Liquid Biopsy Source Research Articles

Abstract B03: Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use

Abstract Introduction: Urine was shown to be a liquid biopsy source with high sensitivity for cfDNA mutation detection in both early- and late-stage cancer patients, outperforming plasma cfDNA and tissue samples in some patients. In addition, obtaining a urine sample is completely noninvasive. Limitations of using urine samples in the clinical setting include their unknown nucleic acid stability and the large total volume of one deposit, and therefore the presence of highly diluted cfDNA. Urine volume concentration, cfDNA stability, cfDNA quantity, and detection of wild-type (wt) EGFR alleles were explored to determine the future suitability of urine cfDNA as a diagnostic or prognostic biosource. Methods: First-void urine samples, with a higher fraction of nucleic acids due to overnight concentration, were collected from 5 healthy donors and 5 cancer patients. Cell-free urine samples were concentrated using Amicon filter tubes to determine differences in cfDNA concentration. In addition, we tested the ability to detect wild-type EGFR exon 19 in cfDNA from concentrated and unconcentrated urine samples using a peptide nucleic acid (PNA) Taqman Assay. Hereafter, stability of urine cfDNA was explored using variable storage temperatures and times and addition of a urine cfDNA preservative directly after sample collection. cfDNA was extracted using the automated QIAsymphony system with the DSP virus/pathogen kit and quantified using the Qubit dsDNA HS Assay in all experiments. Results: Amicon filter-based concentration of urine samples from 15 mL to 1.2 mL yielded a two-fold increase in cfDNA concentration. Importantly, while wild-type EGFR could not be detected in first-void urine using a PNA Taqman Assay, detection was possible using concentrated urine samples. Therefore, in all following experiments, samples were concentrated unless specified otherwise. Stability tests indicated that storage temperature matters when samples are not processed within two hours, with nearly doubled cfDNA concentrations found in samples stored at four degrees for 26 hours, compared to room temperature (RT). Addition of a cfDNA preservative yielded a six-fold higher cfDNA concentration after 26 hours of storage at RT, compared to samples without a preservative added. For this reason, we tested the use of only cfDNA preservative without concentrating the sample and found a four-fold increase in cfDNA concentration when adding only preservative, versus a 15-fold increase when using both after sample storage at RT for 24 hours. Conclusion: Patient urine samples should be either concentrated and processed within two hours, or a urine cfDNA preservative should be added directly after sample collection to prevent cfDNA degradation. Depending on the downstream application, samples where preservative was added may be concentrated to enhance the cfDNA yield, for example, when downstream NGS analysis will be performed. Citation Format: Jillian W. P. Bracht, Niki Karachaliou, Jordi Berenguer, Manuel Fernandez-Bruno, Mónica Garzón, Ana Gimenez-Capitan, Clara Mayo-de-las-Casas, Miguel Angel Molina-Vila, Rafael Rosell. Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use [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 B03.

Sputum supernatant as an alternative source for liquid biopsy in patients with advanced lung cancer.

e21617 Background: With the advancements in the development of targeted therapy, the detection of actionable genes has become routine practice in diagnosing lung cancer. Plasma-based mutation profiling is widely used in clinical settings. Profiling using other body fluids is actively explored. In this study, we investigated the potential of induced sputum obtained from non-small cell lung cancer (NSCLC) patients for mutation profiling. Methods: We performed targeted sequencing on matched FFPE, plasma and induced sputum samples of 41 treatment-naïve advanced NSCLC using a panel consisting of 168 lung cancer-related genes. 10ml of whole blood and 8 ml of induced sputum were obtained from each patient. Induced sputum samples were fractionated into supernatant (sup) and precipitates (ppt). The cohort had a median age of 65 with a majority (32/41) diagnosed with lung adenocarcinoma. 7 of them were diagnosed with squamous cell carcinoma and the remaining 2 patients were diagnosed with neuroendocrine tumor. All patients had advanced disease (stage III or IV). Results: First, we investigated the optimal sputum fraction for mutation profiling by comparing the detection rates and maximum allelic fraction (maxAF) between sputum sup and ppt samples using matched tissue and plasma as references. The detection rate was defined as any mutation detected from the panel used; while maxAF was defined as the highest mutation allelic fraction among all mutations detected. Tissue, plasma, sputum sup and ppt achieved a rate of detection of 100%, 76.9%, 72.4% and 65.7% respectively, revealing comparable detection rates between plasma and sputum sup (p = 0.89). Sup had a significantly higher average maxAF than plasma (p = 0.03) and ppt samples (p < 0.01). Using driver mutations detected from tissue samples as references, sup, ppt and plasma samples achieved a sensitivity of 63.2%, 50% and 67.8%, respectively. Next, we investigated whether induced sputum obtained from smokers would be more informative than non-smokers. Our analysis revealed that sup from smokers had a significantly higher average AF than sup from non-smokers (p < 0.001). Furthermore, there was a statistical significant difference in average AF between sup and ppt obtained from smoker (p < 0.001) but such difference was non-existent in non-smokers (p = 0.46). Conclusions: Our study demonstrated that sputum sup demonstrates a comparable performance as plasma samples and is as a better sampling source than its ppt. Induced sputum from advanced stage NSCLC patients is an alternative media for mutation profiling.

