Tumor Liquid Biopsy Research Articles

Research progress of the clinical applications of tumor liquid biopsy

In recent years, researches about liquid biopsies which contain circulating tumor cells (CTC), circulating tumor DNA (ctDNA) and exosomes in the body fluids of patients with solid tumor have caused great concern. Compared with traditional biopsies, liquid biopsies have many advantages as they are real time, noninvasive and comprehensive. With the progress in the detecting techniques of CTC, ctDNA and exosomes, liquid biopsies can be applied to the early screening, diagnosis, treatment, prognostic evaluation of tumor. This review focuses on the recent advances in detection methods and clinical applications of CTC, ctDNA and exosomes.(Chin J Lab Med, 2018, 41: 621-626) Key words: Liquid biopsy; Neoplastic cells, circulating; Circulating tumor DNA; Exosomes

Abstract 2217: Concordance of PIK3CA mutations in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer: A systematic literature review

Abstract Introduction: This systematic literature review sought to determine the concordance of PIK3CA mutations in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC) patients. Methods: A comprehensive search of PubMed/MEDLINE, EMBASE, Cochrane Central, and select conference abstracts was performed by two independent researchers that included, but was not limited to, keywords: “breast neoplasm,” “PIK3CA protein,” “hormone receptor positive,” and “metastases.” Databases were searched for English-language publications in HR+, HER2- mBC published between January 1993 through August 2017. Studies were included if results reported were specific to patients with HR+, HER2- mBC and reported data comparing PIK3CA mutations from liquid biopsies versus tumor biopsies or primary tumor samples versus metastatic lesion samples. Concordance was assessed between tissue samples (mutations found in primary tumors versus those from metastatic lesions) and between biopsy methods (i.e., tumor biopsy versus liquid biopsy) when the assessment was done at a variety of time points including time of initial diagnosis and time of diagnosis at disease progression. Content analysis was employed to quantify collected data elements. Results: Of 558 studies included for full-text review, four met inclusion criteria (number of patient samples [n]=358). One study (n = 9) found the concordance between the mutation status of primary breast tumors to the mutation status of metastatic lesions to be 100%. Three studies (n = 47; n = 55; n = 247) determined the concordance of PIK3CA mutations between tumor biopsies and liquid biopsies with concordance ranging from 70.4% to 94%. One study found that the concordance of circulating tumor DNA (ctDNA) against tumor biopsy was higher among metastatic lesions only (81.6%) relative to using all tumor samples (either primary or metastatic lesions) (70.4%). Conclusions: Available evidence suggested high level of concordance across sampling methods as well as across time. However, additional evidence is needed to increase study generalizability as well as to assess other possible confounding factors, such as differential timing between tumor and liquid biopsies. Citation Format: Alejandra Aguilar, Joni Dean, Elizabeth Anderson, Lea Mollon, Lisa Davis, Terri Warholak, Ayal Aizer, Emma Platt, Aditya Bardiya, Derek Tang. Concordance of PIK3CA mutations in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer: A systematic literature review [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 2217.

Abstract CT106: Local ablative therapy for oligoprogressive, EGFR-mutant, non-small cell lung cancer (NSCLC) after treatment with osimertinib

