Application Of Liquid Biopsy Research Articles

Abstract 722: Urine for noninvasive liquid biopsy for germline and somatic mutations

Abstract Circulating tumor DNA (ctDNA) from blood and peripheral blood mononuclear cells (PBMCs) are used to determine comprehensive cancer genetics and germline genomics for precision medicine. To evaluate the use of urine samples as a noninvasive alternative to blood samples, we performed a comprehensive characterization of urine DNA as an alternative to PBMC DNA for germline genomics. Whole genome sequencing (WGS) was performed in PBMC and total urine DNA in six normal subjects to compare the quality and comprehensiveness of the genomic data. There was no significant difference between the sequencing data obtained from PBMC and urine DNA upon comparing the variant calling, percent reads passing Phred score Q30 (p>0.05, paired t-test), and coverage of human genome. Similar to plasma cfDNA, mononucleosomal-sized DNA was the most predominant species present in urine cfDNA. Samples from three hepatocellular carcinoma (HCC) patients were further analyzed for insert sizes by NGS, which demonstrated predominantly mononucleosomal-sized DNA fragments, with a series of peaks occurring with 10-bp periodicity, in both urine and plasma cfDNA. With shallow WGS, the genome coverage overlap was 53-61%, and by increasing the depth of coverage the overlap increased from 60% to 74%. Interestingly, 8-10% of the genome covered in plasma DNA was not detected in urine DNA, and 4-8% of the genome covered in urine DNA was not covered in plasma DNA. Next, we compared the detectability of three HCC-associated genetic mutations in the matched urine and plasma DNA obtained from 76 HCC patients including mutations at TP53 codon 249 (TP53 249T), CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124). We observed higher levels of the TP53 mutation, similar levels of CTNNB1 mutation, and lower levels of the TERT mutation in urine cfDNA compared to plasma ctDNA. This suggests a potential for urine as a potential noninvasive source for ctDNA analysis, and a combination of both urine and plasma ctDNA may increase the overall sensitivity of ctDNA detection. In conclusion, our data suggest that (1) total urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (2) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications in other cancers. Citation Format: James V. Vowles, Amy K. Kim, James P. Hamilton, Selena Y. Lin, Fwu-Shan Shieh, Harry Luu, Gabrielle Villafana, Chi-Tan Hu, Ying-Hsiu Su. Urine for noninvasive liquid biopsy for germline and somatic mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 722.

Abstract 3102: Analysis of plasma and serum as a source of circulating tumor DNA for hotspot mutations detection in liquid biopsy

Abstract Blood-based analysis of ctDNA is a promising tool for cancer screening, drug selection, detection of minimum residual disease, monitoring release/recurrence and response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess highly-valuable serum samples collection and comparative studies on the use of serum vs plasma as ctDNA source are still essential. Here we analyzed cell free DNA (cfDNA) from matched plasma and serum samples from healthy donors, colorectal and lung cancer patients to evaluate the differences of serum and plasma on mutation detection efficacy and reproducibility by targeted NGS. We initially addressed the difference of both analytes on the quality of cfDNA recovery and on sequencing performance. We then compared mutation detection using matched samples from healthy individuals spiked-in with standard oligonucleotides including common mutations. The amount of cfDNA was more abundant in serum than plasma and the presence of longer DNA fragments was significantly higher in serum samples. Spiked-in mutated nucleotides were detected in both plasma and serum samples, but allele frequencies (AF) were approximately half in serum samples compared with plasma samples, suggesting ctDNA from serum is more diluted in DNAs of noncancerous origins. We further confirmed this difference in detection efficiency using matched plasma and serum samples from cancer patients and found that mutations with low AF are more frequently missed in serum samples. In addition, concordance with tissue-biopsy mutations found at diagnosis was better in plasma samples. Finally, we examined the use of fragment size selection as an alternative tool to improve mutation detection in serum samples. Our findings clearly demonstrated that the use of serum in retrospective studies should consider the limitations for detecting low AF mutations and indicate the advantages of plasma samples for prospective clinical applications of liquid biopsy. Citation Format: Fabio Pittella-Silva, Yoon Ming Chin, Hui Ting Chan, Satoshi Nagayama, Eisaku Miyauchi, Siew-Kee Low, Yusuke Nakamura. Analysis of plasma and serum as a source of circulating tumor DNA for hotspot mutations detection in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3102.