Evaluation of Circulating Tumor DNA in Patients with Ovarian Cancer Harboring Somatic <i>PIK3CA</i> or <i>KRAS</i> Mutations

PurposeCirculating tumor DNA (ctDNA) is an attractive source for liquid biopsy to understand molecular phenotypes of a tumor non-invasively, which is also expected to be both a diagnostic and prognostic marker. PIK3CA and KRAS are among the most frequently mutated genes in epithelial ovarian cancer (EOC). In addition, their hotspot mutations have already been identified and are ready for a highly sensitive analysis. Our aim is to clarify the significance of PIK3CA and KRAS mutations in the plasma of EOC patients as tumor-informed ctDNA.MethodsWe screened 306 patients with ovarian tumors for somatic PIK3CA or KRAS mutations. A total of 85 EOC patients had somatic PIK3CA and/or KRAS mutations, and the corresponding mutations were subsequently analyzed using a droplet digital polymerase chain reaction in their plasma.ResultsThe detection rates for ctDNA were 27% in EOC patients. Advanced stage and positive peritoneal cytology were associated with higher frequency of ctDNA detection. Preoperative ctDNA detection was found to be an indicator of outcomes, and multivariate analysis revealed that ctDNA remained an independent risk factor for recurrence (p=0.010). Moreover, we assessed the mutation frequency in matched plasma before surgery and at recurrence from 17 patients, and found six patients had higher mutation rates in cell-free DNA at recurrence compared to that at primary diagnosis.ConclusionThe presence of ctDNA at diagnosis was an indicator for recurrence, which suggests potential tumor spread even when tumors were localized at the time of diagnosis.

A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma

Purpose: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. Experimental design: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. Results: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. Conclusion: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

Abstract OT3-18-02: A comparative study of two detection platforms of circulating tumour cells in patients with metastatic breast cancer