Abstract Background: Osimertinib is effective in EGFR-mutant NSCLC in both first- and second-line settings. However, the efficacy of osimertinib is limited by the universal development of resistance. Retrospective studies have shown a survival benefit to local ablative therapy (LAT) in those who develop oligoprogressive disease (progression at a limited number of anatomic sites) on EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. Methods: We initiated a prospective study of LAT in patients with EGFR-mutant NSCLC and oligoprogression during treatment with osimertinib (NCT02759835). Patients with no prior EGFR-TKI therapy (cohort 1) or progression after 1st/2nd generation EGFR-TKI therapy and acquired T790M mutation (cohort 2) start osimertinib. Upon progression, eligible patients with <= 5 progressing sites undergo LAT and resume osimertinib after LAT. Patients previously treated with osimertinib eligible for LAT upon development of oligoprogression are also enrolled (cohort 3). The primary objective is to evaluate the safety and efficacy of reinitiation of osimertinib after LAT (assessed by PFS2). Another goal is to assess mechanisms of resistance to osimertinib by multi-omics analyses of tumor tissue and liquid biopsies. Results: Between 04/2016 and 01/2018, 24 patients were enrolled (cohort 1: 16, cohort 2: 5, cohort 3: 3). Median age was 59.8 (range 36-77). The most common adverse events (AEs) on osimertinib treatment were rash, diarrhea, thrombocytopenia, and QTc prolongation with most of the AEs being grade 1 or 2. Among evaluable patients, confirmed objective response rates prior to LAT in cohorts 1 and 2 were 66.7 % (8/12) and 75% (3/4), respectively, with 11.2 months median PFS (95% CI: 3.6 months-undefined) in cohort 1 and 10.9 months in cohort 2 (95% CI: 6.7-12.8 months). To date, 12 patients had progressive disease, 8 of which had oligoprogression and subsequently underwent LAT (cohort 1: 3; cohort 2: 2; cohort 3: 3). Two patients were treated with combination of surgery and radiotherapy (RT), 4 patients with surgery, and 2 patients with RT. Median PFS2 was 2.3 months (95% CI: 1.0-5.9 months). Whole exome sequencing (WES) was performed on tumor tissues obtained pre-treatment and upon progression on osimertinib. MET amplification, transformation to small cell lung cancer, and EGFR C797S mutation were identified as mechanisms of resistance to osimertinib. Results of molecular analyses of tumor tissue and blood ctDNA will be presented. Conclusions: Patients with EGFR-mutant NSCLC and oligoprogression after EGFR-TKI therapy can be safely treated with LAT. In select patients, this approach could potentially maximize duration of EGFR-TKI treatment and prevent premature switching to other systemic therapies. Citation Format: Chul Kim, Nitin Roper, Chuong Hoang, Laura Wisch, Maureen Connolly, Hsien-Chao Chou, Jun Wei, Manoj Tyagi, Constance M. Cultraro, Liqiang Xi, Maryam Waris, Khoa Dang Nguyen, Eva Szabo, Emerson Padiernos, Aparna Kesarwala, Shaojian Gao, Seth M. Steinberg, Mark Raffeld, Arun Rajan, Javed Khan, Udayan Guha. Local ablative therapy for oligoprogressive, EGFR-mutant, non-small cell lung cancer (NSCLC) after treatment with osimertinib [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 CT106.

Abstract 2225: Limitations on mutation detection for early detection of cancer

Abstract Introduction: Cell-free DNA (cfDNA) has potential utility for early non-invasive detection of cancer. We assess the feasibility of cfDNA mutational assays for early detection based on their physiological and economic requirements. We further review alternative biological signals of early cancer and the potential of machine learning to integrate these signals into reliable diagnostics. Methods: A binomial model was used to assess depth and input requirements, with parameters derived from published data on cfDNA sequencing. Alternative strategies for early detection were assessed by literature review. Results: 30,000x unique coverage is required for 95% sensitivity for 1 mutant read at 0.01% variant allele frequency, requiring 180ng cfDNA input at 50% process efficiency; current sequencing costs and reimbursement levels may make such tests economically infeasible (Table). 5th percentile plasma cfDNA concentration in the screening population is ~2.3 ng/mL, requiring >140mL blood collection for test failure rate <5%. Somatic mutational heterogeneity creates significant specificity challenges. Proteins, RNAs, and compartmentalized macromolecules are more abundant and may overcome cfDNA's issues of quantity, but irrelevant biological processes may confound the data. New techniques in machine learning, including latent variable modeling, may be used to integrate heterogeneous data from multiple sources to overcome these challenges. Conclusions: Mutation-detection assays may not be feasible for early cancer detection due to limited sensitivity arising from low concentrations of cfDNA, heterogeneity leading to specificity challenges, and prohibitive cost. Integrating markers beyond mutations with modern machine learning may provide a potential route to statistically robust biomarker development for early cancer detection. Table. Assay requirements for tumor liquid biopsy and mutation-based early cancer detection. Sequencing cost by panel sizeVAF 95% sensitivityCorrected depthRaw depthInput volume (blood)TEC-Seq1 58 genes 81 kbRazavi et al2 508 genes 2000 KbWES ~20k genes 50,000 KbTumor liquid biopsy0.1%3000x15,000x15 mL$14$340$8300Early cancer detection0.01%30,000x150,000x150 mL$140$3400$83,0001Phallen J, et al. Sci Transl Med. 2017 Aug 16;9(403). 2Razavi P, et al. J Clin Oncol. 2017;35(suppl):abstr 11526.Model parameters: 1) No more than 5% of samples may fail because of insufficient cfDNA quantity; 2) 95% sensitivity to detect one read from any cancer-derived allele, assuming that one is present in the sample; 3) 50% process efficiency: half of the cfDNA molecules in the input blood sample are represented in the sequencer output; 4) 5x oversampling in sequencing for error correction; 5) 100% on-target rate in target enrichment; 6) “$1000 genome” sequencing costs: US $1000/(30 x 3 Gbp) of sequencing bandwidth; 7) only sequencing costs computed; all other costs (labor, equipment, facilities, depreciation, etc.) accounted at $0; 8) panel expansion neither reduces input requirements nor increases sequencing requirements.VAF = variant allele frequency; TEC-Seq = targeted error correction sequencing; WES = whole exome sequencing. Citation Format: Imran S. Haque, Gabriel Otte, Olivier Elemento. Limitations on mutation detection for early detection of cancer [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 2225.

