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Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme μ-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications. [ABSTRACT FROM AUTHOR]

Next-generation deep genome sequencing has only recently allowed us to quantitatively dissect the extent of heterogeneity within a tumour, resolving patterns of cancer evolution. Intratumour heterogeneity and natural selection contribute to resistance to anticancer therapies in the advanced setting. Recent evidence has also revealed that cancer evolution might be constrained. In this review, we discuss the origins of intratumour heterogeneity and subsequently focus on constraints imposed upon cancer evolution. The presence of (1) parallel evolution, (2) convergent evolution and (3) the biological impact of acquiring mutations in specific orders suggest that cancer evolution may be exploitable. These constraints on cancer evolution may help us identify cancer evolutionary rule books, which could eventually inform both diagnostic and therapeutic approaches to improve survival outcomes. [ABSTRACT FROM AUTHOR]

Big Data has emerged in the past few years as a new paradigm providing abundant data and opportunities to improve and/or enable research and decision-support applications with unprecedented value for digital earth applications including business, sciences and engineering. At the same time, Big Data presents challenges for digital earth to store, transport, process, mine and serve the data. Cloud computing provides fundamental support to address the challenges with shared computing resources including computing, storage, networking and analytical software; the application of these resources has fostered impressive Big Data advancements. This paper surveys the two frontiers – Big Data and cloud computing – and reviews the advantages and consequences of utilizing cloud computing to tackling Big Data in the digital earth and relevant science domains. From the aspects of a general introduction, sources, challenges, technology status and research opportunities, the following observations are offered: (i) cloud computing and Big Data enable science discoveries and application developments; (ii) cloud computing provides major solutions for Big Data; (iii) Big Data, spatiotemporal thinking and various application domains drive the advancement of cloud computing and relevant technologies with new requirements; (iv) intrinsic spatiotemporal principles of Big Data and geospatial sciences provide the source for finding technical and theoretical solutions to optimize cloud computing and processing Big Data; (v) open availability of Big Data and processing capability pose social challenges of geospatial significance and (vi) a weave of innovations is transforming Big Data into geospatial research, engineering and business values. This review introduces future innovations and a research agenda for cloud computing supporting the transformation of the volume, velocity, variety and veracity into values of Big Data for local to global digital earth science and applications. [ABSTRACT FROM PUBLISHER]

Background:Circulating DNA can be used to measure the total cell-free DNA (cfDNA) and for detection and quantification of tumor-specific genetic alterations in the peripheral blood, and the broad clinical potential of circulating DNA has attracted increasing focus over the past decade. Concentrations of circulating DNA are high in metastatic colorectal cancer (CRC), and the total levels of cfDNA have been reported to hold strong prognostic value. Colorectal tumors are characterized by a high frequency of well known, clinically relevant genetic alteration, which is readily detected in the cfDNA and holds potential for tailoring of palliative therapy and for monitoring during treatment. This review aims to present the current literature which has specifically reported data on the potential utility of cfDNA and on tumor-specific mutations in metastatic colorectal cancer (mCRC). Method:Methodological, biological and clinical aspects are discussed based on the most recent development in this specific setting, and eligible studies were identified by systematic literature searched from Pubmed and EMBASE in addition to conference papers and communications. Results:The literature regarding cfDNA in CRC is broad and heterogeneous concerning aims, nomenclature, methods, cohorts and clinical endpoints and consequently difficult to include in a single systematic search. However, the available data underline a strong clinical value of measuring both total cfDNA levels and tumor-specific mutations in the plasma of patients with mCRC, pre- and during systemic therapy. Conclusion:This paper had gathered the most recent literature on several aspects of cfDNA in mCRC, including methodological, biological and clinical aspects, and discussed the large clinical potential in this specific setting, which needs to be validated in carefully designed prospective studies in statistically relevant cohorts. [ABSTRACT FROM PUBLISHER]