Abstract 6455: Characterization of extracellular vesicles derived from uveal melanoma cell lines

Abstract Purpose: Extracellular vesicles (EVs) are small (50nm-800nm) membrane-enclosed structures with highly heterogeneous content. Recently, the importance of EVs in tumor progression as well as biomarkers has become apparent in many malignancies. However, there has been very limited research done to investigate EVs derived from uveal melanoma (UM). UM is the most common primary intraocular tumor in adults, and it displays a high frequency of metastases to the liver. The aim of this study was (i) to characterize the protein signatures of UM cell line-derived EVs to determine potential biomarkers, (ii) to determine EV uptake by recipient cell lines, (iii) to analyze the activation of the MAPK pathway in the recipient cells, and to examine the potential of UM EV-educated BRCA1 deficient fibroblasts (Fibro-BKO) to develop tumors in NOD-SCID mice. Methods: EVs were isolated from the conditioned media of UM cell lines (MP41, MP46, 92.1, MEL285, MEL270, OMM2.5) and normal choroidal melanocytes cells using an ultracentrifugation method. Western blots and immunogold-labelling Transmission Electron Microscopy were employed to validate various EV markers (CD63, CD81 and TSG101). Proteomic analysis by mass spectrometry was performed to determine the UM-derived EV proteome signature that could elucidate potential key players in UM metastasis. After exposure to EVs, recipient cells were analysed for the activation of downstream signaling pathways, and were inoculated in NOD-SCID mice to analyze their behaviour. Results: We observed 2069 proteins in EVs using Scaffold Viewer. FUNRich analysis revealed that 95% of these EV proteins were found in the Vesiclepedia database. Gene Ontology analysis confirmed the presence of exosomal markers (CD81, TSG101and Syntenin) as well as cell-cell adhesion-related proteins. The DAVID Functional Annotation chart revealed proteins related to endocytosis, PI3K-Akt signaling pathway and focal adhesion. In addition, we observed high hits in HSP90 and HSP70, well-known biomarkers in UM. Integrin alpha V, which are known for preparing premetastatic niches in the liver environment, were also present. Western blot results confirmed the increase in MAPK pathway in the recipient cells. Furthermore, UM EVs-educated Fibro-BKO transformed and gave rise to tumors in vivo. Conclusion: In conclusion, our study reports an essential step towards understanding protein emission through EVs from UM cells. Our analyses revealed UM EVs cargo candidates that may play a role in pre-metastatic niche formation and metastasis in specific organs, such as the liver. The in vivo study model confirmed that EVs derived from UM are capable of inducing tumorigenesis. We believe that our data pave the way to the application of liquid biopsy to the monitoring of UM-affected patients. Citation Format: Thupten Tsering, Alexander Laskaris, Mohamed Abdouh, Prisca Bustamante Alvarez, Goffredo Arena, Julia Valdemarin Burnier. Characterization of extracellular vesicles derived from uveal melanoma cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6455.

Precision Detection Technology: Equipping Precision Oncology with Wings.

In recent years, precision medical detection techniques experienced a rapid transformation from low-throughput to high-throughput genomic sequencing, from multicell promiscuous detection to single-cell precision sequencing. The emergence of liquid biopsy technology has compensated for the many limitations of tissue biopsy, leading to a tremendous transformation in precision detection. Precision detection techniques contribute to monitoring disease development more closely, evaluating therapeutic effects more scientifically, and developing new targets and new drugs. In the future, the role of precision detection and the joint detection in epigenetics, rare gene detection, individualized targeted therapy, and multigene targeted drug combination therapy should be extensively explored. This article reviews the changes in precision medical detection technology in the era of precision medicine, as well as the development, clinical application, and future challenges of liquid biopsy.