Comparison of detection methods of <em>EGFR</em> T790M mutations using plasma, serum, and tumor tissue in EGFR-TKI-resistant non-small cell lung cancer

BackgroundOsimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor, exerts remarkable effects against EGFR T790M resistance mutation-positive non-small cell lung cancer. Identifying T790M mutation by re-biopsy is essential before prescribing osimertinib. Tissue biopsy is the golden standard for this purpose, but several factors limit its success rate. The liquid biopsy with blood, using circulating tumor DNA, has been an alternative method. However, the true biological meaning and equivalence of liquid biopsy and tumor biopsy are still under investigation. Especially, the usefulness of serum samples to detect T790M mutation is not yet been known.Patients and methodsWe prospectively evaluated the sensitivity, specificity, and parallelism of the detection of EGFR mutations in tissue re-biopsy and liquid biopsy (plasma and serum), simultaneously, from June 2016 to May 2017. EGFR mutations in tumor re-biopsy were evaluated by COBAS ver2 and PNA-LNA PCR clamp method, and those in liquid biopsy were evaluated with COBAS ver2.ResultsFifteen patients were enrolled. In 10 patients whose EGFR mutation was detected in liquid biopsy, the original EGFR mutation (exon 19 del or L858R) was detected in all patients. Detection of EGFR mutation by COBAS ver2 and by PNA-LNA method was almost the same in tissue re-biopsy. The detection rate of T790M was lower than that of the original EGFR mutation in liquid biopsy compared to that in tissue re-biopsy. The detection of T790M in serum exhibited a higher specificity (67%) and positive predictive value (50%) than that in plasma (50% and 40%, respectively). The detection sensitivity was similar in plasma and serum.ConclusionPlasma, serum, and tissue genotyping can have complementary roles for detecting EGFR-T790M using COBAS ver2. Repeated tests with different samples and different methods may improve accuracy of T790M detection and will lead to the maximum benefit for the patient.

The utilization of next-generation sequencing to detect somatic mutations and predict clinical prognosis of Chinese non-small cell lung cancer patients.

PurposeThe development of next-generation sequencing (NGS) has revolutionized the understanding of oncogenesis of multiple types of cancer, including non-small cell lung cancer (NSCLC). However, there has been some debate over the utility of NGS for predicting patient prognosis and determining molecular targeted therapy. Therefore, we sought to demonstrate the numerous applications of NGS in the prognostic predictions and treatment of NSCLC patients.Materials and methodsWe performed NGS on either liquid or tissue tumor biopsies obtained from 53 NSCLC patients. The sequences were analyzed for oncogenic mutations, which were then correlated to clinical prognosis and smoking history.ResultsNGS of tumor biopsies detected both well-known driver mutations as well as rare or novel mutations. EGFR was the most frequently mutated gene, accounting for 32.4% (33/102) of the somatic mutations in this study. The EGFR mutations detected included rare variants such as EGFR exon 19 insertion (K745_E746insIPVAIK) and in cis H835L+L833V. Additionally, novel RET fusion mutations PCM1–RET and ADD3-RET were detected in two adenocarcinoma patients. To demonstrate the functional applications of NGS, we correlated mutations with patient characteristics, outcomes of matched targeted therapy, and outcomes based on allelic frequency of the EGFR-T790M mutation. Finally, we demonstrated that circulating tumor DNA can be used both to measure response to targeted therapy and as a predictor of clinical outcome, by presenting a case study of a single patient.ConclusionWe demonstrated that NGS can be used in multiple applications to effectively identify potential oncogenic driver mutations, guide mutation-targeted therapy decisions, and predict clinical outcomes in Chinese NSCLC patients.

