Plasma Samples Of Cancer Patients Research Articles

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1021.

Optimisation of dispersive liquid-liquid microextraction for plasma sample preparation in bioanalysis of CDK4/6 inhibitors in therapeutic combinations for breast cancer treatment

Cyclin D dependent kinase 4 and 6 (CDK 4/6) inhibitors are novel anticancer drugs used in therapeutic combinations with endocrine therapy for breast cancer treatment. Their determination in patient plasma is of high interest as a prerequisite for possible therapeutic drug monitoring. Dispersive liquid-liquid microextraction (DLLME) shows great potential in bioanalytical sample preparation. Its simplicity and speed, along with the suitability for using small amounts of sample and hazardous solvents are some of its main advantages. However, its application on plasma samples is scarce and requires further development. The aim of this work was to explore the applicability of DLLME in the simultaneous extraction of six drugs of interest from human plasma, with an emphasis placed on achieving high extraction recoveries with low sample and solvent consumption. To tackle the low availability and amount of the plasma sample, as well as the complexity of the biological matrix, three novel DLLME modes are proposed: organic sample DLLME (OrS-DLLME), aqueous sample DLLME (AqS-DLLME), and a modified air-assisted DLLME (AA-DLLME). The extractant and disperser type and volume, volume ratios of all the components in the ternary system, effect of pH and salting out were optimised for all three proposed modes of DLLME. Optimised representative DLLME-HPLC-DAD-FLD method was validated and shown to be linear (R > 0.994), precise (RSD ≤13.8%, interday), accurate (bias −13.1–13.1%, interday) and robust (relative effect −3.34–6.08%). Simultaneous extraction of all six drugs with high recoveries (81.65–95.58%) was achieved. Sample volumes used were as low as 50–100 μL, with necessary organic solvent volumes in μL ranges. Greenness scores obtained using the AGREE software were between 0.63 and 0.66, demonstrating compliance with green analytical chemistry principles. Finally, the validated method was successfully applied on breast cancer patient plasma samples.

Molecularly imprinted electrochemical sensor for the selective and sensitive determination of octreotide in cancer patient plasma sample

In this study, a molecularly imprinted polymer film (P (ANI)@MIP) on the electrode surface was fabricated using aniline as a functional monomer and octreotide (OC) as a template molecule. The developed P (ANI)@MIP was electrochemically electropolymerized on a glassy carbon electrode (GCE) surface. Each step of MIP production was evaluated by viewing the [Fe (CN)6]3-/4- signal obtained using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The P (ANI)@MIP film layer was studied with a scanning electron microscope (SEM), Raman, and contact angle measurements. The parameters consisting of monomer, template ratio, cycle number, removal solution, removal time, and rebinding time were optimized to obtain the best electrochemical sensor. The developed method was validated in line with ICH guidelines. The linear range, LOD, and LOQ were found as 10–80 fM, 0.801 fM, and 2.670 fM, respectively. The selectivity of the method was tested with the response of somatostatin and lanreotide from the same growth hormone family by comparing the OC response. The developed P (ANI)@MIP/GCE sensor is the first reported method for electrochemical analysis of OC. The P (ANI)@MIP/GCE sensor exhibited high sensitivity and selectivity for OC. The novel MIP sensor was used to determine OC in cancer patient plasma samples. The concentration of OC in cancer patients varied between 8.98 ng/mL and 10.10 ng/mL.

Reliability of BRAF mutation detection using plasma sample: A systematic review and meta-analysis