Plasma or Serum: Which Is Preferable for Mutation Detection in Liquid Biopsy?

Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential. We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis. Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples. The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy.

Abstract IA22: Tumor-educated platelets for the detection of cancer

Abstract Blood-based “liquid biopsies” provide a means for minimally invasive molecular diagnostics, overcoming limitations of tissue acquisition. Early detection of cancer, clinical cancer diagnostics, and companion diagnostics are regarded as important applications of liquid biopsies. It has been reported that tumor-educated platelets (TEPs) may enable blood-based cancer diagnostics. Platelets have emerged as central players in the systemic and local responses to tumor growth. Confrontation of platelets with tumor cells via transfer of tumor-associated biomolecules (“education”) is an emerging concept and can result in the sequestration of biomolecules. Moreover, external stimuli induce specific splicing of pre-mRNAs in circulating platelets. Platelets are considered to undergo queue-specific splice events in response to signals released by cancer cells and the tumor microenvironment, such as stromal and immune cells. The combination of specific splice events in response to external signals and the capacity of platelets to directly ingest (spliced) circulating mRNA can provide TEPs with a highly dynamic mRNA repertoire, with potential applicability to cancer diagnostics. We determined the diagnostic potential of TEPs by mRNA sequencing of platelet samples. Blood platelet samples of >1,000 patients with cancer covering multiple tumor types and healthy donors were analyzed. Healthy donors and early- and late-stage cancer patients were distinguished by a self-learning support vector machine (SVM) algorithm, using a set of spliced platelet transcripts with moderate to high expression. These results suggest that blood platelets may advance blood-based “liquid biopsies,” including for the detection of stage I-II tumors. Citation Format: Thomas Wurdinger. Tumor-educated platelets for the detection of cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA22.

Abstract IA11: Clinical applications of liquid biopsy in multiple myeloma

Abstract Multiple myeloma (MM) is characterized by recurrent cytogenetic and molecular abnormalities that have the potential to profoundly impact therapeutic decisions. Conventional karyotyping and/or FISH cytogenetics studies have identified translocations and copy number abnormalities (CNAs) that predict clinical outcomes. More recently, MM whole-exome sequencing studies have uncovered recurrent mutations in genes associated with prognosis and others that are potentially actionable and/or implicated in drug resistance. These observations highlight the need for advanced genomic platforms to inform prognosis and therapeutic decisions. Currently, molecular profiling of MM is performed on bone marrow (BM) aspirates; however, the genetic information obtained from aspirates is confounded by spatial and temporal intratumor genetic heterogeneity. Genomic studies of MM have confirmed the bias introduced by single-site biopsies, demonstrating that not all mutations are shared between targeted biopsies of extramedullary sites and BM. Further, longitudinal whole-genome sequencing studies have not only uncovered tumor heterogeneity at diagnosis but also subclonal competition with a shifting dominance of tumor clones and accumulation of genetic events during progression that are particularly affected by therapeutic pressures. Thus, information acquired from a single biopsy provides a spatially and temporally limited snapshot of a tumor that likely underestimates the complexity of the tumor genomic landscape. Analysis of circulating tumor DNA (ctDNA) and in blood plasma has the potential to overcome these barriers, providing access to the genetic landscape of all cancerous lesions and allowing for noninvasive tracking of genomic evolution over time. Circulating biomarkers can provide a “liquid biopsy” alternative to tissue biopsy, providing new opportunities for treatment tailoring based on real-time monitoring from a simple blood test. Here I will discuss the current liquid biopsy technologies under evaluation for genomic characterization of multiple myeloma. I will also describe the potential clinical applications for liquid biopsies to identify patients for targeted therapies, to track genomic changes, and to follow disease burden in patients with multiple myeloma. Citation Format: Suzanne Trudel, Trevor Pugh. Clinical applications of liquid biopsy in multiple myeloma [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 IA11.

