Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) supports blood-based genomic profiling but is not yet routinely implemented in the setting of a phase I trials clinic. TARGET is a ...molecular profiling program with the primary aim to match patients with a broad range of advanced cancers to early phase clinical trials on the basis of analysis of both somatic mutations and copy number alterations (CNA) across a 641 cancer-associated-gene panel in a single ctDNA assay. For the first 100 TARGET patients, ctDNA data showed good concordance with matched tumor and results were turned round within a clinically acceptable timeframe for Molecular Tumor Board (MTB) review. When a 2.5% variant allele frequency (VAF) threshold was applied, actionable mutations were identified in 41 of 100 patients, and 11 of these patients received a matched therapy. These data support the application of ctDNA in this early phase trial setting where broad genomic profiling of contemporaneous tumor material enhances patient stratification to novel therapies and provides a practical template for bringing routinely applied blood-based analyses to the clinic.
Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are ...inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.
Droplet Digital PCR (ddPCR) is a sensitive platform used to quantify specific nucleic acid molecules amplified by polymerase chain reactions. Its sensitivity makes it particularly useful for the ...detection of rare mutant molecules, such as those present in a sample of circulating free tumour DNA obtained from cancer patients. ddPCR works by partitioning a sample into individual droplets for which the majority contain only zero or one target molecule. Each droplet then becomes a reaction chamber for PCR, which through the use of fluorochrome labelled probes allows the target molecules to be detected by measuring the fluorescence intensity of each droplet. The technology supports two channels, allowing, for example, mutant and wild type molecules to be detected simultaneously in the same sample. As yet, no open source software is available for the automatic gating of two channel ddPCR experiments in the case where the droplets can be grouped into four clusters. Here, we present an open source R package 'twoddpcr', which uses Poisson statistics to estimate the number of molecules in such two channel ddPCR data. Using the Shiny framework, an accompanying graphical user interface (GUI) is also included for the package, allowing users to adjust parameters and see the results in real-time.
twoddpcr is available from Bioconductor (3.5) at https://bioconductor.org/packages/twoddpcr/ . A Shiny-based GUI suitable for non-R users is available as a standalone application from within the package and also as a web application at http://shiny.cruk.manchester.ac.uk/twoddpcr/ .
ged.brady@cruk.manchester.ac.uk or crispin.miller@cruk.manchester.ac.uk.
anthony.chiu@cruk.manchester.ac.uk.
Serial biopsy of pancreatic ductal adenocarcinoma (PDAC), to chart tumour evolution presents a significant challenge. We examined the utility of circulating free DNA (cfDNA) as a minimally invasive ...approach across a cohort of 55 treatment-naïve patients with PDAC; 31 with metastatic and 24 with locally advanced disease. Somatic mutations in cfDNA were detected using next generation sequencing in 15/24 (62.5%) and 27/31 (87%) of patients with locally advanced and metastatic disease, respectively. Copy number changes were detected in cfDNA of 10 patients of whom 7 exhibited gain of chromosome 12p harbouring KRAS as well as a canonical KRAS codon 12 mutation. In multivariable Cox Regression analysis, we show for the first time that patients with KRAS copy number gain and KRAS mutation have significantly worse outcomes, suggesting that this may be linked to PDAC progression. The simple cfDNA assay we describe will enable determination of the presence of KRAS copy number gain and KRAS mutations in larger studies and clinical trials.
VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models ...showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca2+ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca2+ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.
Background: VGF is a neuropeptide involved in chronic pain.
Results: VGF-derived peptide TLQP-21 activates macrophages. We identified gC1qR as a receptor for TLQP-21.
Conclusion: TLQP-21 and gC1qR are involved in chronic pain pathways.
Significance: TLQP-21 and gC1qR may be drug targets for chronic pain treatment.
Poverty and Inequality Ayub, Mahmood
Global journal of emerging market economies,
09/2013, Letnik:
5, Številka:
3
Journal Article
Despite economic growth, a large number of Africans continue to live in poverty and experiences high levels of income and opportunity inequality. Not only does inequality dampen the poverty reduction ...impact of growth, lower the growth rate, hollow out the middle class, encourage corruption and rent seeking, increase crime and violence, and undermine social stability, but it also precludes sustained growth. Access to education, power supply, health services, and water and sanitation are critical to improve the lives of all Africans, especially girls and those in rural areas. In this area, public spending and government transfers can only go so far—the voice of Africa’s poorest citizens needs to be heard by policymakers. While maintaining central government oversights, appropriate steps should be taken to increase participation of the beneficiaries of public services.
Abstract
Motivation
Circulating-free DNA (cfDNA) profiling by sequencing is an important minimally invasive protocol for monitoring the mutation profile of solid tumours in cancer patients. Since the ...concentration of available cfDNA is limited, sample library generation relies on multiple rounds of PCR amplification, during which the accumulation of errors results in reduced sensitivity and lower accuracy.
Results
We present PCR Error Correction (PEC), an algorithm to identify and correct errors in short read sequencing data. It exploits the redundancy that arises from multiple rounds of PCR amplification. PEC is particularly well suited to applications such as single-cell sequencing and circulating tumour DNA (ctDNA) analysis, in which many cycles of PCR are used to generate sufficient DNA for sequencing from small amounts of starting material. When applied to ctDNA analysis, PEC significantly improves mutation calling accuracy, achieving similar levels of performance to more complex strategies that require additional protocol steps and access to calibration DNA datasets.
Availability and implementation
PEC is available under the GPL-v3 Open Source licence, and is freely available from: https://github.com/CRUKMI-ComputationalBiology/PCR_Error_Correction.git.
Supplementary information
Supplementary data are available at Bioinformatics online.
SCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient ...stratification and response monitoring.
We applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA the percentage of genomic regions amplified), Z-score (a measure of standard deviation), and Moran’s I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample.
Genome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival.
We demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.
Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific ...surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.
Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, ...which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours.
In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.
•Demonstrated collection of blood in CellSave tubes preserves cfDNA up to 96 h post draw.•No significant difference between standard EDTA cfDNA and preserved cfDNA.•Preserved cfDNA applicable to both targeted and genome wide NGS analysis.•CTCs and cfDNA can be isolated from same CellSave blood sample.•Comparable copy number aberrations seen in both CTCs and ctDNA from same blood sample.
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