Background:Testing of B-Raf proto-oncogene (BRAF) mutation in tumor is necessary before targeted therapies are given. When tumor samples are not available, plasma samples are commonly used for the testing of BRAF mutation. The aim of this study was to investigate the diagnostic accuracy of BRAF mutation testing using plasma sample of cancer patients.Methods:Databases of Pubmed, Embase, and Cochrane Library were searched for eligible studies investigating BRAF mutation in paired tissue and plasma samples of cancer patients. A total of 798 publications were identified after database searching. After removing 229 duplicated publications, 569 studies were screened using the following exclusion criteria: (1) BRAF mutation not measured in plasma or in tumor sample; (2) lacking BRAF-wildtype or BRAF-mutated samples; (3) tissue and plasma samples not paired; (4) lacking tumor or plasma samples; (5) not plasma sample; (6) not cancer; (7) un-interpretable data. Accuracy data and relevant information were extracted from each eligible study by 2 independent researchers and analyzed using statistical software.Results:After pooling the accuracy data from 3943 patients of the 53 eligible studies, the pooled sensitivity, specificity, and diagnostic odds ratio of BRAF mutation testing using plasma sample were 69%, 98%, and 55.78, respectively. Area under curve of summary receiver operating characteristic curve was 0.9435. Subgroup analysis indicated that BRAF mutation testing using plasma had overall higher accuracy (diagnostic odds ratio of 89.17) in colorectal cancer, compared to melanoma and thyroid carcinoma. In addition, next-generation sequencing had an overall higher accuracy in detecting BRAF mutation using plasma sample (diagnostic odds ratio of 63.90), compared to digital polymerase chain reaction (PCR) and conventional PCR, while digital PCR showed the highest sensitivity (74%) among the 3 techniques.Conclusion:BRAF testing using plasma sample showed an overall high accuracy compared to paired tumor tissue sample, which could be used for cancer genotyping when tissue sample is not available. Large prospective studies are needed to further investigate the accuracy of BRAF mutation testing in plasma sample.

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads.

Simple SummaryGenomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses.DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa.

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

A Disposable Amperometric Immunosensor for the Monitoring of Granzyme B in Blood Plasma Samples of Cancer Patients

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), which participates in inducing apoptosis of target cells for NK cells and cytotoxic CD8+ lymphocytes [1]. GzmB is the major effector of the CTLs and NK cells in its killing attack on cancer cells, although the level of GzmB expression and its cytotoxic potential decrease significantly in the presence of cancer. This decrement is due to a ‘pro-cancer’ environment, where the cancer cells secrete soluble mediators that could inhibit and thus contribute to the decreased GzmB by CD8+ T cells and a reduction in their ability to cause apoptosis of the cancer cells. Once GzmB is secreted from cytotoxic cells, it can be discovered in two different host compartments, namely inside the target cell or extracellularly [2]. Due to this, the change of GzmB level in blood plasma may serve as indication of the patient’s cancer cure progress. Hence, we developed a disposable amperometric GzmB sensor for the blood plasma samples.In developing a robust electrochemical sensing platform, an efficient device is required. To enhance the electrochemical response, catalytic nanoparticles can be used as the electrode material, particularly nanocomposites composed of functionalized conducting polymers. They have been well-thought-of as the candidate of electrode substrate materials for the electrochemical sensing platform due to their ability to augment the electrical conductivity, electron-transfer rate, mechanical strength, surface area, and binding affinity to a specific molecule [3]. Therefore, 3’-(2-aminopyrimidyl)-2,2’:5’,2’’-terthiophene (PATT) bearing an amine group was electro-polymerized onto gold nanoparticles (AuNPs) modified electrode. Then, the stabilized immobilization of antibody on the SPCE/AuNPs/pPATT layer was accomplished through the formation of an amide bond between amine groups of the polymer and carboxylic acid groups of the antibody.In this study, a disposable amperometric immunosensor composed of a sensing probe and a bioconjugate was developed for the detection of GzmB. The sensing probe was fabricated by immobilizing the GzmB monoclonal antibody (Ab1) on the polyPATT/AuNPs layer. The bioconjugate particle was synthesized by self-assembling the monomer mixture of 2,2:5,2-terthiophene-3-(p-benzoic acid) (TBA) and PATT onto AuNPs, then covalently bonding brilliant cresyl blue (BCB) on pTBA and GzmB polyclonal antibody (Ab2) on pPATT layer, respectively. The sensing layers were characterized using the surface analysis, cyclic voltammetry, impedance spectroscopy, and X-ray photoelectron spectroscopy.A redox peak of the bioconjugate (Ab2-SAM-BCB) in the presence of GzmB in the sample was observed at -0.35/-0.37 V, corresponding to the redox reaction of the redox indicator BCB. Optimization of experimental parameters were carried out in terms of temperature (35°C), pH (7.4), the concentration of antibody (300 μg/ml), applied potential (-0.38 (reduction)), and binding time (20 min). At the optimized conditions, the developed immunosensor showed a dynamic range from 3.0 to 50.0 pg/ml and from 50.0 to 1000.0 pg/ml in two slops, with a detection limit of 1.75 ± 0.14 pg/ml. Interference effect, stability, and reproducibility were also evaluated. Reliability of the proposed immunosensor was evaluated through comparison of the results among GzmB concentration of lung cancer patients before and after medicine treatment, and healthy volunteer using human blood plasma samples, where the low level of GzmB was present in the blood plasma of lung cancer patients before medicine treatment (10.50 ± 1.02 pg/ml), which slightly increased level in patients after medicine treatment (15.85 ± 1.99 pg/ml), and the significantly higher level was present in the normal healthy individual (39.62 ± 0.79 pg/ml). Conclusively, the progress of cancer before and after the cure of the patients has been evaluated through monitoring GzmB concentration in the blood plasma.[1] Kurschus, F.C., Jenne, D.E., 2010. Immuno. Rev. 235, 159-171.[2] Boivin, W.A., Cooper, D.M., Granville, D.J., 2009. Lab. Invest. 89, 1195-1220.[3] Naveen, M.H., Gurudatt, N.G., Shim, Y.-B., 2017. Appl. Mater. Today 9, 419–433.