Focused Ultrasound-enabled Brain Tumor Liquid Biopsy

Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500–4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures.

Circulating tumor DNA (ctDNA) in the era of personalized cancer therapy.

The heterogeneity of tumor is considered as a major difficulty to victorious personalized cancer medicine. There is an extremeneed of consistent response evaluation for in vivo tumor heterogeneity anditscoupledconflict mechanisms. In this occasion researchers will be able to keep pace withpredictive, preventive, personalized, and Participatory (P4) medicine for cancer managements. In fact tumor heterogeneity is a central part of cancer evolution,soin order to progress in understanding of the dynamics within a tumor some diagnostic apparatus should be improved. Latest molecular techniques like Next generation Sequencing (NGS) and ultra-deep sequencing could disclose some clones within a liquid tumor biopsy which mainly responsible of treatment resistance. Circulating tumor DNA (ctDNA) as a main component of liquid biopsy is agifted biomarker for cancer mutation tracking as well as profiling. Personalized medicine facilitate learning regarding to genetic pools of tumor and their possible respond to treatment which could be much easier by using of ctDNA.With this information, cliniciansarelooking forward to find the best strategies for prevention, screening, and treatment in the way of precision medicine. Currently, numerous clinical efficacy of such informative improved treatment are in hand. Here we represent the review of plasma-derived ctDNA studies use in personalized cancer managements.

Application and progress of blood-based microRNA detection in tumor liquid biopsy

Application and progress of blood-based microRNA detection in tumor liquid biopsy

NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.

Nanotechnology-Based Strategies for Early Cancer Diagnosis Using Circulating Tumor Cells as a Liquid Biopsy.

Circulating tumor cells (CTCs) are cancer cells that shed from a primary tumor and circulate in the bloodstream. As a form of “tumor liquid biopsy”, CTCs provide important information for the mechanistic investigation of cancer metastasis and the measurement of tumor genotype evolution during treatment and disease progression. However, the extremely low abundance of CTCs in the peripheral blood and the heterogeneity of CTCs make their isolation and characterization major technological challenges. Recently, nanotechnologies have been developed for sensitive CTC detection; such technologies will enable better cell and molecular characterization and open up a wide range of clinical applications, including early disease detection and evaluation of treatment response and disease progression. In this review, we summarize the nanotechnology-based strategies for CTC isolation, including representative nanomaterials (such as magnetic nanoparticles, gold nanoparticles, silicon nanopillars, nanowires, nanopillars, carbon nanotubes, dendrimers, quantum dots, and graphene oxide) and microfluidic chip technologies that incorporate nanoroughened surfaces and discuss their key challenges and perspectives in CTC downstream analyses, such as protein expression and genetic mutations that may reflect tumor aggressiveness and patient outcome.

Abstract 4019: A PCR-bias free capture-based library preparation platform permitting highly accurate and sensitive CNA detection in tumor molecular profiling and liquid biopsy