Abstract Background: The enumeration of circulating tumour cells (CTCs) using the FDA-cleared CellSearch system has repeatedly been proven to bear prognostic significance in patients with localized and metastatic breast cancer (MBC) (Bidard et al. Lancet Oncol 2014; Janni et al. CCR 2016). In addition, CTC count has been suggested to be a valuable alternative for physician-based decision making in choosing between hormone therapy and chemotherapy as frontline treatment for patients with estrogen receptor-positive, HER2-negative MBC (Bidard et al. SABCS 2018). Challenges for the implementation of CTCs as a biomarker or a source of liquid biopsy in clinical practice include that most isolation methods only detect a limited number of CTCs in a blood sample in a subset of patients, even in those with clinically proven metastatic disease, and the limited availability of comparative data on technical variability between different CTC detection assays. Methods: In this study, we compare CTC detection rates in paired 7.5 ml blood samples from patients with MBC starting a new line of treatment using two commercially available CTC detection assays. All patients are recruited at GZA Hospitals, campus Sint-Augustinus, Antwerp, Belgium. The first CTC detection system is the CellSearch system, currently the only FDA-cleared system for the enumeration of CTCs in patients with MBC. This system employs an immunomagnetic CTC enrichment based on the expression of Epithelial Cell Adhesion Molecule (EPCAM) followed by an immunofluorescent CTC detection based on the expression of cytokeratins (CK) in EPCAM+ enriched cells. With this system, only EPCAM/CK double positive cells can be qualified as CTCs. The second system is the Rarecytes system, an automated scanner for immunofluorescently stained cell smears of the entire buffy coat of blood samples. With this system, CTCs will be enriched and detected in a single-step measurement using a cocktail of anti-EPCAM and anti-CK fluorescently labeled antibodies, thereby allowing to detect also single-marker positive CTCs. Based on CTC detection prevalence data in patients with MBC that are available for the CellSearch system and a prespecified hypothesis that with both detection methods a discordant result (relative to a cut-off of &amp;gt;= or &amp;lt;5 CTCs/7.5 ml blood) will be measured in 25% of patients, we aim to include 100 patients with MBC in this benchmarking study in order to be able to detect at least a 15% difference in CTC detection rate in favor of one of both techniques with 80% power at a significance level of 0.05. Concordance/discordance of CTC results between both detection methods will be analyzed both as a dichotomized variable relative to different cut-off values using the McNemar test and Cohen’s kappa test and as a continuous variable using the intraclass correlation coefficient. Expected results and conclusions: Upon submission of the abstract, 30 patients with MBC have been included in this trial after an accrual period of 4 months. Full results are expected mid 2020. Detailed comparison of CTC detection rates between both techniques will not only render analytical data on the performance of the Rarecytes system compared to the CellSearch system - the current gold standard in the field - but might also shed light on the frequency of EPCAM/CK single-positive CTCs in patients with MBC. Conflicts of interest: This study is partially funded by a grant from Rarecyte, Inc. Contact details for collaboration: Dieter Peeters (HistoGeneX NV, Antwerp, Belgium): dieter.peeters@histogenex.com; Andy Buys (Translational Cancer Research Unit, Antwerp, Belgium): andy.buys@gza.be. Citation Format: Dieter Peeters, Andy Buys, Hannah Ledegen, Kathlyn Moehlig, Steffi Oeyen, Dieter Rondas, Alexia De Beukelaer, Steven Van Laere, Liesbet Vervoort, Peter Vermeulen, Christopher Ung, Mark Kockx, Luc Dirix. A comparative study of two detection platforms of circulating tumour cells in 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 OT3-18-02.

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 SY23-01: Blood-borne tumor cell dissemination in cancer patients

Abstract SY23-01: Blood-borne tumor cell dissemination in cancer patients

Low-coverage whole genome sequencing of FFPE-derived-DNA and extracellular vesicle-associated DNA in patients with metastatic cancer.

e14523 Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log2 ratio ≥0.15 and losses &lt; -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.

Review: Peering through a keyhole: liquid biopsy in primary and metastatic central nervous system tumours.

Tumour molecular profiling by liquid biopsy is being investigated for a wide range of research and clinical purposes. The possibility of repeatedly interrogating the tumour profile using minimally invasive procedures is helping to understand spatial and temporal tumour heterogeneity, and to shed a light on mechanisms of resistance to targeted therapies. Moreover, this approach has been already implemented in clinical practice to address specific decisions regarding patients’ follow‐up and therapeutic management. For central nervous system (CNS) tumours, molecular profiling is particularly relevant for the proper characterization of primary neoplasms, while CNS metastases can significantly diverge from primary disease or extra‐CNS metastases, thus compelling a dedicated assessment. Based on these considerations, effective liquid biopsy tools for CNS tumours are highly warranted and a significant amount of data have been accrued over the last few years. These results have shown that liquid biopsy can provide clinically meaningful information about both primary and metastatic CNS tumours, but specific considerations must be taken into account, for example, when choosing the source of liquid biopsy. Nevertheless, this approach is especially attractive for CNS tumours, as repeated tumour sampling is not feasible. The aim of our review was to thoroughly report the state‐of‐the‐art regarding the opportunities and challenges posed by liquid biopsy in both primary and secondary CNS tumours.

