Blood Plasma Of Cancer Patients Research Articles

A novel numerical and artificial intelligence based approach to study anti-angiogenic drugs: Endostatin

Angiogenesis is known as the key factor in vascular tumor growth. In addition to furnishing the tumor with fresh nutrients to survive and spread to the other tissues, tumor-induced vasculature is tortuous and contains leaky vessels. Therefore, anti-angiogenesis is introduced as a way to suppress tumor growth and preclude metastases. Secondly, method of normalization of tortuous tumor-induced vasculature is being used to ease drug delivery to the tumor site. And thirdly, combined therapies of chemotherapy and radiotherapy with anti-angiogenesis agents can help achieve better outcomes in cancer treatment. One endogenous anti-angiogenesis agent that is being used in the aforementioned treatment strategies is known to be endostatin. In this research, a novel formulation is proposed and a computer code is developed to study anti-angiogenesis effects of endostatin. It is shown that endostatin as an endogenous agent can suppress angiogenesis and normalize tumor-induced vasculature which can ease drug delivery to the tumor site. By increasing endostatin concentration to 5 times of its natural concentration in the blood plasma of cancer patients, angiogenesis could be tackled and hindered. Finally, in order to propose a general and simple formulation to predict final microvessel density at different circumstances, a Generalized Regression Neural Network (GRNN) is established. The results of GRNN show that it is able to predict the microvascular density with 87% accuracy. With the aim of GRNN formulation, scientists can observe the vasculature at any desired conditions to get an insight on optimum time for the combined treatment.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Separation of ctDNA by superparamagnetic bead particles in microfluidic platform for early cancer detection

IntroductionConventional biopsy, based on extraction from a tumor of a solid tissue specimen requiring needles, endoscopic devices, excision or surgery, is at risk of infection, internal bleeding or prolonged recovery. A non-invasive liquid biopsy is one of the greatest axiomatic consequences of the identification of circulating tumor DNA (ctDNA) as a replaceable surgical tumor bioQpsy technique. Most of the literature studies thus far presented ctDNA detection at almost final stage III or IV of cancer, where the treatment option or cancer management is nearly impossible for diagnosis. ObjectiveHence, this paper aims to present a simulation study of extraction and separation of ctDNA from the blood plasma of cancer patients of stage I and II by superparamagnetic (SPM) bead particles in a microfluidic platform for early and effective cancer detection. MethodThe extraction of ctDNA is based on microfiltration of particle size to filter some impurities and thrombocytes plasma, while the separation of ctDNA is based on magnetic manipulation to high yield that can be used for the upstream process. ResultBased on the simulation results, an average of 5.7 ng of ctDNA was separated efficiently for every 10 µL blood plasma input and this can be used for early analysis of cancer management. The particle tracing module from COMSOL Multiphysics traced ctDNA with 65.57% of sensitivity and 95.38% of specificity. ConclusionThe findings demonstrate the ease of use and versatility of a microfluidics platform and SPM bead particles in clinical research related to the preparation of biological samples. As a sample preparation stage for early analysis and cancer diagnosis, the extraction and separation of ctDNA is most important, so precision medicine can be administered.

Highly stable surface-enhanced Raman spectroscopy assay on abnormal thrombin levels in the blood plasma of cancer patients.

The accurate discrimination of specific protein levels in the blood of cancer patients is of great importance in clinical diagnostics and prognosis. Here, we report a double-aptamer sandwich strategy on plasmonic magnetic beads (MBs) for a dual-reporter surface-enhanced Raman spectroscopy (SERS) assay for assessing abnormal thrombin (TB) levels in the blood plasma of cancer patients. This SERS assay demonstrated a linear analysis range of 20-400 pM in serum with a limit of detection as low as 20 pM, and to the best of our knowledge, this represents the first attempt to highly stably discriminate an abnormal TB level in the plasma of clinical cancer patients. Two recognition elements of TB aptamers provided high specificity and the dual-reporter assay demonstrated greatly reduced false-positive signals. The sandwich complex produced an efficient SERS "hot spot" to make up for the flaws of the insufficient enhancement of monomer metal nanoparticles. The plasmonic MBs enabled the direct tracking of ultratrace proteins in plasma while avoiding complicated pretreatment with only a few washing steps required. As a preliminary exploration, our report details a new potential tool with high sensitivity, selectivity, and practicality for disease-related protein testing in clinical diagnostics and prognosis.