Abstract Next Generation Sequencing is increasingly implemented as a diagnostic test to identify actionable mutations in cancer patient samples. However, for routine diagnostics, tumor DNA is extracted from formalin-fixed, paraffin-embedded (FFPE) samples, which yields low quantity of damaged DNA. Inability to accurately repair the ends of these DNA fragments impairs adapter ligation by standard double stranded ligation methods. The resulting low yield of adapter-ligated DNA introduces the need for whole-genome PCR amplification prior to target capture. The drawback of such PCR amplification is the introduction of PCR biases, causing reduced sensitivity in the detection of copy number alterations (CNAs), an important biomarker for targeted therapy. To address the need for a library preparation platform that performs well with low quality and quantity DNA, and without relying on massive PCR amplification, we developed an improved, in-solution, version the OS-Seq targeted enrichment assay. OS-Seq circumvents the reliance on PCR amplification by using a single-stranded adapter ligation approach. Damaged bases induced by formalin fixation are removed by excision instead of attempting repair, and then DNA is denatured prior to adapter ligation. This method of adapter ligation result in yields of ~50% for low quality samples, eliminating the need for whole genome PCR. OS-Seq directly uses the adapter-ligated DNA in a linear targeted primer-extension, followed by low-cycle post-capture PCR expansion with Illumina bridge-PCR primers prior to library sequencing. We investigated the PCR duplication rate of the OS-Seq libraries by including an 11-mer random barcode to track unique molecules. We found that most input molecules were present in the sequencing reads at only one copy. Further, we demonstrate a linear correlation between the amount of DNA input (ranging from 1 to 600 ng) and the number of unique molecules sequenced (R2=0.94). Importantly, we show that this low PCR bias allows OS-Seq to detect CNAs in Coriell and Horizon Diagnostic cell lines highly concordant to digital PCR detection (R2=0.96). Further, we present CNA calling on cell line DNA sonicated to 200 bp fragments at 10 ng DNA input, mimicking cell-free DNA. In addition to CNA detection, OS-Seq detects SNVs with a sensitivity of 92-97% and a specificity of 100% down to 5% VAF. In conclusion, the OS-Seq library preparation method relies on single stranded adapter ligation and in-solution target capture, which generates uniform coverage with minimal PCR requirement, resulting in highly sensitive CNA calling. Note: This abstract was not presented at the meeting. Citation Format: Anna Vilborg, Yosr Bouhlal, Ryan Koheler, Daniel Mendoza, Federico Goodsaid, Yannick Pouliot, Austin So, Francisco De La Vega, Hanlee Ji. A PCR-bias free capture-based library preparation platform permitting highly accurate and sensitive CNA detection in tumor molecular profiling and liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2017-4019

EMT: Present and future in clinical oncology.

Epithelial/mesenchymal transition (EMT) has emerged as a key regulator of metastasis by facilitating tumor cell invasion and dissemination to distant organs. Recent evidences support that the reverse mesenchymal/epithelial transition (MET) is required for metastatic outgrowth; moreover, the existence of hybrid epithelial/mesenchymal (E/M) phenotypes is increasingly being reported in different tumor contexts. The accumulated data strongly support that plasticity between epithelial and mesenchymal states underlies the dissemination and metastatic potential of carcinoma cells. However, the translation into the clinics of EMT and epithelial plasticity processes presents enormous challenges and still remains a controversial issue. In this review, we will evaluate current evidences for translational applicability of EMT and depict an overview of the most recent EMT in vivo models, EMT marker analyses in human samples as well as potential EMT therapeutic approaches and ongoing clinical trials. We foresee that standardized analyses of EMT markers in solid and liquid tumor biopsies in addition to innovative tools targeting the E/M states will become promising strategies for future translation to the clinical setting.

The progression of tumor liquid biopsy and its applications in precision medicine

Malignant tumor is the most heterogeneous human disease, and it has been gradually reached the consensus in clinical oncology that the personalized treatment of tumor patients should be directed by molecular diagnostics. However, the ever-changing characteristic of tumor in heterogeneity and the fact that tissue samples are usually unavailable highlight the function and significant of liquid biopsy. The material that could be used in liquid biopsy are mainly the circulating tumor DNA, circulating tumor cells and extracellular vesicles, which though have their own advantages and disadvantages, but could complement to each other, thus accurately and comprehensively reflecting the characteristic of the tumor and gave the best direction for patient's individualized therapy. This article reviewed the recent advances of liquid biopsy and explained its importance in tumor precision medicine.

Personalizing Therapy for Metastatic Prostate Cancer: The Role of Solid and Liquid Tumor Biopsies.

Although biopsies of metastatic prostate cancer are rarely undertaken in the clinical setting, there is increasing interest in developing personalized approaches to therapy by taking into account the genetic and phenotypic changes in an individual tumor. Indeed, analysis of metastatic prostate tumors can predict sensitivity to agents that inhibit DNA repair and resistance to novel hormonal agents, such as abiraterone and enzalutamide, and identify phenotypic changes, such as neuroendocrine differentiation, that have important clinical implications. Although obtaining metastatic tumor tissue is necessary for this genomic and molecular profiling, knowing when to biopsy, selecting the appropriate metastatic lesion, and interpreting the results are major challenges facing clinicians today. In this article, we discuss the rationale for obtaining metastatic tumor tissue, review the bioinformatic approach to analyzing these specimens, discuss the timing and approach to solid and liquid tumor biopsies, review the challenges associated with obtaining and acting on clinically relevant results, and discuss opportunities for the future.

