Liquid Biopsy In Patients Research Articles

Prognostic Value of Novel Liquid Biomarkers in Patients with Metastatic Castration-Resistant Prostate Cancer Treated with Enzalutamide: A Prospective Observational Study.

Several treatment options were recently added for metastatic castration-resistant prostate cancer (mCRPC). However, response to therapy is variable, and biomarkers that can guide treatment selection and response evaluation are lacking. Circulating RNAs are a promising source of biomarkers. We explored messenger RNAs (mRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) as potential biomarkers in liquid biopsies of patients with mCRPC treated with enzalutamide. Forty patients were included in this prospective multicenter observational study. Whole blood was drawn at baseline and 1, 3, and 6 months after start of therapy. Four mRNAs, 6 miRNAs, and 5 lncRNAs were analyzed by quantitative PCR. RNA levels in 30 healthy individuals were used as controls. RNA expression data were analyzed by Kaplan-Meier and Cox regression analyses, and the primary end point was progression-free survival. Clinical factors were included in the multivariable Cox regression analysis. Levels of 2 miRNAs, miR-375 and miR-3687, and 1 lncRNA, N-acetylated alpha-linked acidic dipeptidase like 2 antisense RNA 2 (NAALADL2-AS2), were more than 2-fold higher in patients with mCRPC compared with healthy volunteers. Patients with higher levels of miR-375 or miR-3687 showed a shorter time to progression. Patients with higher levels of NAALADL2-AS2 showed a longer time to progression. In the multivariable Cox regression analysis, higher miR-375, miR-3687 and serum prostate-specific antigen concentrations were shown to be independent predictors for shorter time to progression. We identified miR-3687 as a novel prognostic marker for response in patients with CRPC treated with enzalutamide, and we confirmed the prognostic value of miR-375.

Current concepts and future directions for hemato-oncologic diagnostics.

At present, hemato-oncologic diagnostics is facing dynamic changes. This applies to the exploration and introduction of novel technologies such as next-generation sequencing or digital droplet PCR for myeloid and lymphatic malignancies in laboratory routine, or liquid biopsy for patients with lymphoid malignancies. Targeted therapies such as FLT3 or IDH1/IDH2 inhibitors for acute myeloid leukemia are entering clinical practice. Thus, the demand for hematologic precision diagnostics both at initial diagnosis and during the course of the disease are equally increasing, and a short turn-around time becomes crucial. NGS expands the armamentarium for minimal residual disease diagnostics, but novel questions arise relating to sensitivity, the appropriate time points of this analysis, or the thresholds triggering therapeutic interventions. In this review article, we summarize some of the most relevant current changes and subsequent challenges for diagnostics in various myeloid and lymphatic malignancies. Future directions of hemato-oncologic diagnostics in the next 5-10 years are highlighted.

Detection of COX-2 in liquid biopsy in patients with breast cancer

AimsTo determine the expression of the cyclooxygenase-2 (COX-2) gene in patients with breast cancer attended at the Centro Universitário Saúde ABC/Faculdade de Medicina do ABC (CUS-ABC/FMABC) outpatient clinic. Breast cancer...

ASO Author Reflections: Prognostic Utility of Pre- and Postoperative Circulating Tumor DNA Liquid Biopsies in Patients with Peritoneal Metastases.

ASO Author Reflections: Prognostic Utility of Pre- and Postoperative Circulating Tumor DNA Liquid Biopsies in Patients with Peritoneal Metastases.

Detection of EGFR-Activating and T790M Mutations Using Liquid Biopsy in Patients With EGFR-Mutated Non–Small-Cell Lung Cancer Whose Disease Has Progressed During Treatment With First- and Second-Generation Tyrosine Kinase Inhibitors: A Multicenter Real-Life Retrospective Study