Abstract PR05: Radiation-assisted Amplification Sequencing (RAMP-Seq): Evaluating the use of stereotactic body radiation therapy (SBRT) for enriching circulating tumor DNA in liquid biopsies

Abstract Purpose: To determine if the administration of stereotactic body radiation therapy (SBRT) to known tumor masses temporarily elevates levels of circulating tumor DNA (ctDNA) derived from target or distal masses and to identify an optimal period of peak ctDNA enrichment for liquid biopsy. Experimental Design and Procedures: We used deep, whole-exome sequencing (WES) or 100-gene capture with UMI-based error corrected sequencing of cell-free DNA (cfDNA) of individuals undergoing SBRT as standard-of-care (SOC) to determine (a) whether SBRT treatment induces elevated levels of ctDNA in blood plasma as confirmed by biopsy-proven cases, and (b) the optimal window of ctDNA enrichment for sample collection. We collected blood draws every 24-72 hours from each patient prior to and during SBRT treatment (n=20), as well as for two weeks following the final dose (n=3). CfDNA and genomic DNA were isolated from blood plasma and solid tissue biopsies, respectively, and subjected to whole-exome capture and next-generation sequencing with the Illumina HiSeq 2500 platform. Sequencing data were aligned and mutations were called using BWA MEM and Mutect pipelines as previously described (Butler et al., 2019). Genomic DNA extracted from buffy coat was prepared and used as a matched normal in mutation calling. Variant allele frequencies (VAF) of Mutect calls were compared between baseline and post-treatment samples to determine enrichment levels. WES of bulk tumor DNA from pathology-proven cancer patients was used to confirm that the variants identified in the cfDNA sequencing originated in the target tissue. Data Summary: Preliminary data suggest that SBRT induces enrichment of ctDNA in blood plasma by at least 10-fold within 24-96 hours following treatment. When variants identified in separate biopsies of multiple tumors in a single individual were analyzed after SBRT, we found that increases were not uniform among variant clusters, suggesting an abscopal response from nonradiated tumor tissues found elsewhere in the patient. Conclusions: The ability to detect tumor-derived genomic variants in cfDNA has been a major challenge to liquid biopsy applications, particularly in early-stage cancers and recurrence settings. This novel detection strategy, RAMP-Seq, utilizes highly conformational radiation to induce ctDNA enrichment from tumor-cell death, making previously undetectable levels of ctDNA well within the range of standard sequencing and analysis methods. Such an approach has valuable applications from diagnosis of early-stage cancer to genotyping lesions normally inaccessible by a traditional biopsy. Abscopal effects have also been observed, suggesting that induction of ctDNA enrichment by radiating the target lesion can also result in the increase of ctDNA derived from distal lesions. Taken together, these findings offer promising new approaches to both early- and advanced-stage cancer characterization and intervention. This abstract is also being presented as Poster A44. Citation Format: Christopher Boniface, Kathryn Baker, Chris Dieg, Carol Halsey, Ramtin Rahmani, Mark Deffenbach, Charles Thomas, Garth Tormoen, Nima Nabavizadeh, Paul Spellman. Radiation-assisted Amplification Sequencing (RAMP-Seq): Evaluating the use of stereotactic body radiation therapy (SBRT) for enriching circulating tumor DNA in liquid biopsies [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 PR05.