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach. Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient's circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient's genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques. In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Abstract 735: Sensitive detection of ctDNA in early stage non-small cell lung cancer patients with a personalized sequencing assay

Abstract Introduction Identification of minimal residual disease (MRD) following curative intervention of localized non-small cell lung cancer (NSCLC) holds promise for identifying patients who are at higher risk of relapse and who would benefit from adjuvant treatment. Current routine clinical practice involves serial radiographic imaging following surgery to detect macroscopic disease. Liquid biopsy can identify patients who have MRD without macroscopic disease. Currently available assays have only identified circulating tumor DNA (ctDNA) in a limited number of cases with early stage NSCLC. More sensitive methods are needed to accurately identify the majority of patients who will relapse. Here we evaluate the performance of InVision®MRD, a personalized sequencing assay for plasma cell-free DNA, for detection of ctDNA in patients with early-stage NSCLC undergoing treatment with curative intent. Methods InVision®MRD is a highly sensitive in vitro diagnostic assay, currently available for research use only (RUO), that can detect the presence of tumor DNA traces in cell-free DNA from plasma samples of cancer patients. InVision®MRD identifies tumor-specific variants from exome sequencing of tumor tissue and tracks them in plasma specimens by multiplex PCR and high-depth next-generation sequencing. We evaluated the detection of ctDNA in plasma samples collected from the LUng cancer - CIrculating tumor DNA (LUCID) study, which collected plasma samples from 100 patients with NSCLC stages I-III who underwent radical treatment with curative intent, either surgery or radiotherapy ± chemotherapy. Of patients in the LUCID study, 60% had stage I NSCLC and 40% patients had stage II/III disease, according to TNM 7th edition. Results To evaluate the InVision®MRD assay, a subset of samples from the LUCID study were analyzed. Samples were collected before and after surgery and chemo-radiotherapy from patients with early-stage NSCLC. Using multiplexed analysis of 48 patient-specific variants and high-depth sequencing, ctDNA was detected in 50% of pre-treatment samples analyzed from the first set of 18 patients, at ctDNA fractions ranging from 20 ppm (equivalent to 0.002%) to 19576 ppm (equivalent to 1.958%). Conclusions These findings highlight an opportunity to improve ctDNA detection for early stage NSCLC using a patient-specific plasma sequencing assay. Initial detection rates have reached 50% for patients with early-stage disease prior to treatment, including detection of ctDNA to levels as low as a few parts per million. Together with further data to be presented, this suggests a possible route to improving treatment for early stage NSCLC by detection of residual disease post treatment and for monitoring for early detection of relapse. Citation Format: Katrin Heider, Davina Gale, Andrea Ruiz-Valdepenas, Giovanni Marsico, Garima Sharma, Malcolm Perry, Robert Osborne, Karen Howarth, Tadd Lazarus, Viona Rundell, Jelena Belic, Jerome Wulff, Susan Harden, Doris M. Rassl, Robert C. Rintoul, Nitzan Rosenfeld. Sensitive detection of ctDNA in early stage non-small cell lung cancer patients with a personalized sequencing assay [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 735.