USE IR-SPECTROSCOPY IN TREATMENT ONCOLOGICAL PATIENTS WITH BONE METASTASES BY 153Sm-OXABIFOR

Using infrared spectroscopy, we studied the character of intermolecular interactions in the samples blood plasma of oncological patients with bone metastases in the treatment of their modern β-emitter 153Sm-oxabifor. The therapeutic dose of the 153Sm-oxabifor was 1.0 mCi/kg of the patient's body weight. Analysis of the IR-spectra showed that already after the first course of palliative radionuclide therapy there is a tendency to balance the products of free radical nature and antioxidants, which indicates the effectiveness of the treatment. Molecular changes in the blood plasma of cancer patients with bone metastases registered using infrared spectroscopy are specifc during palliative therapy.

Personalized Treatment Selection and Disease Monitoring Using Circulating Tumor DNA Profiling in Real-World Cancer Patient Management.

Background: Circulating tumor DNA (ctDNA) in the blood plasma of cancer patients is an emerging biomarker used across oncology, facilitating noninvasive disease monitoring and genetic profiling at various disease milestones. Digital droplet PCR (ddPCR) technologies have demonstrated high sensitivity and specificity for robust ctDNA detection at relatively low costs. Yet, their value for ctDNA-based management of a broad population of cancer patients beyond clinical trials remains elusive. Methods: We developed mutation-specific ddPCR assays that were optimized for their use in real-world cancer management, covering 12 genetic aberrations in common cancer genes, such as EGFR, BRAF, KIT, KRAS, and NRAS. We assessed the limit of detection (LOD) and the limit of blank (LOB) for each assay and validated their performance for ctDNA detection using matched tumor sequencing. Results: We applied our custom ddPCR assays to 352 plasma samples from 96 patients with solid tumors. Mutation detection in plasma was highly concordant with tumor sequencing, demonstrating high sensitivity and specificity across all assays. In 20 cases, radiographic cancer progression was mirrored by an increase of ctDNA concentrations or the occurrence of novel mutations in plasma. Moreover, ctDNA profiling at diagnosis and during disease progression reflected personalized treatment selection through the identification of actionable gene targets in 20 cases. Conclusion: Collectively, our work highlights the potential of ctDNA assessment by sensitive ddPCR for accurate disease monitoring, robust identification of resistance mutations, and upfront treatment selection in patients with solid tumors. We envision an increasing future role for ctDNA profiling within personalized cancer management in daily clinical routine.

Circulating RNA in blood plasma as diagnostic tool for clinical oncology

One of the key challenges facing today’s oncology is the discovery of early predictors of malignant neoplasms in patients’ biological samples. Liquid biopsy is a noninvasive diagnostic technique based on the detection and isolation of tumor cells, tumor-derived nucleic acid and exosomes circulating in the blood plasma of cancer patients. There is a plethora of research studies of circulating tumor DNA in patients with MN. The active proliferation of tumor cells occurs in the backdrop of altered gene expression. The presence of tissue-specific transcripts in the circulating RNA fraction suggests that levels of circulating RNA reflect the development of the primary tumor. We think that cell-free RNA circulating in the blood plasma is a promising molecular biomarker for early cancer detection.

Abstract B48: Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA

Abstract In the clinical management of pediatric solid tumors, histologic examination of tumor tissue obtained by an invasive biopsy remains the gold standard to establish a conclusive pathologic diagnosis. However, correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. The DNA methylation pattern of a tumor is known to correlate with the histopathologic diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. It has previously been shown that this methylation pattern can be detected in the cell-free circulating DNA isolated from blood plasma of cancer patients. Here, we provide proof of concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. Using computational deconvolution of the DNA methylation profiles, we were able to obtain the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free circulating DNA. We could demonstrate that the majority of misclassifications were associated with bad sample quality, evidenced by an increase of the test accuracy to 94.3% when only including good-quality samples. Misclassifications were not associated with the extent of the disease. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Our findings show the potential of the approach to tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Given the fact that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in these difficult cases. A preprint of the study manuscript is already available at bioRxiv: https://doi.org/10.1101/795047. Citation Format: Ruben Van Paemel, Andries De Koker, Charlotte Vandeputte, Lieke Van Zogchel, Tim Lammens, Geneviève Laureys, Jo Vandesompele, Gudrun Schleiermacher, Matthieu Chicard, Nadine Van Roy, Ales Vicha, Godelieve A. M. Tytgat, Nico Callewaert, Bram De Wilde, Katleen De Preter. Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA [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 B48.