Abstract IA23: Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling

Abstract Measuring disease biomarkers in biofluids to detect and monitor pathophysiologic events in tissues has enormous appeal. While detecting specific DNA sequences in blood plasma has been successful in informing about the presence of cancers, viruses, and a range of pathogens, this approach does not provide gene expression information, which could reveal more detailed information about disease activity. Measuring cell-free RNA in plasma would provide such information and has the potential to be more sensitive, given that one DNA molecule in a cell gives rise often to thousands or more RNA molecules. However, with the exception of microRNAs, broad sequencing of cell-free messenger RNA (mRNA) profiles in plasma has been generally challenging. We discovered that a major barrier to cell-free RNA sequencing from plasma is that the large majority of mRNAs are both fragmented and have modified phosphorylation states at their 5’ and 3’ ends that make them invisible to standard small RNA-seq methods. We present a modified RNA-seq methodology, called phospho-RNA-seq, which revealed thousands of mRNA in blood plasma. The key to this approach is (i) the incorporation of T4-polynucleotide kinase treatment to change the phosphorylation states at the RNA ends, and (ii) a stringent bioinformatics analysis pipeline to reduce false positive alignments. Phospho-RNA-seq identified cohorts of gene transcripts in plasma, including ones expressed in a tissue-specific manner. As proof-of-concept validation of the approach for biomarker identification, we used phospho-RNA-seq to longitudinally profile plasma specimens collected from patients undergoing hematopoietic stem cell transplantation. We detected bone marrow-enriched and liver-enriched transcript sets in plasma, which tracked with bone marrow recovery and hepatic injury, respectively. By providing expanded access to the transcriptome in plasma and potentially other biofluids, phospho-RNA-seq enables the discovery of new cell-free RNA biomarker signatures for a wide variety of liquid biopsy applications. Citation Format: Maria D. Giraldez, Ryan M. Spengler, Alton Etheridge, Annika J. Goicochea, Missy Tuck, Sung W. Choi, David J. Galas, Muneesh Tewari. Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling [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 IA23.

Required Evidence for Clinical Applications of Liquid Biopsy Using Especially CTCs in Lung Cancer

As therapies have become more and more dependent on tumor as well as patient characteristics, obtaining tumor material has become of great importance. Liquid biopsies hold much potential as shown by a large amount of evidence across several studies. Clinical applications for circulating tumor cells (CTCs) are unfortunately still lacking. In part this is due to a lack of studies comparing liquid biopsies to conventional diagnostics and response measurements as well as studies showing that liquid biopsies can be used to switch therapies leading to improved outcomes. However, liquid biopsies using ctDNA for specific markers such as EGFR, ALK, ROS1 or RET have clinical applications because specific drugs are available.

Clinical performance of a comprehensive novel liquid biopsy test for identifying non-small cell lung cancer (NSCLC) patients for treatment with osimertinib.

9553 Background: Genotyping is required to identify cancer patients (pts) eligible for targeted therapy; however, many do not receive biomarker testing, in part due to limitations associated with tissue-only genotyping practices and the growing list of biomarkers recommended to be tested. Liquid biopsy overcomes many of these limitations but is not yet fully adopted. We report here the clinical performance of a comprehensive liquid biopsy test based on next generation sequencing (NGS) of circulating tumor DNA (ctDNA) for the identification of NSCLC patients with EGFR exon 19 deletions (ex19del) or L858R mutations ( EGFRm) or EGFR T790M, eligible for treatment with osimertinib. Methods: 441 (79%) of 556 pts randomized in FLAURA (NCT02296125; first-line osimertinib vs comparator EGFR TKI in EGFRm NSCLC) and 300 (72%) of 419 pts from AURA3 (NCT012151981; osimertinib vs chemotherapy in NSCLC pts with T790M at progression on EGFR TKI) were retrospectively tested with Guardant360 (G360), a 74-gene ctDNA NGS assay assessing single nucleotide variants, insertion-deletions, amplifications, and fusions in genes relevant to targeted therapy selection as well as microsatellite instability. Progression-free survival (PFS) of pts with EGFRm or T790M detected by G360 was compared to pts detected by the cobas EGFR Mutation Test (cobas) using tissue or plasma with an unadjusted cox model. Results: Treatment with osimertinib was associated with a significant PFS benefit relative to control therapy in NSCLC pts with EGFRm (FLAURA) and T790M (AURA3) detected using G360 (Table). Observed clinical benefit for pts with EGFRm or T790M detected by G360 was similar to that for pts with EGFRm or T790M identified by cobas using tissue or plasma specimens. Conclusions: This analysis demonstrates that G360 accurately identifies pts for osimertinib therapy while simultaneously providing comprehensive genotyping for other therapeutic molecular targets. The application of NGS liquid biopsy has the potential to increase rates of pts genotyped and access to precision medicine. [Table: see text]

Detection of actionable gene mutations in breast cancer by amplicon-based next-generation sequencing liquid biopsy.