Abstract 3097: Analytical development of the RaDaRTM assay, a highly sensitive and specific assay for the monitoring of minimal residual disease

Abstract Background 5%-30% of patients with primary non-metastatic cancer eventually relapse and die of metastatic disease, even though no macroscopic disease remains after initial curative-intent treatment. Adjuvant therapy is often administered to target minimal residual disease (MRD) that may be present but does not change outcome for most patients. Detecting MRD by liquid biopsy analysis after initial treatment and in advance of overt relapse could help identify patients who may benefit from adjuvant therapy or enrolment in clinical trials. Methods We developed InVision®MRD, a highly sensitive and specific method for detection of trace levels of tumor DNA in plasma cell-free DNA. Tumor-specific variants are identified using whole exome sequencing of tumor tissue samples, and a bespoke process designs primer panels targeting mutated loci. A multiplex high-fidelity PCR using InVision® technology captures and amplifies up to 48 patient–specific tumor variants in cell-free DNA, followed by high depth next generation sequencing. Proprietary algorithms compare patient-specific sequencing data to controls,and integrate information across variants and replicates to detect trace levels of tumor-derived signal. The method is currently available for research use only (RUO). Results We evaluated the performance of the InVision®MRD assay using samples from cancer patients and cell-line dilutions. We diluted three different tumor cell-lines into their matched normal counterparts. Primer panels were designed against 48 variants, specific to the tumor-cell line. Dilutions were amplified by multiplex PCR and sequenced on the Illumina NovaSeq to depth >100,000 reads per locus. Using 48 variants, the InVision®MRD assay had a mean sensitivity of 100% at 20 parts per million (20 ppm, equivalent to 0.002%) and sensitivity of 70% at 10 ppm (0.001%) with an overall specificity of 100%. To evaluate the effect of the number of variants on sensitivity and specificity we performed bootstrapping experiments selecting subsets of variants. When using only 16 variants, the I InVision®MRD assay had a mean sensitivity of 95% at 40 ppm (0.004%), 64% at 20 ppm (0.002%) and 22% at 10 ppm (0.001%) with specificity 99.99%. We tested the InVision®MRD assay on samples from cancer patients and detected tumor DNA in patient plasma and diluted samples with estimated tumor fractions at and below 20 ppm (0.002%). Conclusions The InVision®MRD assay provides a highly sensitive and specific method to detect MRD in plasma samples of cancer patients. Our results demonstrate excellent sensitivity with analysis of 16 variants which is further enhanced when 48 variants are analyzed. The high sensitivity of our approach is key to providing early information to guide treatment decisions after initial treatment by surgery or chemo/radiotherapy. The approach is generalisable to multiple tumor types and specimen types. Citation Format: Giovanni Marsico, Garima Sharma, Malcolm Perry, Sophie Hackinger, Tim Forshew, Karen Howarth, Jamie Platt, Nitzan Rosenfeld, Robert Osborne. Analytical development of the RaDaRTM assay, a highly sensitive and specific assay for the monitoring of minimal residual disease [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 3097.

Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy.

Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.

Immunoavidity-Based Capture of Tumor Exosomes Using Poly(amidoamine) Dendrimer Surfaces.

Tumor-derived blood-circulating exosomes have potential as a biomarker to greatly improve cancer treatment. However, effective isolation of exosomes remains a tremendous technical challenge. This study presents a novel nanostructured polymer surface for highly effective capture of exosomes through strong avidity. Various surface configurations, consisting of multivalent dendrimers, PEG, and tumor-targeting antibodies, were tested using exosomes isolated from tumor cell lines. We found that a dual layer dendrimer configuration exhibited the highest efficiency in capturing cultured exosomes spiked into human serum. Importantly, the optimized surface captured a > 4-fold greater amount of tumor exosomes from head and neck cancer patient plasma samples than that from healthy donors. Nanomechanical analysis using atomic force microscopy also revealed that the enhancement was attributed to multivalent binding (avidity) and augmented short-range adhesion mediated by dendrimers. Our results support that the dendrimer surface detects tumor exosomes at high sensitivity and specificity, demonstrating its potential as a new cancer liquid biopsy platform.

A Dual Antibody-modified Nanochannel Biosensor for Capture and Identification of Exosomes

Since exosomes carry the genetic information of their parental cells, there is an increasing need for new technologies capable of detecting exosomes with high sensitivity and accuracy. In this study, we propose a dual antibody-modified nanochannel biosensor for rapid, sensitive analysis of exosomes. Two antibodies (anti-CD63 and anti-EpCAM) were simultaneously immobilized on the surface of the porous anodic alumina (PAA) nanochannel to combine with proteins overexpressed on the exosomal membrane. As such, exosomes could be efficiently captured by the dual antibody-modified nanochannel. The captured exosomes effectively covered the nanochannel inlet, leading to remarkable block of ion flow through the channel. Based on this principle, the captured exosomes could be sensitively and accurately detected using a picoammeter. It was found that the use of two antibodies greatly increased the capture efficiency of exosomes compared to that of a single antibody. The results showed that exosomes down to 7.3 × 103 particles/mL were successfully detected by the nanochannel. Moreover, exosomes were successfully detected from plasma samples using a nanochannel biosensor modified with dual antibody, and the results showed significant differences in the exosomes content in plasma samples of cancer patients and healthy people, so as to initially distinguish cancer patients from healthy people.