Microfluidic systems offer precisely controlled experimental conditions for efficient study of mammalian cells, ranged from a single cell to matrixbased three-dimensional (3D) in vitro culture models. droplet-based microfluidic system is a robust and highly reproducible device enabling encapsulation of a cell or cells within well-confined microenvironment. Recently, such droplet-based cell culture models have drawn much attention due to their unique properties such that conventional culture systems couldn't provide. The encapsulation of cells in specifically designed aqueous phase of a microfluidic system can provide profound understand of cell to cell and cell to extracellular matrix interactions, also can be used to regulate various cell behaviors. A droplet-based cell culture system allows better control over confinement for culturing, maintaining, and analyzing cells, such as high-throughput screening. In this review, we discuss recent researches on microdroplet-based 3D cell culture models, and advanced applications of microfluidic systems. [ABSTRACT FROM AUTHOR]

Emulsions are metastable dispersions. Their lifetimes are directly related to the dynamics of surfactants. We design a microfluidic method to measure the kinetics of adsorption of surfactants to the droplet interface, a key process involved in foaming, emulsification, and droplet coarsening. The method is based on the pH decay in the droplet as a direct measurement of the adsorption of a carboxylic acid surfactant to the interface. From the kinetic measurement of the bulk equilibration of the pH, we fully determine the adsorption process of the surfactant. The small droplet size and the convection during the droplet flow ensure that the transport of surfactant through the bulk is not limiting the kinetics of adsorption. To validate our measurements, we show that the adsorption process determines the timescale required to stabilize droplets against coalescence, and we show that the interface should be covered at more than 90% to prevent coalescence. We therefore quantitatively link the process of adsorption/desorption, the stabilization of emulsions, and the kinetics of solute partitioning-- here through ion exchange--unraveling the timescales governing these processes. Our method can be further generalized to other surfactants, including nonionic surfactants, by making use of fluorophore-surfactant interactions. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Laboratory Medicine / Laboratoriums Medizin is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Laboratory Medicine / Laboratoriums Medizin is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: BRAF is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA. Methods: The level of BRAF ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients. Results: The castPCR assay was performed using a mixture of BRAF DNA and BRAF DNA and found to be able to detect BRAF at a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF mutation. BRAF ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAF ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF ctDNA preceded exacerbation of clinical symptoms. Conclusion: The castPCR assay can detect and quantitate small amounts of BRAF ctDNA in samples containing large amounts of BRAF cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients. [ABSTRACT FROM AUTHOR]

Global land cover (LC) maps have been widely employed as the base layer for a number of applications including climate change, food security, water quality, biodiversity, change detection, and environmental planning. Due to the importance of LC, there is a pressing need to increase the temporal and spatial resolution of global LC maps. A recent advance in this direction has been the GlobeLand30 dataset derived from Landsat imagery, which has been developed by the National Geomatics Center of China (NGCC). Although overall accuracy is greater than 80%, the NGCC would like help in assessing the accuracy of the product in different regions of the world. To assist in this process, this study compares the GlobeLand30 product with existing public and online datasets, that is, CORINE, Urban Atlas (UA), OpenStreetMap, and ATKIS for Germany in order to assess overall and per class agreement. The results of the analysis reveal high agreement of up to 92% between these datasets and GlobeLand30 but that large disagreements for certain classes are evident, in particular wetlands. However, overall, GlobeLand30 is shown to be a useful product for characterizing LC in Germany, and paves the way for further regional and national validation efforts. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K,EZH2Y641N, andMYD88L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rareXPO1,EZH2, andMYD88mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments. [ABSTRACT FROM AUTHOR]

Background: Circulating cell-free DNA (cfDNA) is emerging as a surrogate sample type for mutation analyses. To improve the clinical utility of cfDNA, we developed a sensitive peptide nucleic acid (PNA)-based method for analyzing EGFR and KRAS mutations in the plasma cfDNA of patients with advanced non-small cell lung cancer (NSCLC).Methods: Baseline tissue and plasma samples were collected from treatment-naïve advanced NSCLC patients participated in a randomized phase II study, which was registered with ClinicalTrials.gov at Feb. 2009 (NCT01003964). EGFR and KRAS mutations in the plasma cfDNA were analyzed retrospectively using a PNA clamping-assisted fluorescence melting curve analysis. The results were compared with those obtained from tissue analysis performed using the direct sequencing. Exploratory analyses were performed to determine survival predicted by the plasma and tissue mutation status.Results: Mutation analyses in matched tissue and plasma samples were available for 194 patients for EGFR and 135 patients for KRAS. The mutation concordance rates were 82.0 % (95 % confidence interval [CI], 76.5-87.4) for EGFR and 85.9 % (95 % CI, 80.1-91.8) for KRAS. The plasma EGFR mutation test sensitivity and specificity were 66.7 % (95 % CI, 60.0-73.3) and 87.4 % (95 % CI, 82.7-92.1), respectively, and the plasma KRAS mutation test sensitivity and specificity were 50.0 % (95 % CI, 41.6-58.4) and 89.4 % (95 % CI, 84.2-94.6), respectively. The predictive value of the plasma EGFR and KRAS mutation status with respect to survival was comparable with that of the tissue mutation status.Conclusions: These data suggest that plasma EGFR and KRAS mutations can be analyzed using PNA-based real-time PCR methods and used as an alternative to tumor genotyping for NSCLC patients when tumor tissue is not available. [ABSTRACT FROM AUTHOR]