1035 Background: The PI3K-inhibitor, alpelisib, was approved for the treatment of hormone receptor (HR)-positive, HER2-negative breast cancers with PIK3CA mutations based on findings from the SOLAR-1 trial. PIK3CA-positivity in tumor tissue was 29% (341/1172), but was only 15% (177/1172) in plasma cell-free DNA (cfDNA). The lower mutation detection rate observed in cfDNA may limit the clinical application of liquid biopsy in breast cancer. Amplicon-based next-generation sequencing (NGS) approach may confer improved sensitivity, allowing more effective profiling. We conducted a study to evaluate the use of this technology. Methods: Plasma cfDNA from 113 breast cancer patients (82.3% metastatic) underwent real-world testing in a CAP and ISO15189 accredited central laboratory. We analysed genetic alterations in cfDNA using an amplicon-based NGS technology. The presence of PIK3CA and other mutations relevant to breast cancer were correlated to molecular subtypes and treatment histories. Results: At least one mutation was detected in 70.8% of cases. Mutations were more frequent in metastatic cases (77.4%) compared to non-metastatic cases (27.3%). Across all patients, mutations in PIK3CA (33.6%), TP53 (32.7%), ESR1 (22.1%), GATA3 (7.1%) and ERBB2 (7.1%) were most frequently detected, in accordance with tumor tissue genotyping studies. PIK3CA mutations were more common in HR+ HER2- patients (44.4% vs 28.6% of other patients). Among PIK3CA-mutant cases, multiple PIK3CA mutations were present in 18.4% of cases, and hotspot mutations H1047R (34.2%), E542K (26.3%) and E545K (15.8%) were most frequent. An association was seen between PIK3CA mutation and prior treatment with CDK4/6 inhibitors (palbociclib, ribociclib) or mTOR inhibitor (everolimus), with 58% of PIK3CA-mutant cases having received these treatment previously compared to only 20% of PIK3CA-wild type (wt) cases. In addition, 75% of previously treated ESR1-mutant cases had specifically received hormonal treatment, compared to 60 % of ESR1-wt cases that received any treatment. Conclusions: We report similar PIK3CA mutation frequencies (~30%) with amplicon-based NGS on cfDNA compared to tumor tissue testing in breast cancer. Importantly, other driver mutations were also observed at similar frequencies as external tissue studies, implying high sensitivity as the primary reason for performance. This supports the clinical utility of an amplicon-based NGS-based approach to liquid biopsy for the sensitive detection of actionable mutations in breast cancer.

Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification.

The invasive nature and the pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids has recently gained significant traction in the liquid biopsy field. EVs offer an essential "snapshot" of their precursor cells in real time and contain an information-rich collection of nucleic acids, proteins, lipids, and so on. The analysis of protein phosphorylation, as a direct marker of cellular signaling and disease progression could be an important stepping stone to successful liquid biopsy applications. Here we introduce a rapid EV isolation method based on chemical affinity called EVtrap (extracellular vesicle total recovery and purification) for the EV phosphoproteomics analysis of human plasma. By incorporating EVtrap with high-performance mass spectrometry (MS), we were able to identify over 16 000 unique peptides representing 2238 unique EV proteins from just 5 μL of plasma sample, including most known EV markers, with substantially higher recovery levels compared with ultracentrifugation. Most importantly, more than 5500 unique phosphopeptides representing almost 1600 phosphoproteins in EVs were identified using only 1 mL of plasma. Finally, we carried out a quantitative EV phosphoproteomics analysis of plasma samples from patients diagnosed with chronic kidney disease or kidney cancer, identifying dozens of phosphoproteins capable of distinguishing disease states from healthy controls. The study demonstrates the potential feasibility of our robust analytical pipeline for cancer signaling monitoring by tracking plasma EV phosphorylation.