Tracking Drug-Induced Epithelial-Mesenchymal Transition in Breast Cancer by a Microfluidic Surface-Enhanced Raman Spectroscopy Immunoassay.

Epithelial-mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E-cadherin and N-cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed "SERS immunoassay") based on sensitive and simultaneous detection of soluble E-cadherin (sE-cadherin) and soluble N-cadherin (sN-cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface-enhanced Raman scattering readout to enable accurate detection of sE-cadherin and sN-cadherin from as low as 10 cells mL-1 . This assay enables tracking of a concurrent decrease in sE-cadherin and increase in sN-cadherin in breast cancer cells undergoing drug-induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE-cadherin and sN-cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10–500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods’ reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

Abstract 939: Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients

Abstract Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA. The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots. The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965). This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients. Citation Format: Samuel Woodhouse, Vincent Plagnol, Karen Howarth, Stefanie Lensing, Matt Smith, Michael Epstein, Mikidache Madi, Sarah Smalley, Catherine Leroy, Jonathan Hinton, Frank de Kievit, Esther Musgrave-Brown, Colin Herd, Katherine Baker-Neblett, Will Brennan, Peter Dimitrov, Nathan Campbell, Nitzan Rosenfeld, James Clark, Davina Gale, Jamie Platt, John Calaway, Greg Jones, Tim Forshew. Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients [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 939.

Abstract LB-223: Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples

Abstract The increased reliance on liquid biopsy next generation sequencing assays for oncology clinical decisions has highlighted the importance of understanding the factors that impact the analytical validation and clinical utility of these tests. Although recent studies have been published examining the concordance between tumor and plasma sequencing data from the same patient, there has been a lack of insight into the intra-assay variability within these tests. We have previously performed a replicate study across multiple ctDNA sequencing vendors and found a high degree of variability among assays particularly at low allele fractions (AF<1%). To further extend our analysis, we examined the variability of ctDNA sequencing results from replicate patient plasma samples with the same vendor assay. The cohort consisted of 25 total samples: seven cancer patient plasma samples, representing triplicate 2 ml baseline plasma samples, plus four healthy normal samples, representing two different pools of six subjects in duplicate. The plasma names were blinded and shipped to Guardant Health for Guardant360 panel testing and both the raw data and reported variant calls were returned to AstraZeneca for comparative analysis. A total of 17 unique variants were called in this study, of which four variants were concordant for all three replicate plasmas and 13 variants were reported in only one or two replicates. Examining the raw data for all samples, the majority of the discordant variants were able to be identified at low levels. Plasmas had low amounts of ctDNA, and two patients had low unique molecule counts, with allele fractions ranging from 0.10-1.13%. Interestingly, the reported yield of the extracted DNA did not correlate with the ability to detect low frequency somatic variants. Data will be presented showing the unblinded comparisons between the replicate samples for both the cancer patient plasma data and the pooled normal plasmas. This study highlights the importance of reproducibility studies for ctDNA samples with variants at low allele fractions to discern between false negative and false positive finding. Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Barrett Nuttall, Tristan J. Lubinski, Fakhera Afshar, Amelia Raymond, Carl Barrett. Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples [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 LB-223.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment

BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Development of quantitative immunochromatographic assay for rapid and sensitive detection of carbohydrate antigen 19-9 (CA 19-9) in human plasma

A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle (GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that has been associated with cancers (such as pancreatic and colorectal cancers) and various non-cancerous diseases. The principle is based on sandwich-type immunoreactions between gold nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9 conjugates and the components of the running buffer were optimized to obtain the best sensitivity and reproducibility of the assay. The detection limit of the assay was determined to be 5UmL−1 (S/N=3) with a linear range of 5UmL−1–100UmL−1. CA 19-9 concentrations in healthy human and pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative immunochromatographic assay (QIA), and the results were in accordance with that obtained with enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical application and biomedical diagnosis, particularly in limited resource settings.