker was 21 (range: 5–66) days, whereas the median time from blood draw to the liquid-biopsy results was 10.5 (7–19) days. The median time between the availability of liquid-biopsy findings and that of the last biomarker was 5 days. Treatment changes following the liquid-biopsy results were observed in 3 (7%) patients.ConclusionPerforming liquid-biopsy upfront is feasible and accurate and allows a shorter time for treatment in NSCLC, especially when tumor tissue is scarce.

Demonstration of the value of liquid biopsy to identify metastatic breast cancer: Comparison and combination of cell and cell-free platforms.

e13004 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy analysis method including CTC characterization and ctDNA alterations in late-stage metastatic breast cancers (MBC). Methods: Blood samples were collected for cell and cell-free DNA analysis from 57 pts with MBC. 44 pts were progressing on treatment and 13 were responding to treatment. Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, and slides were bio-banked. Immunofluorescent staining and subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed using a validated NGS panel to detect class 1A ctDNA alterations. Results: Within this cohort of 57 MBC pts, CTCs, scLST+CTCs and ctDNA alterations were detected in (77%, 49% and 33%, respectively). All MBC pts on active therapy (23%) had no CTC detected. No CTCs, scLST+CTCs or ctDNA were detected in the HD cohort, suggesting high specificity (100%, 100% and 100%, respectively). The presence of CTCs or of scLST+CTCs had low concordance with the presence of ctDNA+ alterations in MBC pts, suggesting that these platforms provide independent and complementary value when identifying MBC. To test this hypothesis, we designed combination models which added ctDNA to cell analysis methods ([CTC+][ctDNA+] and [scLST+CTC+][ctDNA+]). Adding ctDNA to either cell analysis method increased sensitivity by 5% and 12%, respectively, over the individual methods and retained 100% specificity. Conclusions: Here we demonstrate the value of comprehensive cell and cell-free liquid biopsy analysis for the detection of MBC pts. Our data suggest there may be clinical utility of adding cell-free to cell analysis for the identification of MBC. Analysis of the comparative and combined utility of these liquid biopsy methods in earlier settings is ongoing.

A phase Ib, open-label, multi-cohort study of metronomic capecitabine plus camrelizumab for treatment of refractory solid tumor: McCrest trial.

e16079 Background: The efficacy of immune checkpoint inhibitors (ICIs) monotherapy for refractory solid tumor is relatively poor. Combination therapy is an effective strategy to improve its efficacy. Metronomic chemotherapy that can modulate tumor immunomicroenvironment is a potential partner for ICIs, which is also well-tolerated and low-cost. Methods: This was a phase Ib, open-label, single-center, multi-cohort study designed to evaluate the safety and efficacy of camrelizumab combined with metronomic capecitabine for refractory solid tumors. Patients who were disease progression after standard treatment could be enrolled. PD-L1 status was not compulsory. Capecitabine was administrated orally at 500mg bid every day, with camrelizumab at 200mg IV on Day 1 of a 14-day cycle. Primary endpoint was adverse events (AEs). Secondary endpoints including progression-free survival (PFS), overall survival (OS), objective response rate (ORR) and disease control rate (DCR). The exploratory endpoint was also set to identify the potential molecular alterations during treatment by liquid biopsy analysis. Results: As of December 12, 2021, 19 patients were enrolled including gastric cancer (8), esophageal cancer (6), pancreatic cancer (2), cholangiocarcinoma (2) and duodenal cancer (1). Ten patients had 2 or more prior lines of therapy. The common treatment-related AEs including reactive cutaneous capillary endothelial proliferation (10/19), fatigue (9/19), leukemia (5/19), anemia (5/19), hyponatremia (4/10) and blood bilirubin increased (4/19). Grade 3 AEs including fatigue (2/19) and bronchitis (1/19), and no grade 4 AEs was observed. Three patients suffered severe AEs, including bowel perforation (1), bowel obstruction (1) and bleeding (1), which were all treatment unrelated. There were 4 patients still under treatment, and median number of treatment cycle was 4 (1-16 cycles). The median PFS and OS was 2.1 months (95%CI 1.9-2.3 months) and 5.1 months (95%CI 1.6-8.5 months) respectively. Partial response was observed in 1 esophageal cancer patient with PD-L1 TPS &lt; 1. For 12 patients with assessable lesions, the DCR was 41.6% (5/12). Peripheral blood samples were collected during treatment, and molecular alterations after administration of metronomic capecitabine and camrelizumab were under analysis. Conclusions: The combination of metronomic capecitabine plus camrelizumab was well tolerated in patients with refractory solid tumors. Its efficacy should be assessed in further study. Clinical trial information: NCT04508686, NCT04510818, NCT04932187.

Improved sensitivity in identification of first recurrences of metastatic breast cancer with a combination of cell and cell-free liquid biopsy analysis.