ABSTRACT Microalgae have emerged as a promising source for producing future renewable biofuels. Developing better microalgal strains with faster growth and higher oil production rates is one of the major routes towards economically viable microalgal biofuel production. In this work, we present a droplet microfluidics-based microalgae analysis platform capable of measuring growth and oil content of various microalgal strains with single-cell resolution in a high-throughput manner. The platform allows for encapsulating a single microalgal cell into a water-in-oil emulsion droplet and tracking the growth and division of the encapsulated cell over time, followed by on-chip oil quantification. The key feature of the developed platform is its capability to fluorescently stain microalgae within microdroplets for oil content quantification. The performance of the developed platform was characterized using the unicellular microalga Chlamydomonas reinhardtii and the colonial microalga Botryococcus braunii. The application of the platform in quantifying growth and oil accumulation was successfully confirmed using C. reinhardtii under different culture conditions, namely nitrogen-replete and nitrogen-limited conditions. These results demonstrate the capability of this platform as a rapid screening tool that can be applied to a wide range of microalgal strains for analyzing growth and oil accumulation characteristics relevant to biofuel strain selection and development. Biotechnol. Bioeng. 2016;113: 1691-1701. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf) DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccf DNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccf DNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccf DNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccf DNA and did not respond to treatment with epidermal growth factor receptor ( EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccf DNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. [ABSTRACT FROM AUTHOR]

The papers in this special section focus on Big Data, with particular emphasis on the exploitation of data collected via industrial sensor networks (be it wireless or not) for Internet-based industrial applications. The papers explore the handling and analysis of the collected data. It is expected that analyzing the data can improve the reliability of industrial systems by predicting the occurrence potential risks and then rectification can be made accordingly. Such risks are inevitably linked to uncertain factors hidden in the systems that can be revealed by the data analysis process. [ABSTRACT FROM PUBLISHER]

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. [ABSTRACT FROM AUTHOR]

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. [ABSTRACT FROM AUTHOR]

Personalized medicine shows promise for maximizing efficacy and minimizing toxicity of anti-cancer treatment. KRAS exon 2 mutations are predictive of resistance to epidermal growth factor receptor-directed monoclonal antibodies in patients with metastatic colorectal cancer. Recent studies have shown that broader RAS testing (KRAS and NRAS) is needed to select patients for treatment. While Sanger sequencing is still used, approaches based on various methodologies are available. Few CE-approved kits, however, detect the full spectrum of RAS mutations. More recently, "next-generation" sequencing has been developed for research use, including parallel semiconductor sequencing and reversible termination. These techniques have high technical sensitivities for detecting mutations, although the ideal threshold is currently unknown. Finally, liquid biopsy has the potential to become an additional tool to assess tumor-derived DNA. For accurate and timely RAS testing, appropriate sampling and prompt delivery of material is critical. Processes to ensure efficient turnaround from sample request to RAS evaluation must be implemented so that patients receive the most appropriate treatment. Given the variety of methodologies, external quality assurance programs are important to ensure a high standard of RAS testing. Here, we review technical and practical aspects of RAS testing for pathologists working with metastatic colorectal cancer tumor samples. The extension of markers from KRAS to RAS testing is the new paradigm for biomarker testing in colorectal cancer. [ABSTRACT FROM AUTHOR]

Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) is a novel assay format that allows for the efficient enrichment and sensitive detection of all mutations in a region of interest using a chemically modified blocking oligonucleotide, which impedes the amplification of wild-type sequences. The assay is compatible with DNA extracted from tissue and cell-free circulating DNA. The main features of E-ice-COLD-PCR are the simplicity of the setup and the optimization of the assay, the use of standard laboratory equipment and the very short time to results (~4 h including DNA extraction, enrichment and sequence-based identification of mutations). E-ice-COLD-PCR is therefore a highly promising technology for a number of basic research as well as clinical applications including detection of clinically relevant mutated subclones and monitoring of treatment response or disease recurrence. [ABSTRACT FROM PUBLISHER]

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains. [ABSTRACT FROM AUTHOR]

Screening methods for the most frequent diagnosed malignant tumor, colorectal cancer (CRC), have limitations. Circulating cell-free DNA (cfDNA) analysis came into focus as a potential screening test for CRC. Detection of epigenetic and genetic alterations of cfDNA as DNA methylation or DNA mutations and related ribonucleic acids may improve cancer detection based on unique, CRC-specific patterns. In this review the authors summarize the CRC-specific nucleic acid biomarkers measured in peripheral blood and their potential as screening markers. Detection of DNA mutation has inadequate sensitivity; however, methylated DNA can be established with higher sensitivity from CRC plasma samples. The ribonucleic acid based miRNA studies represented higher sensitivity for CRC as compared with mRNA studies. Recently, isolation of cfDNA has become automated, highly reproducible and a high throughput method. With automated possible diagnostic tools, a new approach may be available for CRC screening as liquid biopsy. [ABSTRACT FROM PUBLISHER]

Head and neck cancer patients often present with advanced metastatic disease resulting in a poor 5-year survival. Therefore, there is a need for non-invasive diagnostic tools that could complement conventional imaging to inform clinicians of patient outcomes and treatment responses. A liquid biopsy addresses this unmet clinical need; a simple peripheral blood draw could provide information about the disseminated disease in terms of circulating tumor cells and circulating tumor DNA. Moreover, detectable tumor DNA in the saliva of head and neck cancer patients could signify the early signs of the disease and present an opportunity for clinical intervention. This review provides an overview of the current literature with regard to the feasibility of such a test in the head and neck cancer field and highlights the need for such a test. [ABSTRACT FROM PUBLISHER]

Purpose. The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC). Experimental design. Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues. Results. cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%)were positive for the T790M mutation, with fractions of T790M (1) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance. Conclusion. Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs. [ABSTRACT FROM AUTHOR]

Formed from a reciprocal translocation t(9:22)(q34;q11) of genetic material between the long arms of human chromosomes 9 and 22, the constitutively active breakpoint cluster region (BCR) Abelson 1 (ABL) tyrosine kinase BCR-ABL is known to be causative of chronic myelogenous leukemia (CML). In 98 % of CML patients harboring the t(9:22)(q34;q11) translocation, known as the Philadelphia chromosome, the chimeric BCR-ABL oncogene is created through cleavage of the BCR gene within its major breakpoint region (M- BCR) and breakage of the ABL gene within a 100-kbp region downstream of exon 2a. Clinical detection of the fused BCR-ABL oncogene currently relies on direct visualization by fluorescence in situ hybridization (FISH), a relatively tedious assay that typically offers a detection limit of ca. 2 %. Here, we describe a novel assay that uses droplet digital PCR (ddPCR) technology to reliably measure M- BCR status and the presence of BCR-ABL. When applied to cell-line models of CML, the assay accurately quantifies BCR-ABL frequency to a detection limit of 0.25 %. It therefore offers improved specificity relative to FISH, and may allow identification of variant translocation patterns, including derivative chromosome 9 deletions. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Background: Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods: We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results: Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion: The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. [ABSTRACT FROM AUTHOR]

In this review, we describe key components of a computational infrastructure for a precision medicine program that is based on clinical-grade genomic sequencing. Specific aspects covered in this review include software components and hardware infrastructure, reporting, integration into Electronic Health Records for routine clinical use and regulatory aspects. We emphasize informatics components related to reproducibility and reliability in genomic testing, regulatory compliance, traceability and documentation of processes, integration into clinical workflows, privacy requirements, prioritization and interpretation of results to report based on clinical needs, rapidly evolving knowledge base of genomic alterations and clinical treatments and return of results in a timely and predictable fashion. We also seek to differentiate between the use of precision medicine in germline and cancer. [ABSTRACT FROM AUTHOR]

Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR. [ABSTRACT FROM AUTHOR]

Background: Stool DNA testing represents a potential noninvasive approach to detect upper gastrointestinal (UGI) neoplasms. However, little is known about fecal recovery efficiency of DNA exfoliated from UGI tumors.Aims: The purpose of this study was to establish a human ingestion model that quantitatively approximates daily cellular shedding from UGI neoplasms and to estimate fecal DNA marker recovery rates.Methods: Healthy volunteers (n = 10) ingested two scheduled doses of raw salmon, 0.3 and 30 g, simulating the mass exfoliated daily from 1 to 4.5 cm lesions. To approach a steady-state, each dose was ingested over three consecutive days in randomized order. Following defecation of an indicator dye ingested with test meals, stools were collected over 48 h. Ingested salmon DNA was captured from stools using probes targeting pathognomonic Salmonidae sequences (SlmII). Captured DNA was quantified using PCR primers to generate 178, 138, 88 and 55 bp amplicons.Results: SlmII sequences were recovered from all stools following salmon ingestion; recovery was proportional to amount ingested (p = 0.004). Fecal recovery of ingested salmon varied inversely with amplicon size targeted; mean recovery rates of SlmII were 0.49, 0.91, 3.63, and 7.31 copies per 100,000 copies ingested for 178, 134, 88, and 55 bp amplicons, respectively (p < 0.0001). Longer oro-anal transit was associated with reduced recovery.Conclusions: While recovery efficiencies are low, ingested cellular DNA simulating daily amounts shed from UGI tumors can readily be detected in stool. Assay of shorter-fragment analyte increases recovery. This ingestion model has potential value in studying the effects of perturbations relevant to the fecal recovery of DNA exfoliated from UGI tumors. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method. [ABSTRACT FROM AUTHOR]

Background: Noninvasive plasma-based detection of EGFR mutations using digital PCR promises a fast, sensitive and reliable approach to predicting the efficiency of EGFR-TKI. However, the low throughput and high cost of digital PCR restricts its clinical application., Methods: We designed a digital PCR assay, which can simultaneously detect 39 mutations of exons 18-21 of the EGFR gene. To assess overall performance, retrospective FFPE tissues from 30 NSCLC patients and plasma from 33 NSCLC patients were collected and analysed., Results: The LoD of the EGFR mutations was as low as 0.308 copies/μL, and the linear correlation between the detected and expected values at different concentrations (0.01-10%) was low as well. Compared to ARMS-PCR in FFPE, the accuracy values of the dEGFR39 assay in plasma from 33 patients was 87.88% (29/33, 95% CI 72.67-95.18%). While monitoring the 33 patients, the EGFR mutation load as assessed by dEGFR39 was associated with the objective response to treatment. Thirteen samples from eight patients were identified by dEGFR39 to harbour the T790M mutation over time; of these patients, only nine (69%) were detected using SuperARMS., Conclusion: Our results indicate that dEGFR39 assay is reliable, sensitive and cost-efficient. This method is beneficial for profiling EGFR mutations for precision therapy and prognosis after TKI treatment, especially in patients with insufficient tissue biopsy samples.

Uncovering and quantifying the laws of the evolutionary dynamics of cancer, in particular in the context of specific genetic lesions and in individual patients, has the potential to revolutionize precision oncology. Recent technological advances in the study of human cancer have increased access to in vivo human data and have thereby facilitated the confirmation or refutation of existing theoretical models. In this Perspective, we discuss recent work at the intersection of quantitative mathematical models of cancer evolution and patient data that provides insights into different stages of tumor evolution, including premalignant and malignant progression and response to therapy., (© 2020. Springer Nature America, Inc.)

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene. [ABSTRACT FROM AUTHOR]

We describe the design, fabrication and use of a dual-layered microfluidic device for ultrahigh-throughput droplet trapping, analysis, and recovery using droplet buoyancy. To demonstrate the utility of this device for digital quantification of analytes, we quantify the number of droplets, which contain a β-galactosidase-conjugated bead among more than 100,000 immobilized droplets. In addition, we demonstrate that this device can be used for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction of β-galactosidase and its fluorogenic substrate FDG, between droplets. [ABSTRACT FROM AUTHOR]