BackgroundIn advanced epidermal growth factor receptor (EGFR)-mutated non–small-cell lung cancer (NSCLC) patients whose disease has progressed during treatment with first- and second-generation tyrosine kinase inhibitors (TKIs), liquid biopsy (LB) is routinely used to evaluate the presence of EGFR T790M as an acquired resistance mechanism. The objective of this study was to assess a real-life picture of EGFR T790M detection in LB. Materials and MethodsLiquid biopsies performed between June 2016 and October 2018 for advanced EGFR-mutated NSCLC at disease progression during treatment with first- and second-generation TKIs were retrospectively evaluated in 5 Italian centers. Circulating tumor DNA was extracted from plasma and tested with different commercial kits. The detection rate in LBs and the patients’ characteristics were correlated. ResultsWe enrolled 120 consecutive patients. The overall T790M detection rate observed using LB was 25.8%. Fifty-four of 89 (60.7%) patients with negative LB results underwent tissue rebiopsy, and 56% were positive for T790M. The overall rate of T790M positivity in the study cohort was 49.2%. LB performed before formal tumor progression according to Response Evaluation Criteria In Solid Tumors criteria was negative for T790M in all patients (n = 21; P = .012). T790M positivity was statistically significantly higher in cases of disease progression at extrathoracic metastatic sites (P = .008) and, specifically, in the case of worsening bone disease (P = .003). ConclusionOur study shows that the detection of T790M-positive patients whose disease progressed during treatment with first- and second-generation TKIs in real life was according to the literature. However, this result was obtained with a specific clinical course (repeat LBs and tissue rebiopsy), thus implying the necessity for multidisciplinary management.

Abstract P5-06-28: Optimization and validation of PIK3CA mutation detection with droplet digital PCR in liquid biopsies of patients with metastatic breast cancer

Abstract Background Approximately 40% of oestrogen receptor positive (ER+) metastatic breast cancer harbour PIK3CA hotspot mutations, leading to an over-activated PI3K pathway. The PI3Kα-selective inhibitor, alpelisib, is FDA approved and currently available in early access program for patients with ER+ HER2-negative metastatic disease, if an activating PIK3CA mutation is confirmed. Mutational analysis on liquid biopsies may increase the number of eligible patients given the challenges of tissue procurement in metastatic setting. We optimized and validated ddPCR assays for PIK3CA hotspot mutations in cell-free DNA (cfDNA) of metastatic breast cancer patients. Patients and Methods We prospectively collected two blood samples (cfDNA Collection tubes, Roche Diagnostics) of 20 metastatic breast cancer patients at progression (N=12) or during therapy (N=8). PIK3CA mutation status was previously demonstrated by Next Generation Sequencing (NGS) performed in 4 patients on primary tumor tissue and in 16 patients on metastatic tumor tissue; 8 patients tested positive and 12 negative. Tumors were ER+ HER2-negative, triple-negative and HER2-positive in 14, 5 and one patients, respectively. After plasma-isolation, cfDNA was manually extracted with Cobas® cfDNA Sample Preparation Kit (2mL plasma) and semi-automatically with Maxwell® RSC ccfDNA Plasma Kit (2mL and 4mL plasma). Inter-run variability, intra-run variability, precision and robustness of the assays, and concordance between NGS and ddPCR for the detection of PIK3CA hotspot mutations on tumor tissue and in plasma, respectively, were assessed. Results All 20 samples were successfully processed with ddPCR. The highest cfDNA yield was obtained by Maxwell 4mL extraction method (median: 0.483 ng/µL; range: 0.140 - 10.500 ng/µL). The per-mutation sensitivity and specificity was 87.5% and 95.8%, respectively. Two samples showed discordant results between PIK3CA mutations detected by NGS on tumor tissue and by ddPCR in plasma. One sample tested positive for p.(H1047R) on tumor tissue, and for p.(E542K), p.(E545K) and p.(H1047R) in cfDNA. The other switched between p.(E545K) mutation on tumor tissue and p.(E542K) in cfDNA. Tumor tissue of samples for which the results of the NGS test were discordant with the ddPCR test on plasma, was tested again using ddPCR on the tissue. This still showed discordances in PIK3CA mutations which can be explained by tumor heterogeneity and the lower detection limit of ddPCR versus NGS. Conclusion Detection of PIK3CA hotspot mutations with ddPCR in cfDNA is feasible. We observed good concordance between NGS and ddPCR for the detection of PIK3CA hotspot mutations on tumor tissue and in plasma, respectively. In 10% of cases, discordant PIK3CA mutation status in tissue versus plasma was detected. Further investigation of different metastatic lesions in these cases is ongoing. Citation Format: Laurence Slembrouck, Demi Renders, Sara Vander Borght, Patrick Neven, Giuseppe Floris, Lien Spans, Hans Wildiers, Kevin Punie, Ann Smeets, Ines Nevelsteen, Ignace Vergote, Adriaan Vanderstichele, Isabelle Vanden Bempt. Optimization and validation of PIK3CA mutation detection with droplet digital PCR in liquid biopsies of patients with metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-28.