e13003 Background: Liquid biopsy analysis detecting circulating tumor cells (CTC) or circulating tumor DNA (ctDNA) is a non-invasive approach that may provide clinically actionable information for patients’ (pts) treatment decisions when conventional biopsy is otherwise inaccessible or infeasible. Here we report a liquid biopsy method including CTC characterization &amp; ctDNA alterations in pts symptomatic of first metastatic recurrence with a history of primary breast cancer (BC). Methods: Blood samples from 34 symptomatic BC pts were collected for cell &amp; cell-free DNA analysis before undergoing contemporaneous tissue biopsy &amp; initiating treatment for metastatic BC (MBC). Blood was also collected from 25 blood donors (HD) with no known cancer history. After plasma isolation, nucleated cells were plated, &amp; slides were bio-banked. Immunofluorescent staining &amp; subsequent imaging were performed on replicate slides. CTCs were identified using Epic Sciences digital imaging &amp; machine learning algorithms trained to identify breast cancer cells. Single-cell (sc) isolation for genomic quantification of large-scale transitions (scLST) was used to further characterize individual CTCs. cfDNA from bio-banked plasma was analyzed to detect Class IA alterations in ctDNA. Results: Within this cohort of 34 patients, CTCs &amp; ctDNA alterations were detected in 80% &amp; 58%, respectively while no CTCs &amp; ctDNA were detected in the HD cohort suggesting high specificity (100% &amp; 100%). In this study, 26 pts were confirmed MBC (M1-BC) by tissue biopsy, 3 pts were non-MBC by tissue biopsy but still diagnosed as metastatic (Mx-BC), &amp; 5 pts were confirmed non-MBC (M0-BC). To benchmark the cellular (CTCs &amp; scLST+CTCs) &amp; genomic (ctDNA) individual platforms, we designed thresholds of CTCs &gt; 4, scLST+CTC+, &amp; ctDNA+ that optimally identified M1-BC pts with high specificity (100%, 100%, &amp; 100%) but low sensitivity (46%, 46%, &amp; 50%). CTCs &gt; 4 &amp; scLST+CTC+ cutoffs had low concordance with the presence of ctDNA alterations, suggesting these platforms provide independent &amp; complementary value when identifying MBC. To test this hypothesis, we designed combination models [CTC &gt; 4][ctDNA+] &amp; [scLST+CTC+][ctDNA+] that significantly improved sensitivity (75% &amp; 67%) over individual platforms while maintaining high specificity (100% &amp; 100%) when identifying the first recurrence of metastasis in BC pts. Conclusions: To our knowledge, this is the first report showing the additive &amp; complementary value of models combining cellular &amp; genomic liquid biopsy platforms for the detection of the first MBC recurrence. Our data suggest the clinical utility of combined CTC &amp; ctDNA platforms for the identification of MBC when tissue biopsy is inaccessible or infeasible.

Cell-free DNA in sports medicine: implications for clinical laboratory medicine

Abstract Background Physical activity can have a strong impact on the concentration of several promising candidate biomarkers, including cell-free DNA (cfDNA). Content This narrative review describes the current understanding of how physical strain leads to increases of cfDNA and discusses how this interferes with attempts to standardize cfDNA analysis in clinical laboratory medicine. Summary In general, all cells of the human body can release DNA, whereas neutrophils are described as the major source releasing cfDNA under resting conditions. Event at low physical load, cfDNA is rapidly released by immune cells. We recently, identified neutrophils as the major cell-type contributing to cfDNA increases during acute exercise. Both, endurance and strength training can affect the signal-to-noise ratio of liquid biopsy (LB) analysis, affecting the clinical validity between minutes up to several days. Furthermore, we discuss why physical distress of various kinds in a perioperative cancer setting can improve or compromise signal-to-noise. Therefore, physiological events including, but not limited to, activation of blood cells can provoke pre-analytical challenges for ultra-sensitive detection of cfDNA in LB settings. Outlook We discuss why future attempts to standardize liquid biopsy may therefore profit from a deeper understanding of the physiological release mechanisms of cfDNA.

LCMC LEADER neoadjuvant screening trial: LCMC4 evaluation of actionable drivers in early-stage lung cancers.

TPS8596 Background: Comprehensive genomic profiling (CGP) has transformed the care of patients with advanced non-small cell lung cancer (NSCLC), giving many patients access to precision targeted treatment and immunotherapy with remarkable improvements in outcomes. Studies show that patients with lung cancers with oncogenic drivers are the least likely group to benefit from checkpoint inhibitors and are better served by enrollment in studies of targeted therapies. Early-stage NSCLC is now poised to benefit from these precision approaches with the regulatory approval of the first tyrosine kinase inhibitors and checkpoint inhibitors for the adjuvant treatment of resected NSCLC, each requiring testing for precision biomarkers. Neoadjuvant precision therapy for NSCLC has the potential to further improve treatment outcomes. Methods: The LCMC4 Evaluation of Actionable Drivers in EaRly Stage Lung Cancer (LEADER) Neoadjuvant Screening Trial (NCT04712877) is a collaborative diagnostic study developed by the Lung Cancer Mutation Consortium (LCMC), supported by the Thoracic Surgery Oncology Group and the Lung Cancer Research Foundation. The primary objective is to determine the proportion of patients with stage IA2-III lung cancers who possess actionable oncogenic drivers, defined as 1 of 11 actionable genomic alterations: mutations in EGFR, BRAFV600E, MET exon 14, KRAS G12C, and HER2, rearrangements in ALK, RET, NTRK, and ROS1, and amplification of MET and HER2. The study will also assess the feasibility of CGP to detect actionable oncogenic drivers in patients with suspected early-stage lung cancers scheduled to undergo biopsies to establish the diagnosis of lung cancer. The protocol will enroll 1000 patients with operable stage IA2-III (TNM 8th edition) lung cancer who will undergo CGP utilizing the Foundation Medicine 324 gene assay as well as paired liquid biopsy analysis. Results will enable selection of neoadjuvant therapy and enrollment onto independent therapeutic trials with genomically matched neoadjuvant treatment, standard therapies, or other trials if no driver is detected. The approach will be considered feasible if &gt;35% of non-squamous NSCLCs have 1 of the 11 actionable alterations. Tumor mutational burden and PD-L1 IHC will be assessed. Plasma specimens collected pre- and post neoadjuvant treatment and post-surgery will be used for research to study the ability of circulating tumor DNA to assess neoadjuvant treatment response and minimal residual disease. 26 academic sites in the US plan to enroll patients. Clinical trial information: NCT04712877.