CD40 DNA hypermethylation in primary gastric tumors; as a novel diagnostic biomarker

AimsGastric cancer (GC) remains one of the deadliest malignancies worldwide due to its poor prognosis. DNA methylation changes, as an early event during tumor progression, constitute attractive markers for cancer diagnostics. In the current study, CD40 DNA methylation was investigated in GC as a novel epigenetic biomarker. Main methodsWe first analyzed DNA methylation microarrays from the Gene Expression Omnibus database on GC samples to evaluate the potential diagnostic value of CD40 methylation. Moreover, using q-MSP, in a set of internal samples including GC primary tumors and adjacent normal specimens, CD40 DNA methylation levels were determined. The Cancer Genome Atlas (TCGA) data on GC was also analyzed for further validation. Key findingsOur results illustrated significant CD40 hypermethylation in GC samples compared to normal specimens which was significantly correlated with the clinical stage of malignancy. Besides, the high accuracy of CD40 methylation as a diagnostic biomarker in GC was confirmed using the ROC curve analysis with an AUC value of 0.9089. Also, gene set enrichment analysis showed that CD40 is mainly involved in biological processes regulating immune response activation in GC. Further analysis of other prevalent cancer entities in TCGA showed that CD40 hypermethylation is a common event during tumor progression and could be considered as a potential biomarker for the detection of breast, colorectal, and prostate cancers as well. SignificanceThe finding of this study suggests that CD40 methylation as a potential pan biomarker could be a valuable target for liquid biopsy application of human cancers.

THE ROLE OF LIQUID BIOPSY IN THE CHOICE OF TACTICS FOR THE TREATMENT OF NON-SMALL CELL LUNG CANCER

This article describes the potential uses of liquid biopsy for non-small cell lung cancer as a method for determining the mutational profile, as well as minimal residual disease after radical surgical treatment. This paper describes the advantages and disadvantages of this method compared to tissue biopsy. Presented potential applications of liquid biopsy to personalize treatment.

Circulating tumor cells as Trojan Horse for understanding, preventing, and treating cancer: a critical appraisal.

Circulating tumor cells (CTCs) are regarded as harbingers of metastases. Their ability to predict response to therapy, relapse, and resistance to treatment has proposed their value as putative diagnostic and prognostic indicators. CTCs represent one of the zeniths of cancer evolution in terms of cell survival; however, the triggers of CTC generation, the identification of potentially metastatic CTCs, and the mechanisms contributing to their heterogeneity and aggressiveness represent issues not yet fully deciphered. Thus, prior to enabling liquid biopsy applications to reach clinical prime time, understanding how the above mechanistic information can be applied to improve treatment decisions is a key challenge. Here, we provide our perspective on how CTCs can provide mechanistic insights into tumor pathogenesis, as well as on CTC clinical value. In doing so, we aim to (a) describe how CTCs disseminate from the primary tumor, and their link to epithelial-mesenchymal transition (EMT); (b) trace the route of CTCs through the circulation, focusing on tumor self-seeding and the possibility of tertiary metastasis; (c) describe possible mechanisms underlying the enhanced metastatic potential of CTCs; (d) discuss how CTC could provide further information on the tissue of origin, especially in cancer of unknown primary origin. We also provide a comprehensive review of meta-analyses assessing the prognostic significance of CTCs, to highlight the emerging role of CTCs in clinical oncology. We also explore how cell-free circulating tumor DNA (ctDNA) analysis, using a combination of genomic and phylogenetic analysis, can offer insights into CTC biology, including our understanding of CTC heterogeneity and tumor evolution. Last, we discuss emerging technologies, such as high-throughput quantitative imaging, radiogenomics, machine learning approaches, and the emerging breath biopsy. These technologies could compliment CTC and ctDNA analyses, and they collectively represent major future steps in cancer detection, monitoring, and management.