Emergence of RAS mutations in patients with metastatic colorectal cancer receiving cetuximab-based treatment.

208 Background: In management of patients with RAS wild-type mCRC, anti-EGFR therapies have demonstrated clinical benefit. However, the correlation between emergence of RAS mutations in circulating tumor DNA (ctDNA) and secondary resistance to anti-EGFR therapies requires further elucidation. In this study, we aim to examine evolutionary changes in RAS mutations through liquid biopsy in patients with mCRC during and after anti-EGFR therapy (NCT03401957). Methods: A total of 39 intention-to-treat (ITT) patients diagnosed with RAS wild-type mCRC, from Cathay General Hospital (CGH) and Kaohsiung Medical University Hospital (KMUH), were analyzed in this study. Of them, 24 were male and 15 were female, with median age of 55 years. Patients received cetuximab-based FOLFIRI or FOLFOX regimen as first-line treatment. Blood samples from enrolled patients were collected before and every 3 months during cetuximab-based treatment and also at disease progression. These blood samples were evaluated for RAS resistance mutations using MassARRAY platform. The primary endpoint is percentage of RAS mutations detected in ctDNA from patients during cetuximab treatment. The correlation between the tumor response of these patients and the emergence of RAS mutations in ctDNA is further analyzed. Results: Between January 2018 and July 2019, 3 (8.3%) of 36 per-protocol (PP) patients subsequently with newly detected ctDNA KRAS/NRAS mutations in their blood samples during the treatment period, and all of three patients developed progressive disease (PD). Of 36 PP patients, 19 (52.8%) patients had complete/partial response (CR/PR), 13 (36.1%) patients had stable disease (SD) and 4 (11.1%) patients had PD. The disease control rate was 88.9% in the present study. Three of 4 PD patients were detected with ctDNA KRAS/NRAS mutations in their blood sample, with time to onset of newly detected ctDNA RAS at 3 and 6 months posttreatment, respectively. Conclusions: In current study, the frequency of RAS mutations in blood samples during the treatment period was 8.3% and all of these 3 patients developed PD. The results of this study will offer substantial, valuable information for anti-EGFR therapeutic strategy in patients with mCRC.

Liquid biopsy in peritoneal fluid and plasma as a prognostic factor in advanced colorectal and appendiceal tumors after complete cytoreduction and hyperthermic intraperitoneal chemotherapy.

Background:Positive cytology has been identified as an independent negative prognostic factor in patients with peritoneal metastases (PM) of colorectal origin. Liquid biopsy in plasma may detect increasing levels of circulating tumor DNA (ctDNA) and could help predict systemic relapse in patients with colorectal cancer, but little is known about the role of liquid biopsy in peritoneal fluid. The aim of this study was to evaluate the prognostic value of peritoneal fluid and plasma liquid biopsy in patients undergoing complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CC-HIPEC).Methods:A longitudinal prospective study was designed in patients with KRAS-mutated colorectal or appendiceal primary tumor, including PM of colorectal origin, pseudomyxoma peritonei and patients at high risk of developing PM (selected for second-look surgery). Eleven patients were recruited according to inclusion and exclusion criteria. ctDNA from plasma and peritoneal fluid before and after HIPEC was studied by droplet digital PCR looking for KRAS mutation. A close follow-up was scheduled (mean of 28.5 months) to monitor for systemic and peritoneal recurrences.Results:All patients with positive plasma postHIPEC had systemic relapse and four patients died as a result, while those with negative plasma postHIPEC did not relapse. Patients with negative peritoneal ctDNA after CC-HIPEC did not present peritoneal relapse. Of six patients with positive peritoneal ctDNA postHIPEC, two presented peritoneal recurrence and four systemic relapses.Conclusions:Treatment with CC-HIPEC does not always neutralize ctDNA in peritoneal fluid, and its persistence after treatment may predict adverse outcome. Despite being a proof of concept, an adequate correlation between liquid biopsy in plasma and peritoneal fluid with both systemic and peritoneal relapse has been observed.