First-void urine as a non-invasive liquid biopsy source to detect vaccine-induced human papillomavirus antibodies originating from cervicovaginal secretions

BackgroundMonitoring HPV antibodies non-invasively would be a major advantage for large epidemiological studies and follow-up of vaccinees. ObjectivesThis study investigated the presence of HPV-specific antibody transudates from systemic circulation in first-void urine of (un)vaccinated subjects and the agreement with paired sera. Study designIn this case-control study, 55 paired first-void urine and serum samples were included from 19- to 26-year-old women, unvaccinated (n = 19) or vaccinated (n = 36) with the bi- or quadrivalent HPV vaccine during adolescence (NCT02714114). Human IgA, total human IgG, and HPV6/11/16/18-Ig(M/G/A) were measured in paired samples. ResultsSignificant positive Spearman rank correlations (rs) were found in HPV-specific antibody levels between paired samples (HPV6: rs = 0.777; HPV11: rs = 0.757; HPV16: rs = 0.876; HPV18: rs = 0.636 (p < 0.001)). In both first-void urine and serum, significantly higher HPV6/11/16/18 antibody levels were observed in vaccinated compared with unvaccinated women (p ≤ 0.017). ConclusionsThe present study provides the first proof that vaccine-induced HPV antibodies are detectable in the first-void urine of young women. Moreover, significant positive correlations were observed between HPV6/11/16/18-antibodies in first-void urine and paired sera. Further optimization and validation are required to demonstrate its potential use in epidemiological studies and follow-up of HPV vaccination.

Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid

BackgroundExosomes are a subset of extracellular vesicles 30–200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF.MethodsThe main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a highly sensitive next-generation sequencing. The signatures of CSF exosomal mRNAs were then compared between four normal healthy donors and four sporadic amyotrophic lateral sclerosis patients to identify disease-related biomarkers. Differentially expressed genes were identified by DESeq2.ResultsRNA sequencing from CSF exosomes was successfully performed, that was demonstrated by the high pearson’s product-moment correlation coefficient (r = 0.993) in the technical replicates. Also, position coverage analysis revealed that most detected mRNAs retained their integrity throughout their full-length in CSF exosomes. In CSF exosomes from normal healthy donors, an average of 14,807 genes were detected, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. In comparison with exosomal mRNAs in CSF from four patients with amyotrophic lateral sclerosis, 543 genes were significantly changed, as represented by CUEDC2. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis.ConclusionWe successfully established the methodology of comprehensive analysis of exosomal mRNAs in human CSF. It was shown to be useful to identify disease biomarkers for central nervous system. Several genes, such as CUEDC2, in CSF exosomes were suggested to be candidate disease biomarkers for amyotrophic lateral sclerosis.

Unique genomic profiles obtained from cerebrospinal fluid cell-free DNA of non-small cell lung cancer patients with leptomeningeal metastases

ABSTRACTBackground: Leptomeningeal metastases (LM), associated with poor prognosis, are frequent complications of advanced non-small cell lung cancer (NSCLC) patients, especially in patients with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the mutational landscape of LM has not been comprehensively investigated in large cohorts and the underlining biology of LM remains elusive. Some studies have explored the potential of cerebrospinal fluid (CSF) in reflecting the molecular profile of LM but with limited number of patients enrolled.Methods: In this study, we performed capture-based targeted sequencing using a panel consisting of 168 lung cancer-related genes on matched CSF and plasma samples from 72 advanced NSCLC patients with confirmed LM to interrogate the potential of CSF as a source of liquid biopsy.Results: We revealed a rate of detection of 81.5% and 62.5% for CSF and plasma, respectively (p = 0.008). The maximum allelic fraction (MaxAF) was also significantly higher in CSF (43.6% vs. 4.6%) (p < 0.001). CSF, harboring a unique genomic profile by having a significant number of CSF-specific mutations, primarily copy number variations, is superior to plasma in reflecting the mutational profile of LM. Further pathway enrichment analysis revealed that most of CSF-specific mutations participated in pathways relevant to the tumorigenesis and the development of metastases. Moreover, our data also revealed that TP53 loss of heterozygosity (LOH) predominantly existed in CSF (p < 0.001).Conclusions: Collectively, we demonstrated that CSF provides a more comprehensive profile of LM than plasma in a large cohort, thus can be used as an alternative source of liquid biopsy for LM patients.