Perspectives of the Application of Liquid Biopsy in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common gastrointestinal tumors and the second leading cause of cancer death worldwide. Since traditional biopsies are invasive and do not reflect tumor heterogeneity or monitor the dynamic progression of tumors, there is an urgent need for new noninvasive methods that can supplement and improve the current management strategies of CRC. Blood-based liquid biopsies are a promising noninvasive biomarker that can detect disease early, assist in staging, monitor treatment responses, and predict relapse and metastasis. Over time, an increasing number of experiments have indicated the clinical utility of liquid biopsies in CRC. In this review, we mainly focus on the development of circulating tumor cells and circulating tumor DNA as key components of liquid biopsies in CRC and introduce the potential of exosomal microRNAs as emerging liquid biopsy markers in clinical application for CRC.

Circulating tumor DNA and liquid biopsy in oncology.

Techniques for analyzing circulating tumor DNA (ctDNA) to detect, characterize and monitor cancer have matured rapidly. An increasing body of clinical evidence is demonstrating the capabilities of this technology as a diagnostic test. The full potential of ctDNA liquid biopsy in the diagnosis, characterization and management of solid and hematological malignancies will be uncovered through interventional clinical trials evaluating clinical utility. In this Review, we discuss the current landscape of ctDNA liquid-biopsy applications across the cancer continuum and highlight opportunities for clinical investigation.

Application of liquid biopsy for surgical management of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. Although drug development over the past decade has gradually improved the prognosis of PDAC, the prognosis remains extremely poor. The predominant determinant of a poor prognosis is that patients are already at the advanced stage when they are diagnosed. Therefore, it is essential to detect early‐stage PDAC to ensure a good prognosis. However, in general, being asymptomatic at the early stage makes the detection of early‐stage PDAC very difficult. Recently, much attention has been focused on the utility of a liquid biopsy as a biomarker. Theoretically, early‐stage tumors can be detected even under asymptomatic conditions. A number of studies on liquid biopsies have been reported so far. Several biomarkers, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCS), and exosomes, are used in liquid biopsies, with the potential to be applied to the clinical setting. Each biomarker is reported to have different utilities, such as the detection of early‐stage disease, the differential diagnosis of PDAC from other types of pancreatic tumors, the prediction of the prognosis or risk of recurrence, and monitoring the treatment response. In this review, we focus on ctDNA, CTCS, and exosomes as representative liquid biopsy biomarkers and describe the specific functions of each biomarker in the treatment of PDAC. Furthermore, we discuss the application of liquid biopsies, especially for the surgical management of PDAC.

Current and future applications of liquid biopsy in nonsmall cell lung cancer from early to advanced stages.

Liquid biopsy refers to the analysis of any tumour-derived material circulating in the blood or any other body fluid. This concept is particularly relevant in lung cancer as the tumour is often difficult to reach and may need an invasive and potentially harmful procedure. Moreover, the multitude of anticancer drugs and their sequential use underline the importance of conducting an iterative assessment of tumour biology. Liquid biopsies can noninvasively detect any targetable genomic alteration and guide corresponding targeted therapy, in addition to monitoring response to treatment and exploring the genetic changes at resistance, overcoming spatial and temporal heterogeneity.In this article, we review the available data in the field, which suggest the potential of liquid biopsy in the area of lung cancer, with a particular focus on cell-free DNA and circulating tumour cells. We discuss their respective applications in patient selection and monitoring through targeted therapy, as well as immune checkpoint inhibitors. The current data and future applications of liquid biopsy in the early stage setting are also investigated.Liquid biopsy has the potential to help manage nonsmall cell lung cancer throughout all stages of lung cancer: screening, minimal residual disease detection to guide adjuvant treatment, early detection of relapse, systemic treatment initiation and monitoring of response (targeted or immune therapy), and resistance genotyping.