Concordance of epidermal growth factor receptor mutation detection in bodily fluids other than blood with tissue biopsy: A retrospective analysis

Background: The concept of liquid biopsy as a surrogate for tumor tissue is not only restricted to blood-derived samples but also includes other malignant bodily fluids such as cerebrospinal fluid, pleural fluid, pericardial fluid, and ascites. The application of liquid biopsy in patients with metastatic lung cancer, with disease progression on epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment, is unexplored in India.Objective: We aimed to determine the presence of EGFR gene mutations in various bodily fluids of patients with EGFR-mutant progressive metastatic lung adenocarcinoma.Materials and Methods: In this retrospective study conducted at the Tata Memorial Hospital in Mumbai, India, data from 11 patients with advanced EGFR-mutant lung adenocarcinoma were analyzed. Between December 2017 and June 2018, the cell-free DNA (cfDNA) obtained from the malignant bodily fluids and plasma of 11 patients were tested for the presence of EGFR-sensitizing mutations using real-time reverse transcription-polymerase chain reaction. The status of EGFR-sensitizing mutations in plasma and other bodily fluids was compared with that of the baseline tissue biopsy. In addition, the status of the acquired T790M resistance mutation in exon 20 at disease progression was determined using plasma and other bodily fluids.Results: Of the 11 patients, at disease progression, EGFR sensitizing mutations (exon 19 deletion and exon 21 L858R insertion) could be detected in the plasma of 7 (63%) and in other bodily fluids of all the 11 (100%) patients. Thus, the concordance for EGFR-sensitizing mutation detection in the plasma and other bodily fluids with the baseline formalin-fixed paraffin-embedded tissue was 63% and 100%, respectively. In addition, the acquired T790M resistance mutation was detected in the plasma of 3 (27.3%) patients and other bodily fluids of 9 (81.8%) patients.Conclusion: The rate of detection of the T790M mutation is higher in the free DNA obtained from malignancy-involved bodily fluid supernatant as compared to the plasma cfDNA. Our results suggest that cfDNA obtained from bodily fluids other than plasma can be used for the detection of EGFR mutations and that the mutation status may be a useful predictor of the response to TKIs.

Results of Liquid Biopsy Studies by Next Generation Sequencing in Patients with Advanced Stage Non-small Cell Lung Cancer: Single Center Experience from Turkey.

Several studies demonstrated the utility of plasma-based cell-free circulating tumor DNA (ccfDNA) in determination of mutations in non-small cell lung cancer (NSCLC). We aimed to report our results of next generation sequencing (NGS) using liquid biopsy in patients with NSCLC. Patients with advanced stage NSCLC were enrolled and their genomic profiling results were recorded. Next generation sequencing targeted panel includes 19 hot-spot genes. The plasma was separated from the peripheral blood sample and ccfDNAs were isolated for NGS. We performed genomic profiling in 100 patients (20 females and 80 males) with a median age of 59.3 (range 26-79). A second liquid biopsy was performed in eight patients who developed progressive disease after the first treatment. The study population had adenocarcinoma (AC) (n = 73), squamous cell carcinoma (SCC) (n = 14), or NSCLC-NOS (not otherwise specified) (n = 13). In the SCC group, three of 14 patients had variants on EGFR and MET genes. In the AC and NSCLC-NOS groups, 39 out of 86 patients (45.3%) had variants. The most common one was in the EGFR gene (n = 27, 31.4%) including seven mutations related to drug resistance and two were polymorphisms. Three patients had both driver and resistance mutations (EGFR T790M, n = 2; KRAS exon 2 G12S and MET exon 14 E1012K, n = 1). Fifteen patients (17.4%) had an activating EGFR mutation and eight patients (9.3%) had variants in the KRAS gene. We reported our results regarding genomic profiling related to treatment using liquid biopsy in patients with NSCLC. Advantages of this method are the non invasiveness and reproducibility.

High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery.