Urinary Exosomal and cell-free DNA Detects Somatic Mutation and Copy Number Alteration in Urothelial Carcinoma of Bladder

Urothelial bladder carcinoma (UBC) is characterized by a large number of genetic alterations. DNA from urine is a promising source for liquid biopsy in urological malignancies. We aimed to assess the availability of cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in urine as a source for liquid biopsy in UBC. We included 9 patients who underwent surgery for UBC and performed genomic profiling of tumor samples and matched urinary cfDNA and exoDNA. For mutation analysis, deep sequencing was performed for 9 gene targets and shallow whole genome sequencing (sWGS) was used for the detection of copy number variation (CNV). We analyzed whether genetic alteration in tumor samples was reflected in urinary cfDNA or exoDNA. To measure the similarity between copy number profiles of tumor tissue and urinary DNA, the Pearson’s correlation coefficient was calculated. We found 17 somatic mutations in 6 patients. Of the 17 somatic mutations, 14 and 12 were identified by analysis of cfDNA and exoDNA with AFs of 56.2% and 65.6%, respectively. In CNV analysis using sWGS, although the mean depth was 0.6X, we found amplification of MDM2, ERBB2, CCND1 and CCNE1, and deletion of CDKN2A, PTEN and RB1, all known to be frequently altered in UBC. CNV plots of cfDNA and exoDNA showed a similar pattern with those from the tumor samples. Pearson’s correlation coefficients of tumor vs. cfDNA (0.481) and tumor vs. exoDNA (0.412) were higher than that of tumor vs. normal (0.086). We successfully identified somatic mutations and CNV in UBC using urinary cfDNA and exoDNA. Urinary exoDNA could be another source for liquid biopsy. Also, CNV analysis using sWGS is an alternative strategy for liquid biopsy, providing data from the whole genome at a low cost.

Exosomal RNA-profiling of pleural effusions identifies adenocarcinoma patients through elevated miR-200 and LCN2 expression

HypothesisThe inherent challenges associated with tissue biopsies from lung have spurred an interest in the use of liquid biopsies. Pleural effusions are one source of liquid biopsy. Recently, extracellular vesicles of endocytic origin, exosomes, have attracted interest as liquid biopsy of tumors as they are thought to be a mirror of their tumor of origin. Here, we aimed to analyze if RNA profiling of exosomes isolated from pleural effusions could differentiate patients with lung adenocarcinoma from patients with benign inflammatory processes. MethodsExosomes were isolated from 36 pleural effusions from patients with adenocarcinoma (n = 18) and patients with benign inflammatory processes (n = 18). The two groups were balanced with respect to age and smoking history but with a gender bias towards males in the benign group. Profiling was conducted using RT-qPCR arrays covering 754 microRNAs and 624 mRNAs followed by statistical ranking of differentially regulated transcripts between the two patient cohorts. ResultsRNA profiling revealed differential expression of 17 microRNAs and 71 mRNAs in pleural effusions collected from patients with lung adenocarcinoma compared to pleural effusions from benign lung disease. Overall, top differentially expressed microRNAs, including miR-200 family microRNAs, provided a stronger diagnostic power compared to top differentially expressed mRNAs. However, the mRNA transcript encoding Lipocalin-2 (LCN2) displayed the strongest diagnostic power of all analyzed transcripts (AUC: 0.9916). ConclusionsOur study demonstrates that exosomal RNA profiling from pleural effusions can be used to identify patients with lung adenocarcinoma from individuals with benign processes and further proposes miR-200 microRNAs and LCN2 as diagnostic markers in lung cancer liquid biopsies.

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.

Abstract 1943: A microRNA signature in urinary exosomes for diagnosis of prostate cancer