Personalizing Therapy for Metastatic Prostate Cancer: The Role of Solid and Liquid Tumor Biopsies

Although biopsies of metastatic prostate cancer are rarely undertaken in the clinical setting, there is increasing interest in developing personalized approaches to therapy by taking into account the genetic and phenotypic changes in an individual tumor. Indeed, analysis of metastatic prostate tumors can predict sensitivity to agents that inhibit DNA repair and resistance to novel hormonal agents, such as abiraterone and enzalutamide, and identify phenotypic changes, such as neuroendocrine differentiation, that have important clinical implications. Although obtaining metastatic tumor tissue is necessary for this genomic and molecular profiling, knowing when to biopsy, selecting the appropriate metastatic lesion, and interpreting the results are major challenges facing clinicians today. In this article, we discuss the rationale for obtaining metastatic tumor tissue, review the bioinformatic approach to analyzing these specimens, discuss the timing and approach to solid and liquid tumor biopsies, review the challenges associated with obtaining and acting on clinically relevant results, and discuss opportunities for the future.

Establishment of a digital PCR platform for detection of EGFR T790M mutation in plasma circulating DNA from NSCLC patients

Objective Digital PCR (dPCR) was established to detect plasma epidermal growth factor receptor (EGFR) T790M mutation of non-small cell lung cancer (NSCLC) patients and was evaluated in terms of analytical performance and clinical application significance. Methods The specific primers and probes for EGFR T790M mutation and wildtype were designed to establish dPCR platform. Limit of blank, sensitivity and linearity of dPCR were evaluated by the detection of plasmids with different concentrations to set up optimal reporting system and reanalyzing process.The mutation of EGFR T790M in plasma and tissue samples from 10 patients with advanced NSCLC resistant to EGFR-TKI therapy who were enrolled in Zhongshan Hospital Fudan University from January 2014 to October 2015 were analyzed by dPCR and amplification refractory (ARMS), respectively. The consistency was evaluated between dPCR and ARMS by Chi-square test. The correlation of T790M abundance detected by dPCR between plasma and tissue samples was also analyzed by Peasrson correlation analysis. Results Limit of blank and sensitivity of dPCR was 10 copies and 0.01%, respectively. dPCR was evaluated as linear in the range of 0.01%-100%(Y=1.226X-3.984, R2=0.999). The consistency between dPCR and ARMS of tissue samples was good (kappa=0.80), while the positive rates of plasma T790M detected by dPCR was significantly higher than ARMS (50% vs 20%, P<0.05). It was found that T790M abundance detected by dPCR was highly correlated between lung cancer tissue and plasma(R=0.923, P<0.05)using Pearson correlation analysis. Conclusions A new method of dPCR with high sensitivity and absolute quantification is established for the detection of EGFR T790M mutation in plasma from advanced NSCLC patients, which brings tumor liquid biopsy into real. It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in patients with advanced NSCLC resistant to EGFR-TKI.(Chin J Lab Med, 2016, 39: 170-175) Key words: Polymerase chain reaction; Lung neoplasms; Receptor, epidrmal growth factor; DNA mutational analysis

Diagnostic implications of intrapatient genetic tumor heterogeneity

ABSTRACTThe complex genetic composition of neuroblastoma emphasizes the importance of conscientious and meticulous diagnosis. Clones with amplification or segmental chromosomal aberrations sometimes remain hidden. Several determinations should be performed when sufficient tumor material is available to establish the final diagnosis by combining the results of different techniques on tumor fragments or liquid biopsies.

Abstract P3-10-03: Etirinotecan pegol target-specific pharmacodynamic biomarkers in circulating tumor cells from patients with metastatic breast cancer in the phase 3 BEACON study