Purpose: Expediting the diagnosis of pancreatic ductal adenocarcinoma (PDAC) would benefit care management, especially for the start of treatments requiring histological evidence. This study evaluated the combined diagnostic performance of circulating biomarkers obtained by peripheral and portal blood liquid biopsy in patients with resectable PDAC. Experimental design: Liquid biopsies were performed in a prospective translational clinical trial (PANC-CTC #NCT03032913) including 22 patients with resectable PDAC and 28 noncancer controls from February to November 2017. Circulating tumor cells (CTCs) were detected using the CellSearch® method or after RosetteSep® enrichment combined with CRISPR/Cas9-improved KRAS mutant alleles quantification by droplet digital PCR. CD63 bead-coupled Glypican-1 (GPC1)-positive exosomes were quantified by flow cytometry. Results: Liquid biopsies were positive in 7/22 (32%), 13/22 (59%), and 14/22 (64%) patients with CellSearch® or RosetteSep®-based CTC detection or GPC1-positive exosomes, respectively, in peripheral and/or portal blood. Liquid biopsy performance was improved in portal blood only with CellSearch®, reaching 45% of PDAC identification (5/11) versus 10% (2/22) in peripheral blood. Importantly, combining CTC and GPC1-positive-exosome detection displayed 100% of sensitivity and 80% of specificity, with a negative predictive value of 100%. High levels of GPC1+-exosomes and/or CTC presence were significantly correlated with progression-free survival and with overall survival when CTC clusters were found. Conclusion: This study is the first to evaluate combined CTC and exosome detection to diagnose resectable pancreatic cancers. Liquid biopsy combining several biomarkers could provide a rapid, reliable, noninvasive decision-making tool in early, potentially curable pancreatic cancer. Moreover, the prognostic value could select patients eligible for neoadjuvant treatment before surgery. This exploratory study deserves further validation.

Phase II Study Evaluating the Mechanisms of Resistance on Tumor Tissue and Liquid Biopsy in Patients With EGFR-mutated Non-pretreated Advanced Lung Cancer Receiving Osimertinib Until and Beyond Radiologic Progression: The MELROSE Trial

BackgroundOsimertinib, a third-generation tyrosine kinase inhibitor, is a new therapeutic option in epidermal growth factor receptor (EGFR)-mutated non-pretreated advanced non–small-cell lung cancer (NSCLC). The tumor escape mechanisms after first-line treatment with osimertinib are partially known; most of the data being obtained by analysis of circulating tumor DNA (ctDNA) from the FLAURA phase III trial. Study DesignThe MELROSE study, a French multicentric, open label, phase II trial (ClinicalTrials.govNCT03865511) plans to enroll 150 patients with treatment-naive advanced EGFR-mutated (L858R or exon 19 deletion) NSCLC, age ≥ 18 years, with an Eastern Cooperative Oncology Group performance status 0 or 1. All patients will receive osimertinib at the dose of 80 mg/d. Tumor assessment according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria will be performed every 3 months, with brain and thoracoabdominal computed tomographic scan. The continuation of osimertinib is at the discretion of the referring physician, particularly if clinical benefit is observed. The primary objective is the genetic tumor profile, both on tissue biopsy and ctDNA analyses, at the time of disease progression. Other endpoints include kinetic studies of ctDNA, biological progression-free survival (bPFS) (time from first study dose to first biological event on ctDNA), median PFS according to RECIST criteria 1.1 (called radiological [r] PFS), and median clinical (c) PFS (time from the first study dose to off-osimertinib). This study started in April 2019, and 18 centers in France are participants.

P1.01-21 Sputum Can Serve as an Alternative Source for Liquid Biopsy in Patients with Lung Cancer

P1.01-21 Sputum Can Serve as an Alternative Source for Liquid Biopsy in Patients with Lung Cancer

Abstract 1362: Genome-wide sequencing of cell-free DNA enables detection of copy number alterations in cancer patients where tissue biopsy is not feasible

Abstract In instances where tissue biopsy is not medically prudent or tumor tissue material is insufficient for molecular testing, alternative methods are needed. Since cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this scenario. Here we present data from 91 patients with known neoplasms whose tumors were not able to be tested using conventional tissue biopsy. The cfDNA from each patient was assayed with low-coverage (~0.3X) genome-wide sequencing and copy number alteration (CNA) events were identified and characterized using analytical methods originally developed for noninvasive prenatal testing (NIPT). To quantify the level of CNAs present in the plasma of cancer patients, we utilized the genomic instability number (GIN). First, the technical variability of the GIN was evaluated in a large sample cohort. We therefore processed genome-wide sequencing results from 27,742 pregnant women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs. Utilizing these data, we established a threshold whereby we could differentiate technical noise from biological signal when CNAs were present down to a 1% ctDNA fraction with 99.7% specificity. Applied broadly, CNAs were detected in 32 of the 91 patients with difficult-to-biopsy cancers evaluated (35%) with detection frequencies tightly linked to cancer type. Importantly, there was a significant reduction in progression-free survival for the first treatment after liquid biopsy for patients with an elevated GIN relative to those without, suggesting a prognostic link between GIN and patient outcome across a wide variety of tumor types. In addition, samples with an elevated GIN also showed a significant concomitant reduction in cfDNA fragment length, consistent with previous reports suggesting that cfDNA derived from tumors is shorter in length than cfDNA from healthy tissue. Finally, we characterized the genomic location of the detected CNAs to potentially enable the utility and interpretation of these results clinically. Collectively, the detected CNAs covered more than 95% of all autosomes, highlighting a benefit of using genome-wide sequencing. Taken together, these data demonstrate a proof-of-concept for using low-coverage, genome-wide sequencing of cfDNA in patients where tissue biopsy is not feasible or medically recommended. Citation Format: Taylor J. Jensen, Aaron M. Goodman, Shumei Kato, Christopher K. Ellison, Kimberly Kelly, Lisa Kim, Gregory A. Daniels, Kerry Fitzgerald, Erin McCarthy, Prachi Nakashe, Amin R. Mazloom, Graham McLennan, Eyad Almasri, Daniel S. Grosu, Marcia Eisenberg, Razelle Kurzrock. Genome-wide sequencing of cell-free DNA enables detection of copy number alterations in cancer patients where tissue biopsy is not feasible [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1362.