Abstract MicroRNAs constitute a class of small cellular noncoding RNAs (typically 19-23 nt) which function as post-transcriptional regulators of gene expression. The involvement of microRNA in both normal development as well as disease is well established and microRNAs are considered important biomarkers in several diseases including cancer. Surprisingly cell free microRNAs are also found in several biofluids where they have been shown to be very stable, most likely because they are protected from degradation by protein complexes and other particles including exosomes. Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy. We have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes. Our developed technologies was applied to cell-free urine from three different cohorts including individuals with prostate cancer (PCa) and benign prostate hyperplasia, to identify several differentially regulated microRNAs in urine from PCa bearing individuals. PCa specific signatures were obtained by different combinations of these differentially regulated microRNAs. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer. Data showing that small miRNA signatures (down to two miRNAs) hold strong diagnostic potential in PCa will be presented. Citation Format: Thorarinn Blondal, Anne I. Rasmussen, Anni R. Thomsen, Michael Borre, Jacob Fredsøe, Ditte Andreasen, Torben Falck Ørntoft, Karina D. Sørensen, Peter Mouritzen. A microRNA signature in urinary exosomes for diagnosis of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1943.

Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.

Blood-based Tumor Markers in Lung Cancer

近年来，“液态活检”概念异军突起，不仅可以辅助诊断某些类型实体肿瘤，而且能够监测复发，评估疗效及肺癌分子表达，更是无创肿瘤筛查方法。其来源主要分为循环肿瘤细胞（circulating tumor cells, CTCs），循环肿瘤DNA（circulating tumor DNA, ctDNA）等。本文讨论了肺癌领域中CTCs、ctDNA及其他肿瘤标志物的生物学特征、检测方法及临床应用，总结了保证特异性前提下的高敏感度、便于临床应用、可重复性好三个重要评判标准的“液态活检”技术，尤其是肺癌Ⅰ期敏感度可达67%的靶向PCR CTC技术的应用，以期使“液态活检”真正从科研探索走向临床应用。

Abstract 3987: Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy

Abstract MicroRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). MicroRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including complexing with RNA binding proteins or HDL and through active exosome transport. Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy. Cell free urine samples are an obvious liquid biopsy source for microRNA markers in prostate cancer and could serve as diagnostic tools as well as treatment response markers. Yet microRNA levels in urine samples are extremely sparse and cannot be detected reliable by conventional sample preparation methods. It has been reported that tumour derived exosomes carrying genetic information specific for prostate cancer can be measured in urine samples following prostate massage. However, a methodology eliminating the need for prostate massage of every patient to be tested in a clinical setting is highly desirable. The purpose of this study was to combine a simple exosomal enrichment method with our highly sensitive LNA™-based qPCR platform for detection of microRNAs and apply this to a cohort consisting of more than 300 cell free urine samples from prostate cancer cases and controls. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer, showing miRNA signatures of strong diagnostic potential. Analysis is pending and data will be presented. Citation Format: Thorarinn Blondal, Anni R. Thomsen, Jörg Krummheuer, Michael Borre, Jacob Fredsøe, Christa Haldrup, Ditte Andreasen, Maria W. Teilum, Niels Tolstrup, Karina D. Sørensen, Torben F. Ørntoft, Peter Mouritzen. Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2015-3987

Bone marrow as a reservoir for disseminated tumor cells: a special source for liquid biopsy in cancer patients.

Besides circulating tumor cells, disseminated tumor cells (DTCs) in bone marrow (BM) might be used as a 'liquid biopsy' to obtain information helpful to steer therapies in individual patients. Moreover, the molecular characterization of DTCs may provide important insight into the biology of cancer metastasis. BM is a frequent site of metastasis in breast, prostate and lung cancer, and it might represent a sanctuary site for DTCs derived from various additional types of epithelial tumors. Highly sensitive and specific immunocytological and molecular methods enable the detection of DTCs in BM of cancer patients at the single-cell level years before the occurrence of metastases. This information might be useful to assess individual prognosis and stratify patients at risk to systemic adjuvant anti-cancer therapies. Although most data on the prognostic value of DTCs are available for breast cancer, several single institution studies including patients with colon, lung, prostate, esophageal, gastric, pancreatic, ovarian and head and neck carcinomas have also documented an association between the presence of DTCs at primary surgery and subsequent metastatic relapse. Most DTCs are in a dormant (that is, non-proliferative) stage, frequently express HER2 and display a cancer stem cell and immune escape phenotype. Here, we summarize the current knowledge about specific biological properties of DTCs in BM, and discuss the clinical relevance of DTC detection in cancer patients with regard to an improved individualized therapeutic management. This will stimulate further technical developments that may make BM sampling more acceptable for the clinical management of patients with solid tumors.