Abstract Background: Etirinotecan pegol (EP) is the first long-acting topoisomerase 1-inhibitor providing sustained level of active metabolite that concentrate in the tumor for an entire chemotherapy cycle. In a Phase 2 study (Lancet Oncology 2013), EP demonstrated a 29% overall response rate in patients with metastatic breast cancer, leading to the Phase 3 global BEACON study in this population. Detection of circulating tumor cells (CTCs) using immunomagnetic EpCAM-based methods have been conceptually accepted as a "liquid tumor biopsy". To circumvent the limited recovery of CTCs these methods provide for molecular profiling applications, we isolated CTCs based on an antibody-independent, continuous flow dielectrophoresis (DEP) technology (ApoStream®). We developed quantitative multiplex immunofluorescent assays for target-specific pharmacodynamics (PD) biomarkers for EP in CTCs isolated pre- and post-treatment. Here we present the distribution of the PD biomarkers in CTC samples collected from BEACON patients. Methods: Donation of blood samples for CTC analysis was voluntary. BEACON patients participating in the substudy had serial (pre-dose, Cycle 2 Day 1, Cycle 4 Day 1, End of Treatment) 7.5-mL whole blood samples drawn in EDTA tubes and shipped ambient to ApoCell (Houston, TX) within 96 hrs for processing. PBMCs were separated by Ficoll® gradient, and CTCs were isolated using ApoStream® technology. Isolated cells were deposited on three slides and stained for DAPI, CD45, and cytokeratin markers, as well as 2-3 of the seven PD markers, and analyzed by iCys® laser scanning cytometer equipped with image analysis software. CTC count, mean fluorescence intensity (MFI) of PD biomarkers, and % marker-positive cells were analyzed for their distribution. Results: 75% of the 852 BEACON patients participated in the CTC substudy, yielding 649 pre-treatment CTC samples. As of Mar14, 538 Cycle 2 Day 1, 281 Cycle 4 Day 1, and 394 End of Treatment samples were collected. 98% of pre-treatment blood samples were successfully processed. 97% had detectable CTCs, with a median of 504 CTCs (interquartile range: 128-1432). PD markers could be analyzed in 83-92% of samples after implementing a minimum requirement of 10 CTCs analyzed. Staining was positive for Top1, Top2, μH2Ax, Rad51, ABCG2, Ki67, and TUNEL in 82%, 71%, 14%, 30%, 20%, 56%, and 94% of samples, respectively. Percent of marker-positive cells varied from 0.2-69%, with 6- to 37-fold interquartile ranges. Median MFI in these cells varied between 25x103 and 100x103, with 1.2- to 1.6-fold interquartile ranges. Conclusions: CTC collection was successfully incorporated into the BEACON study, with 75% patient participation. CTC detection rate using ApoStream® was high, permitting evaluation of biomarkers at baseline and over time. EP target-specific PD biomarkers can be measured in CTCs isolated from patients participating in BEACON and will be assessed for their potential to predict clinical response. Final BEACON efficacy and safety results are expected in early 2015, which will allow further analysis of CTCs with patient outcome (response, PFS, OS) and change in CTCs at time of progression. Citation Format: Edith A Perez, Katie Caygill, Alison L Hannah, Javier Cortes, Ahmad Awada, Joyce O'Shaughnessy, Christopher Twelves, Hope Rugo, Seock-Ah Im, Kenna Anderes, Darren W Davis, Ute Hoch. Etirinotecan pegol target-specific pharmacodynamic biomarkers in circulating tumor cells from patients with metastatic breast cancer in the phase 3 BEACON study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-10-03.

Nanostructure embedded microchips for detection, isolation, and characterization of circulating tumor cells.

ConspectusCirculating tumor cells (CTCs) are cancer cells that break away from either a primary tumor or a metastatic site and circulate in the peripheral blood as the cellular origin of metastasis. With their role as a “tumor liquid biopsy”, CTCs provide convenient access to all disease sites, including that of the primary tumor and the site of fatal metastases. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status without the flaws and limitations encountered in performing conventional tumor biopsies. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies.Inspired by the nanoscale interactions observed in the tissue microenvironment, our research team at UCLA pioneered a unique concept of “NanoVelcro” cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of Velcro: when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding. Through continuous evolution, three generations (gens) of NanoVelcro CTC chips have been established to achieve different clinical utilities. The first-gen NanoVelcro chip, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Side-by-side analytical validation studies using clinical blood samples suggested that the sensitivity of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch. In conjunction with the use of the laser microdissection (LMD) technique, second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation. The individually isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger sequencing and next-generation sequencing, NGS) to verify the CTC’s role as tumor liquid biopsy. Created by grafting of thermoresponsive polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive NanoVelcro) have demonstrated the capture and release of CTCs at 37 and 4 °C, respectively. The temperature-dependent conformational changes of polymer brushes can effectively alter the accessibility of the capture agent on SiNS, allowing for rapid CTC purification with desired viability and molecular integrity.This Account summarizes the continuous evolution of NanoVelcro CTC assays from the emergence of the original idea all the way to their applications in cancer research. We envision that NanoVelcro CTC assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.