Emergence of RAS mutations in patients with metastatic colorectal cancer receiving cetuximab-based treatment: a study protocol

BackgroundIn the management of patients with RAS wild-type metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor (EGFR) therapies have demonstrated a clinical benefit, with longer survival. However, the correlation between the emergence of circulating RAS mutations and secondary resistance to anti-EGFR therapies requires further elucidation. In this study, we aim to examine evolutionary changes in RAS mutations through liquid biopsy in patients with mCRC during and after anti-EGFR therapy.MethodsA total of 120 patients diagnosed with RAS wild-type mCRC will be enrolled in this study. Patients will receive a cetuximab-based infusional 5-fluorouracil regimen as first-line treatment. Cetuximab-based treatment is expected to continue until disease progression, intolerable toxic effects, or withdrawal of consent. Blood samples from enrolled patients will be collected before and then every 3 months during cetuximab-based treatment and also at disease progression. These blood samples will be evaluated for RAS resistance mutations by using the MassARRAY platform. The primary endpoint is the percentage of RAS mutations detected in circulating DNA from patients during cetuximab treatment. The correlation between the tumor response and survival outcomes of these patients and the emergence of circulating RAS mutations will be further analyzed.DiscussionLiquid biopsy is a powerful technology that can represent tumor heterogeneity in a relatively noninvasive manner. Because RAS mutations play a major role in resistance to anti-EGFR therapy for mCRC, examining evolutionary changes in these mutations during such treatment through liquid biopsy would be useful. After comprehensively analyzing the emergence of circulating RAS mutations and its clinical relevance in this study, our results should provide practical guidance on anti-EGFR therapy for mCRC.Trial registrationThe date of trial registration (NCT03401957) in this study was January 17, 2018.

Re-biopsy and liquid biopsy for patients with non-small cell lung cancer after EGFR-tyrosine kinase inhibitor failure.

BackgroundRe‐biopsy is important for exploring resistance mechanisms, especially for non‐small cell lung cancer (NSCLC) patients who develop resistance to EGFR‐tyrosine kinase inhibitors (TKIs). Liquid biopsy using circulating tumor DNA has come into use for this purpose. This retrospective study investigated the status of re‐biopsy and liquid biopsy in NSCLC patients with EGFR mutations and evaluated their effect on clinical strategies and prognosis.MethodsFive hundred fifty‐five NSCLC patients with resistance to EGFR‐TKIs were included and divided into three groups: re‐biopsy, liquid biopsy, and no re‐biopsy. Amplification refractory mutation system (ARMS) PCR or super ARMS PCR was used to detect EGFR mutations.ResultsThree hundred eight (55.5%) patients underwent re‐biopsy; 45.5% (140/308) were positive for T790M. The most common re‐biopsy procedure was computed tomography‐guided percutaneous core needle biopsy (60.1%), followed by effusion drainage (29.5%) and superficial lymph node biopsy (6.5%). One hundred eighteen (21.3%) patients underwent liquid biopsy; the T790M detection rate was 41.5% (49/118.) Of the 308 patients who underwent re‐biopsy, 69 were examined for EGFR mutations with plasma. The concordance rate of T790M detection between tissue and plasma was 66.7%. A statistical difference in further treatment after EGFR‐TKI failure was observed among all groups (P = 0.014). Patients in the biopsy groups were more likely to receive third‐generation EGFR‐TKIs. Multivariate analysis showed that re‐biopsy had a significant impact on overall survival (P < 0.001).ConclusionRe‐biopsy plays a pivotal role in the management of patients with NSCLC and resistance to EGFR‐TKIs. Liquid biopsy may be an alternative if difficulties performing re‐biopsy exist.