Separation, Purification, and Detection of cfDNA in a Microfluidic Device

Cell free DNA (cfDNA) is degraded DNA fragments found in the blood plasma of cancer patients. While cfDNA is a good marker for early diagnostics and cancer prognosis, the extraction of cfDNA from whole blood and sample preparation for later sequencing is still challenging. Here, we presented a microfluidic device for the removal of cells from a cfDNA sample in a first step. In a second step, carboxylated magnetic beads were used for cfDNA extraction and purification. Lastly, cfDNA was amplified using a low-power, plasmonic polymerase chain reaction (PCR) system. Using fluorescent-labeled beads, we demonstrated that the separation efficiency for cells was 99% and the mixing efficiency for purification of cfDNA was 94%. Captured cfDNA could be successfully amplified by PCR, as demonstrated by gel electrophoresis. We confirmed that the limit of detection of our microfluidic system was 10 ng/mL.

Rezidüel Tümör Tespiti İçin Bir Sıvı Biyopsi Yöntemi Olarak Alelspesifik Emülsiyon PCR (asePCR)

AimThis study aimed to develop an allele-specific emulsion polymerase chain reaction (asePCR)technique for detection of cell-free circulating tumor DNA (ctDNA) in the blood plasma of cancer patients. Materials and MethodsGenomic DNA extracted from normal (wild-type) and thyroid cancer (mutant) cell lines (MCF10A and B-CPAP) were used to obtain specific dilution series (spike-in) of mutant DNA with wild-type DNA. Two sequential PCR steps (regular and asePCR) were performed with specifically designed primers for each step to detect the spiked-in mutant DNA fraction within the wild-type normal DNA. The DNA products amplified by regular PCR were used as templates in the asePCR where specific reverse primers were designed to amplify either the mutant or wild-type allele while a common forward primer was covalently bound to the beads. The asePCR reaction mix was encapsulated in water-in-oil emulsion compartments and incubated in a thermocycler with specific PCR program settings. At the end of asePCR reaction, oil compartments were broken and DNA-coated beads were evaluated by FACS to determine the mutant and wild-type fractions for each sample.ResultsThe asePCR technique successfully detected the mutant DNA in the background of wildtype DNA (0% mutant) as a proof-of-principle. A strong correlation (r2>0.99) was found between the expected mutant fraction and the fraction measured by asePCR. asePCR was sensitive and specific enough to consistently detect various low mutant DNA fractions in the background of wild-type DNA with no false positives in the wild-type sample.Discussion and Conclusion Our asePCR can be used to detect mutant ctDNA in the plasma of cancer patients as a liquid biopsy method for tumor response evaluation and early detection of possible relapses.

Comparative sub-population analysis of exosomes from blood plasma of cancer patients

To increase the sensitivity and specificity of the developed methods for diagnosis of oncological diseases using exosomes of blood, a stage of pre-selection of tumor exosomes from a common pool of circulating microvesicles is required. In the present work, universal proteins have been identified, their expression has been increased in the exosomes of patients with colorectal cancer, head and neck squamous cell carcinomas, and lung cancer. The use of antibodies against major exosomal proteins will further develop a simple and high-performance method of affinity isolation of tumor exosomes.

Development of a biosensor for electrochemical detection of tumor-associated proteins in blood plasma of cancer patients by aptamers.

A molecular biosensor based on DNA aptamers (aptasensor) for the diagnosis of lung cancer in blood plasma samples was designed. To create the aptasensor, the aptamer 17_80, obtained in the study of postoperative material, was used. The affinity and binding selectivity of the aptamer 17_80 to the lung tumor tissue was confirmed on histological sections of postmortem samples of lung tissue. Using affinity enrichment and mass spectrometry, a possible target molecule of the aptamer 17_80, vimentin, was found.

The plasma DNA concentration as a potential breast cancer screening marker.

Increased levels of plasma DNA have frequently been noticed in the blood plasma of cancer patients. The possibility of using plasma DNA level as the indicator of tumor stage in breast cancer was investigated in plasma samples obtained from 100 breast cancer patients and 100 healthy women who were included as controls. Circulatory plasma free DNA was extracted from plasma samples and quantified by fluorometer. The median concentration of plasma DNA in the plasma samples from breast cancer patients classified by TNM staging system as stage I, II, III, IV and breast surgical patients were 0.5, 235, 422, 1,280 and 0.5ng/ml, respectively. The level of plasma DNA in the stage II- IV group was significantly higher than those in the surgical group with breast cancer and control group (P value <0.001). The plasma DNA concentration in stage II, III and IV of breast cancer were higher when compared with healthy group. These tumor size, TNM stage and metastasis were significantly correlated with plasma DNA. The cut point of 120ng/ml was early screening and treatment follow up breast cancer.