Abstract P4-01-07: A comprehensive liquid biopsy in patients undergoing neoadjuvant therapy

Abstract Background: Precision medicine is revolutionizing breast cancer (BC) care. Comprehensive liquid biopsies are a tool for personalized care in patients with locally advanced breast cancer (LABC). Identifying robust biomarkers as part of a comprehensive liquid biopsy to predict response to treatment is of immense clinical interest. Methods: After obtaining IRB approval, serial blood samples were collected from patients with LABC undergoing neoadjuvant therapy. Paired biopsies were collected prior to treatment and were sent to Foundation Medicine for next-generation sequencing (NGS). We used a sized-base microfilter technology to capture circulating tumor cells (CTCs) and circulating cancer associated fibroblasts (cCAFs). Patients with one or more CTCs or cCAFs were deemed positive for these tests. Additionally, in collaboration with Foundation Medicine, we extracted circulating tumor DNA (ctDNA) and we analyzed it using the FoundationACT platform. Patients with a detectable genomic alteration in their plasma were considered as having a positive ctDNA test. Our primary objective is to determine if a comprehensive liquid biopsy can serve as a prognostic marker of pathologic complete response (pCR). Results: For this analysis we describe our findings in the initial blood draw of the first 18 patients enrolled. The mean age is 54 years (38-70). All patients who had their tumors sequenced had a detectable mutation. Consistent with the findings of others, we found TP53 mutations to be the most prevalent at 83.3%. We found that 44% of patients had ctDNA, 68.4% had cCAFs and 78.9% had CTCs. Many patients also had clusters of cells, consisting of one cell type, or co-clusters, consisting of both. 38.9% had CTC clusters, 16.7% had cCAF clusters and 16.7% had co-clusters (CTCs and cCAFs together). Some patients with CTCs did not have cCAFs and vice versa. The number of CTCs and cCAFS did not correlate with stage of disease or receptor status. Conclusions: We describe a comprehensive liquid biopsy combining a sized-based microfilter technology for CTC and cCAFs identification and the FoundationACT platform for ctDNA analysis is feasible and these biomarkers can be detected in patients with LABC prior to the initiation of neoadjuvant therapy. Our study is accruing rapidly, and we will update our results with the longitudinal collection and the prognostic value of a comprehensive liquid biopsy at the time of the meeting. Citation Format: Sandoval Leon AC, Medina Saenz K, Miller P, Benson A, Calfa C, Mahtani R, Slingerland J, Perez A, Vogel C, Valdes-Albini F, El-Ashry D, Lippman M. A comprehensive liquid biopsy in patients undergoing neoadjuvant therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-07.

Recent Advances in Liquid Biopsy in Patients With Castration Resistant Prostate Cancer.

Management of localized and advanced prostate cancer benefits from several therapeutic options with a surprising improvement in terms of clinical outcome. The selection of patients more likely to benefit from a specific approach still remains a key issue as well as the early identification of patients with aggressive disease which could benefit from a more aggressive treatment strategy. The lack of reliable bio-marker in castration resistant setting able to monitor response to treatment and early inform about tumor progression is an emerging issue. Accordingly, circulating DNA and circulating tumor cells appears a promising and attractive approach despite to date practical applications of these techniques are few and not validated. The aim of this review of the literature is to explore current knowledge on liquid biopsy in prostate cancer focusing on possible future applications.

Abstract 990: Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance

Abstract Small cell lung cancer (SCLC) accounts for 14% of lung cancer diagnoses in the United States and is characterized by rapid onset of chemoresistance and poor clinical outcomes. Once considered a homogeneous disease, recent analyses of SCLC have identified intratumoral heterogeneity (ITH) with respect to NOTCH signaling, ASCL1/NEUROD1 balance and MYC amplification - all of which are potential mechanisms underlying SCLC's aggressive and refractory biology. Unfortunately, patient-derived models of SCLC with which to better characterize the molecular profiles of refractory SCLC are scarce. To address this, we generated circulating tumor cell-derived xenograft (CDX) models from liquid biopsies of patients with treatment-naïve or relapsed SCLC. Each CDX model underwent pathological review to confirm tumors were consistent with SCLC based on histology and standard immunohistochemical markers (e.g., TTF1, chromogranin A, synaptophysin, NCAM). Sequencing of these models revealed mutations typical of SCLC (e.g. TP53, RB1), which were maintained in vivo over multiple passages. Importantly, each model's in vivo response to cisplatin matched the patient's platinum response at the time of CDX generation. At the proteomic level, platinum-resistant models exhibited mTOR activation, increased SOX2 and ATM, and reduced E-cadherin, suggesting a shift toward EMT and cancer stem cell expansion may contribute to resistance. To investigate ITH, we analyzed single-cell gene expression profiles by RNAseq using a droplet-based Chromium Single Cell system that analyzed a filtered subsample of 2000 cells per tumor. Consistent with SCLC, all CDX models contained large numbers of cells expressing neuroendocrine-specific genes (SYP, CHGA). However, Principle Component Analysis revealed that cells from chemosensitive CDX models had distinct expression profiles from resistant models. Using our published EMT gene signature, we found that resistant models had higher proportions of mesenchymal (vs. epithelial) cells. Several other distinctions between sensitive and resistant models were detected at the single-cell level but not in bulk RNA and protein analyses, suggesting that single-cell resolution can identify occult platinum-resistant subpopulations. For example, higher proportions of ASCL1- and DLL3-expressing cells were associated with platinum sensitivity, whereas a shift toward predominant NEUROD1-expression was observed with resistance. Cells expressing each of these three genes were identified across all tumors, suggesting platinum-sensitive and resistant subpopulations are ubiquitous but that even subtle shifts in the fractional distribution of these subsets can exert significant impact on response. These data support further use of single-cell analysis to explore the role of ITH as a driver of drug resistance in SCLC. Citation Format: C. Allison Stewart, Carl M. Gay, Yuanxin Xi, Siva V., Junya Fujimoto, Pan Tong, Lixia Diao, Lerong Li, Mohan Bolisetty, Neda Kalhor, Patrice Lawson, Mayra Vasquez, Hai Tran, Ignacio I. Wistuba, Bonnie Glisson, Jianjun Zhang, Stephen G. Swisher, Jack A. Roth, John V. Heymach, Paul Robson, Jing Wang, Lauren A. Byers. Single-cell profiling of small cell lung cancer circulating tumor cell-derived xenograft models reveals intratumoral heterogeneity among mediators of chemoresistance [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 990.

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: a new medium of liquid biopsy

BackgroundLeptomeningeal metastases (LM) are more frequent in non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the purpose of this study was to explore the potential role of cerebrospinal fluid (CSF) as a source of liquid biopsy in patients with LM. Patients and methodsPrimary tumor, CSF, and plasma in NSCLC with LM were tested by next-generation sequencing. In total, 45 patients with suspected LM underwent lumbar puncture, and those with EGFR mutations diagnosed with LM were enrolled. ResultsA total of 28 patients were enrolled in this cohort; CSF and plasma were available in 26 patients, respectively. Driver genes were detected in 100% (26/26), 84.6% (22/26), and 73.1% (19/26) of samples comprising CSF cell-free DNA (cfDNA), CSF precipitates, and plasma, respectively; 92.3% (24/26) of patients had much higher allele fractions in CSF cfDNA than the other two media. Unique genetic profiles were captured in CSF cfDNA compared with those in plasma and primary tissue. Multiple copy number variations (CNVs) were mainly identified in CSF cfDNA, and MET copy number gain identified in 47.8% (11/23) of patients was the most frequent one, while other CNVs included ERBB2, KRAS, ALK, and MYC. Moreover, loss of heterozygosity (LOH) of TP53 was identified in 73.1% (19/26) CSF cfDNA, which was much higher than that in plasma (2/26, 7.7%; P<0.001). There was a trend towards a higher frequency of concomitant resistance mutations in patients with TP53 LOH than those without (70.6% versus 33.3%; P=0.162). EGFR T790M was identified in CSF cfDNA of 30.4% (7/23) of patients who experienced TKI progression. ConclusionCSF cfDNA could reveal the unique genetic profiles of LM and should be considered as the most representative liquid biopsy medium for LM in EGFR-mutant NSCLC.