Extracellular DNA oxidation stimulates activation of NRF2 and reduces the production of ROS in human mesenchymal stem cells

Introduction: Human blood normally contains circulating cell-free DNA (cirDNA). Cell-free DNA (cfDNA) present in cell culture medium is termed extracellular DNA (ecDNA). Its concentration, GC content and oxidation level depend on physiological state of the organism. cirDNA could probably be one of the aggressive factors encountered by therapeutic stem cells. The authors hypothesize that oxidized cirDNA could influence their survival rate. They aimed to uncover the effects of oxidized ecDNAs, including ecDNA of cultivated primary tumor cells and cirDNA from blood plasma of cancer patients on mesenchymal stem cells (MSCs).Areas covered: Increased concentrations of cfDNA stimulate a rapid increase in reactive oxygen species (ROS) synthesis and up-regulate antioxidant response genes (NRF2, KEAP1, SOD1, BRCA1, BCL2) in MSCs. This response is more prominent when cfDNA contains higher proportions of 8-oxo-dG. Within an hour, oxidized DNA induces a decrease in ROS production while NRF2 mRNA levels continue to augment and the NRF2 protein translocates into the nucleus. Additionally, oxidized DNA up-regulates PPRAG2 with no apparent induction of adipogenesis. This kind of response is specific for MSCs.Expert opinion: Oxidized cfDNA up-regulates NRF2 and PPARG2 and reduces ROS production in MSCs. These effects should be taken into account when considering therapeutic applications of stem cells.

Correlation of histone methyl marks with circulating nucleosomes in blood plasma of cancer patients

Circulating DNA is present in plasma/serum, mainly complexed with histones as nucleosomes. The detection of circulating nucleosomes (cNUCs) in the peripheral blood may be a diagnostic modality for cancer-associated changes of modified histone tails in blood circulation. In the present study, we investigated the correlation between the trimethylation of H3 lysine 9 (H3K9me3) and H4 lysine 20 (H4K20me3), which are hallmarks of pericentric heterochromatin, and cNUCs in healthy subjects and patients with colorectal cancer (CRC) and multiple myeloma (MM). The plasma concentration of cNUCs was measured using the Cell-Death Detection ELISA kit. Histone methylation marks were detected using chromatin immunoprecipitation (ChIP), followed by quantitative PCR with pericentric satellite 2 as the target sequence. The results showed a high variation in the concentrations of cNUCs, with healthy subjects exhibiting the lowest levels (median 0.194), the CRC patients intermediate (median 0.25) and the MM patients the highest levels (median 0.648). However, the differences between the groups did not reach statistical significance (p>0.05). Analysis using the Pearson's correlation test revealed a significant positive correlation between the concentration of cNUCs and H3K9me3 and H4K20me3 in the whole study group (N=57, p<0.001 for both histone marks). A study of the correlation between cNUCs and histone marks in the individual study groups demonstrated the correlation between cNUCs and H3K9me3 in CRC patients to be weak (p=0.046), indicating that circulating H3K9me3 may be modified in CRC patients. The histone marks were normalized using the values of cNUCs. In agreement with the weak correlation between cNUCs and H3K9me3 in CRC patients, H3K9me3 levels (median 0.047) were lowest in this group compared with the other two groups (0.06 in healthy subjects, 0.2 in MM patients, p = not significant). For H4K20me3, the median values were 0.022 in healthy subjects, 0.052 in CRC patients and 0.056 in MM patients. In conclusion, our findings indicate a marked correlation between cNUCs and histone methyl marks.

Deoxyribonuclease Activity and Circulating DNA Concentration in Blood Plasma of Patients with Prostate Tumors

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten-labeled 974-bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 +/- 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 +/- 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10-234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13-339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0-0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.

Circulating DNA and DNase Activity in Human Blood

The concentration of circulating DNA (cirDNA) and deoxyribonuclease activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concentration of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass-milk protocol. A 1-kbp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers was used as a substrate for DNase. DNase activity was estimated from the data of immunochemical detection of the nonhydrolyzed amplicon. The average concentration of cirDNA in the plasma of healthy donors was low (34 +/- 34 ng/mL), and was accompanied with high DNase activity (0.356 +/- 0.410 U/mL). The increased concentrations of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concentration of cirDNA.

Whole Genome Amplification of Plasma-Circulating DNA Enables Expanded Screening for Allelic Imbalance in Plasma

Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by approximately 1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens.