Before starting cancer treatment with fluoropyrimidine-based chemotherapies, it is highly recommended to check for dihydropyrimidine dehydrogenase (DPD) deficiency by measuring uracilemia (or calculating the dihydrouracil:uracil ratio). This article discusses some of the ways of doing this.
Background
Approved for treatment of humans 60 years ago, fluoropyrimidinebased chemotherapies remain important antineoplastic agents. They are widely used in Europe, for example in France 100¦000 patients are medicated with this group of anticancer drugs.
Indeed, 5-fluorouracil (5-FU) and its oral pre-prodrug capecitabine are the backbone in the treatment of colorectal, pancreatic, gastric, breast, head and neck cancers. They work by interfering with enzymes (principally thymidylate synthase) involved in producing new DNA, thereby blocking the growth of cancer cells. They are administered by injection or by mouth. However, the use of fluoropyrimidines is associated with an important risk of toxicity, mainly due to deficiency of the enzyme involved in its catabolism, dihydropyrimidine dehydrogenase (DPD).
In France, health authorities recommend the determination of uracil concentration to guide dosing of fluoropyrimidines. Numerous liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been proposed but they include complex liquid–liquid or solid-phase extraction procedures.
Prescribers may be unaware that their patients lack functional DPD (encoded by the DPYD gene) and hence cannot break down fluorouracil, resulting in its build-up. This can lead to severe and life-threatening side effects such as neutropenia, neurotoxicity, severe diarrhea and stomatitis.
Up to 15% of patients exhibit a partial deficiency, whereas 0.1–0.5% may have a complete deficiency. Consequently, a 5-FU dose can lead to severe or lethal toxicity, and it is therefore highly recommended to screen for DPD status to determine a safe dose for the patient.
This deficiency may be detected either by genotyping (an approach that explores the polymorphisms of the DPYD gene) or by phenotyping, which consists of measuring uracilemia or calculating the 5,6-dihydrouracil:uracil (UH2:U) ratio.
Brief methodological overview
- The genotyping approach explores four variants known for reducing DPD activity (DPYD*13, DPYD*9A, DPYD*2A, and 2846A>T) and has the advantage of producing a fast and relatively inexpensive response by using automated techniques. Its specificity is very good, but its sensitivity is poor (not all DPD deficiencies are detected by genotyping).
- DPD is essential for converting endogenous U to UH2. Therefore, uracilemia or the UH2:U ratio reflect the level of DPD activity. Measurement of these components is feasible in plasma by liquid chromatography with photodiode array detection (LC-DAD) and LC-MS but requires complex sample preparation with protein precipitation, liquid–liquid extraction (LLE) or solid-phase extraction. Up to now, only analytical methods with multiple manual steps involving centrifugation, filtration and evaporation have been reported. Although results are satisfactory, the methods are time-consuming and tedious.
In genotyping, genes causing the deficiency are focused on, whereas with LC-MS/MS, the activity of DPD is estimated by measuring the ratio of the compounds UH2 and U. The first method looks only at the cause, whereas the second, safer method, looks at the result considering all deficiency cases while reducing toxic risks.
Need for accuracy, reliability and robustness
Proposed threshold values of 16 and 150 ng/mL for uracilemia characterize a partial or complete DPD deficiency, respectively. Inaccurate quantification of these threshold values may totally influence patient care and medical decisions. Analytical methods must therefore be accurate, reliable and robust. Automation is undoubtedly the best solution for reduction of errors while ensuring best reproducibility, robustness and reliability.
In this context, Shimadzu has developed a fully-automated procedure for the measurement of U and UH2 in human plasma. It is known as indirect phenotyping and provides faster testing as well as greater accuracy, safety and standardization. It is a method where the extraction is carried out by a programmable liquid handler directly coupled to a LC-MS/MS system.
The Centre Hospitalier Universitaire de Limoges (CHU Limoges), France, has been involved in proposing a method combining accuracy and time-efficiency. They suggested a new solution based on a novel sample preparation system, coupling an HPLC instrument and a triplequadrupole mass spectrometer.
Extraction is performed by an automated sample preparation system, the Clinical Laboratory Automation Module (CLAM)-2030 (Shimadzu Corporation) coupled to an LC-MS/MS system. Responding to the needs of clinical research sites, the CLAM-2030 provides stable data acquisition, lower running costs and improved work efficiency. It can be connected to four models of triple-quadrupole liquid chromatography mass spectrometers. Once the primary (or secondary) tube is loaded onto the automated system, no further human intervention is required as the CLAM-2030 resulting in high standardization.
The system was used in positive electrospray ionization mode. Acquisition method targeted multiple reaction monitoring (MRM) transitions for uracil, dihydrouracil, uracil-13C, 15N2 and dihydrouracil-13C, 15N2. The workflow procedure is summarized in Figure 1.
The CLAM-2030 targets pharmaceutical and medical departments as well as biological analysis labs. It is a technological key system applied in Shimadzu’s European Innovation Center (EuIC) programme. The EuIC merges the cutting-edge analytical technologies of Shimadzu with game-changing topics and expertise in markets and science covered by opinion leaders, strategic thinkers and scientific experts in order to create new solutions for tomorrow. In France, the CHU University Hospital is a cooperation partner of the EuIC.
The CLAM-2030 module automates everything from the preparation of urine, blood, and other biological samples to measurement via liquid chromatography mass spectrometry (LC-MS). Within a few minutes, the CLAM-2030 preparation module completes the blood-sample preparation process including the addition of reagents, mixing of the solution and the addition of a deproteinization liquid, compared to the 15–20 minutes that this process conventionally takes. Further, if the samples and reagents are placed and positioned in special containers for automatic conveyance to the LC-MS by an autosampler, the module can perform all of the processes automatically, on weekends and overnight.
Quick results
By overlapping sample treatment, a result is obtained every 14 minutes after the first sample. This method is fully validated according to ISO 15189 requirements. The result of the validation study are summarized in Table 1. A 5 ng/mL limit of quantification is obtained for both U and UH2 with good linearity (R² >0.995). At 16 ng/mL (threshold value) the inaccuracy and coefficients of variation were less than 5% for intra- and inter-assay tests, clearly sufficient to avoid misdiagnosing the level of DPD activity.
The method has been applied successfully in 64 consecutive patients tested at the CHU Limoges, and its results were similar to those of a classic LC-MS method (LLE for sample preparation) used routinely until then. For each patient, the same diagnosis (absence or presence of DPD deficiency) was given and the Bland–Altman plot (Fig. 2) shows good agreement between the two methods.
Conclusion
As DPD deficiency screening in patients given fluoropyrimidine-based chemotherapy is now highly recommended, most labs in charge of the measurement of U (and UH2) will or are already facing an increase in this activity. Shimadzu therefore proposes a fully-automated solution ensuring an accurate and robust measurement without requiring precious laboratory staff time. The simplicity of operation and the minimization of user involvement in the sample preparation process will help obtain high throughput for the monitoring of 5-FU and capecitabine treatments.
Beckman Coulter partners with South West London Pathology to reduce the impact of sepsis
, /in E-News /by 3wmediaThe clinical diagnostics, company Beckman Coulter has implemented DxH 900 haematology analysers and the Early Sepsis Indicator across the South West London Pathology (SWLP) network. SWLP is an award-winning NHS pathology partnership set up by St. George’s University Hospitals NHS Foundation Trust, Croydon Health Services NHS Trust and Kingston Hospital NHS Foundation Trust. The installation enables SWLP laboratories to provide a single, integrated pathology service to more than 3.5 million people across South West London via three hospitals, 200 GP practices and 30 community healthcare sites.
Beckman Coulter’s DxH 900 haematology analysers enable clinical laboratories like SWLP to perform complete blood count and white blood cell differential tests. Demonstrating an industry-leading 93% first-pass yield, the DxH 900 reduces the number of manual slide reviews, helping to generate reportable results as quickly as possible. In addition, the DxH 900 features the Early Sepsis Indicator, the only CE marked and FDA-cleared haematologic biomarker that aids the diagnosis of sepsis in adult patients.
Commenting on the implementation, Simon Brewer, Managing Director at South West London Pathology, said: “Emergency departments across our network see 370,000 patients a year. And, with conditions like sepsis becoming more and more prevalent, it is mission critical to have the tools and technology to identify, diagnose, and begin treatment as early as possible.”
Life science start-up report reveals boom in new life science ventures
, /in E-News /by 3wmediaBioCity, a life science incubator and business collective has released its biennial publication, the UK Life Science Start-Up Report, an in-depth analysis of emerging businesses within the life sciences across the UK.
The report looks at the prevalence of life science start-ups in the UK over the past five years and the broader landscape in which they operate to also asses the quality of UK life science start-ups.
The report documents an unprecedented period of growth for the life sciences, thanks in part to a change in the funding landscape, expressed in a four-fold increase to £2.8 billion of investment in early-stage ventures, compared to the previous five-year period.
Multiple factors are highlighted as driving this expansion, but of greatest impact was Industry news January 2020 11 | the emergence of a number of significant venture funds able and willing to make very large investments in early stage businesses. Also identified as a contributing factor is the increasing use of smaller companies and academia as sources of innovation by large pharma companies aiming to counteract falling R&D productivity. Simultaneously, many universities such as Bristol, Newcastle and Aberdeen introduced a gear change in spin-out formation.
Author of the report, Dr Glenn Crocker said: “Both the number of companies starting up and the amount invested in them has taken off. We have seen a 50% increase in the number of companies and a four-fold increase in investment going into them; this will likely result in a substantial increase in the demand for space. We estimate that this cohort of businesses alone could require 1.4 million sq ft of specialist facilities over the next five years. One consequence of this demand growth is that real estate investors are being increasingly attracted to the sector.”
AI can be used to detect and grade prostate cancer
, /in E-News /by 3wmediaResearchers at Karolinska Institutet have developed a method based on artificial intelligence for histopathological diagnosis and grading of prostate cancer. The AI-system has the potential to solve one of the bottlenecks in today’s prostate cancer histopathology by providing more accurate diagnosis and better treatment decisions. The study shows that the AI-system is as good at identifying and grading prostate cancer as world-leading uro-pathologists.
“Our results show that it is possible to train an AI-system to detect and grade prostate cancer on the same level as leading experts,” says Martin Eklund, associate professor at the Department of Medical Epidemiology and Biostatistics at Karolinska Institutet who led the study. “This has the potential to significantly reduce the workload of uro-pathologists and allow them to focus on the most difficult cases.”
A problem in today’s prostate pathology is that there is a certain degree of subjectivity in the assessments of the biopsies. Different pathologists can reach different conclusions even though they are studying the same samples. This leads to a clinical problem where the doctors must pick treatment based on ambiguous information. In this context, the researchers see significant potential to use the AI-technology to increase the reproducibility of the pathological assessments.
To train and test the AI system, the researchers digitized more than 8000 biopsies taken from some 1200 Swedish men in the ages of 50–69 to high-resolution images using digital pathology scanners. About 6,600 of the samples were used to train the AI system to spot the difference between biopsies with or without cancer. The remaining samples, and additional sets of samples collected from other labs, were used to test the AI system. Its results were also compared against the assessments of 23 world-leading uro-pathologists. The study was conducted in collaboration with researchers at Tampere University in Finland.
The findings showed that the AI-system was almost near-perfect in determining whether a sample contained cancer or not, as well as in estimating the length of the cancer tumour in the biopsy. When it comes to determining the severity of the prostate cancer, the so-called Gleason score, the AI system was on par with the international experts.
“When it comes to grading the severity of the prostate cancer, the AI is in the same range as international experts, which is very impressive, and when it comes to diagnostics, to determine whether or not it is cancer, the AI is simply outstanding,” says Lars Egevad, professor in pathology at Karolinska Institutet and co-author of the study.
The initial findings are promising but more validation is needed before the AI system may be rolled out broadly in clinical practice, according to the researchers. That is why a multicenter study spanning nine European countries is currently underway with completion slated by the end of 2020. That study, which is financed by EIT Health, aims to train the AI-system to recognize cancer in biopsies taken from different laboratories, with different types of digital scanners and with very rare growth patterns. In addition, a randomized study starting in 2020 will examine how the AI-model may be implemented in Sweden’s health care system.
“AI-based evaluation of prostate cancer biopsies could revolutionize future health care,” says Henrik Grönberg, professor in cancer epidemiology at Karolinska Institutet and head of the Prostate Cancer Center at St Göran Hospital in Stockholm. “It has the potential to improve the diagnostic quality, and thereby secure a more equitable care at a lower cost.” Karolinska Institute
Johnson & Johnson aims to produce a billion doses of COVID-19 vaccine
, /in Corona News, E-News /by 3wmediaJohnson & Johnson has announced the selection of a lead COVID-19 vaccine candidate on which it expects to initiate human clinical studies by September at the latest with the first batches of the vaccine available for emergency use authorization in early 2021.
In addition, the company announced the significant expansion of the existing partnership between the Janssen Pharmaceutical Companies of Johnson & Johnson and the Biomedical Advanced Research and Development Authority (BARDA).
Johnson & Johnson also said the company will rapidly scale up its manufacturing capacity with the goal of providing a global supply of more than one billion doses of the vaccine.
Through the new partnership, BARDA and Johnson & Johnson together have committed more than $1 billion of investment to co-fund vaccine research, development, and clinical testing. The company says will use its validated vaccine platform and is allocating resources, including personnel and infrastructure globally, as needed, to focus on these efforts.
BARDA is part of the Office of the Assistant Secretary for Preparedness and Response (ASPR) at the U.S. Department of Health and Human Services.
Commenting on the initiative, Alex Gorsky, Chairman and Chief Executive Officer, Johnson & Johnson, said: “The world is facing an urgent public health crisis and we are committed to doing our part to make a COVID-19 vaccine available and affordable globally as quickly as possible. As the world’s largest healthcare company, we feel a deep responsibility to improve the health of people around the world every day. Johnson & Johnson is well positioned through our combination of scientific expertise, operational scale and financial strength to bring our resources in collaboration with others to accelerate the fight against this pandemic.”
The company’s expansion of its manufacturing capacity will include the establishment of new U.S. vaccine manufacturing capabilities and scaling up capacity in other countries. The additional capacity will assist in the rapid production of a vaccine and will enable the supply of more than one billion doses of a safe and effective vaccine globally.
Paul Stoffels, M.D., Vice Chairman of the Executive Committee and Chief Scientific Officer, Johnson & Johnson, said: “We are very pleased to have identified a lead vaccine candidate from the constructs we have been working on since January. We are moving on an accelerated timeline toward Phase 1 human clinical trials at the latest by September 2020 and, supported by the global production capability that we are scaling up in parallel to this testing, we expect a vaccine could be ready for emergency use in early 2021.” In addition to the vaccine development efforts, BARDA and Johnson & Johnson have also expanded their partnership to accelerate Janssen’s ongoing work in screening compound libraries, including compounds from other pharmaceutical companies. The company’s aim is to identify potential treatments against the novel coronavirus. Johnson & Johnson and BARDA are both providing funding as part of this partnership. These antiviral screening efforts are being conducted in partnership with the Rega Institute for Medical Research (KU Leuven/University of Leuven), in Belgium.
UK consortium set to trial COVID-19 adenoviral vaccine candidate
, /in Corona News, E-News /by 3wmediaA research consortium led by the Jenner Institute, Oxford University is set to begin fast-tracked clinical trials for a COVID-19 vaccine.
The adenoviral vaccine candidate, ChAdOx1 nCov-19 (ChAdOx1) is one of five frontrunner vaccines in development around the world, and expected to be the UK’s first COVID-19 vaccine.
Developed at the Jenner Institute, ChAdOx1 is one of the most promising vaccine technologies for COVID-19 as it can generate a strong immune response from one dose.
Cobra Biologics (Cobra), an international CDMO for biologics and pharmaceuticals, issued a statement 31 March saying they had joined the consortium to assist with the rapid development scale-up and production of the vaccine.
The ChAdOx1 consortium includes the University of Oxford Jenner Institute, University of Oxford Clinical Biomanufacturing Facility, the Vaccines Manufacturing and Innovation Centre (VMIC), Advent Srl, Pall Life Sciences, Cobra Biologics and Halix BV.
The consortium is currently recruiting individuals from a range of ages in the UK to trial the vaccine’s efficacy, in April 2020 – a crucial step in the vaccine’s development. Cobra is actively planning for a fast set-up phase to facilitate the efficient production of a GMP working cell bank and then 200L GMP viral vaccine. The consortium partners expect to develop and manufacture the vaccine candidate in multiple batches, to support a 1 million dose scale batch size, by mid 2020.
For more information about the trial, visit: www.covid19vaccinetrial.co.uk
Siemens Healthineers awarded FDA approval for RAPIDPoint 500e Blood Gas Analyzer
, /in Corona News, E-News /by 3wmediaSiemens Healthineers’s latest critical care testing solution, the RAPIDPoint® 500e Blood Gas Analyzer, has received clearance from the U.S. FDA, and is now available in the U.S., Europe and countries requiring the CE mark. The analyser generates blood gas, electrolyte, metabolite, CO-oximetry, and neonatal bilirubin results, which are used to diagnose and monitor critically ill patients in the intensive care unit, operating room, or emergency room.
The RAPIDPoint 500e Blood Gas Analyzer is an essential instrument supporting COVID-19 response efforts, where blood gas testing plays a critical role in managing infected patients and monitoring their respiratory distress. Routine blood gas testing is also performed when patients require mechanical ventilation. Arterial blood gas tests provide the status of a patient’s oxygenation levels and enable healthcare providers to determine whether adjustments to ventilator settings or other treatments are required.
“The RAPIDPoint 500e Blood Gas Analyzer has become a trusted instrument in Europe’s endeavour to combat COVID-19 and to help address an unprecedented demand for blood gas testing in affected respiratory patients,” said Christoph Pedain, Head of Point of Care Diagnostics, Siemens Healthineers.
“Point-of-care teams monitoring respiratory conditions in critical care settings need a blood gas testing solution that delivers fast, accurate results and increases workflow efficiencies. A safe operating environment amid growing concerns about cybersecurity threats in healthcare is also important.”
The analyser elevates confidence in patient results with Integri-sense Technology, a comprehensive series of automated functional checks designed to deliver accurate test results at the point-of-care. Additionally, the RAPIDPoint 500e Analyzer integrates seamlessly into hospital networks with the Siemens Healthineers Point of Care Ecosystem, which offers convenient, remote management of operators and devices located across multiple sites.
Commenting on the device, Dr. Daniel Martin, Royal Free Hospital, London, said: “As an ICU physician, I know that the values I am handed during an emergency allow me to confidently make life-saving decisions. The RAPIDPoint system is easy to use and allows me to not worry about the machine and focus my attention on my patients.”
PCR Biosystems scales up production to meet global demand for COVID-19 diagnostic test
, /in Corona News, E-News /by 3wmediaUK-based PCR Biosystems issued a statement 1 April saying they are continuing to scale up operations to ensure the critical enzyme mix for COVID-19 tests remain available to the UK and global healthcare systems as demand for testing rises.
To meet current and upcoming requirements and ensure supply chain security, PCR Biosystems has already significantly increased – and will continue to increase – manufacture of qPCRBIO Probe 1-Step Go and all other critical reagents for rapid and sensitive RT-qPCR, the company said.
The company noted it has capacity to manufacture enough reagent daily for 4 million reactions – which is sufficient for millions of diagnostic tests.
qPCRBIO Probe 1-Step Go is a universal probe kit designed for fast and sensitive probe-based RT-qPCR. It is PCR Biosystems’s recommended product for COVID-19 diagnostic tests, supporting the detection, quantification and typing of the SARS-CoV-2 virus. All that’s required is the addition of specific primers and probes, together with the swab extract and water. qPCRBIO Probe 1-Step Go is compatible with all qPCR instruments and is engineered for use on a wide range of probe technologies including TaqMan®, Scorpions® and molecular beacon probes. In March 2020, PCR Biosystems introduced bulk pack sizes of this key product, to further support customers in high-throughput COVID-19 testing.
Alex Wilson, Co-Founder of PCR Biosystems, explained: “These are unprecedented times, and, as a global PCR company, we are ideally placed to support the scientific and healthcare communities in their response to COVID-19. When the enormity of COVID-19 testing requirements became apparent, we immediately started scaling up production of the critical components. We already have capacity to supply 4 million reactions’ worth of reagent every day – and we have the option to scale up further if needed to ensure we can always meet global demand.”
For more information on PCR Biosystems’s reagents, visit: www.pcrbio.com
Oxford-based businesses collaborate to scale up production of SARS-CoV-2 antigens
, /in Corona News, E-News /by 3wmediaOXGENE and The Native Antigen Company are collaborating to scale up production of SARS-CoV-2 reagents by combining OXGENE’s proprietary Adenoviral Protein Machine Technology with The Native Antigen Company’s antigen development expertise. Together, they aim to scale their antigen manufacturing capabilities to deliver high-purity, recombinant proteins for the development of diagnostics and vaccines.
Unlike the PCR tests that are currently being used, these diagnostics will be able to confirm past infections and determine levels of immunity to SARS-CoV-2. This could be invaluable for disease modelling and public health policy, as true transmission rates and case fatality rates can be determined. These tests could also be instrumental for the diagnosis of healthcare workers who have been exposed to the virus to ensure that they have developed natural immunity before returning to work, and to help measure patient immune responses for the rapid development of a SARS-CoV-2 vaccine.
The Native Antigen Company was one of the first recognised suppliers of SARS-CoV-2 antigens in February 2020, demonstrating their ability to rapidly support the diagnostic and vaccine industries with high-quality infectious disease reagents.
OXGENE’s Protein Machine Technology allows for the scalable production of viral proteins in mammalian cells using their proprietary adenoviral expression vector. Through genetic modification, the adenovirus is ‘tricked’ into making SARS-CoV-2 proteins rather than its own, thereby harnessing the innate power of highly scalable viral protein production.
Commenting on the collaboration, Dr Ryan Cawood, Chief Executive, OXGENE, said: “Our novel Protein Machine Technology represents a significant development in the rapid and scalable generation of high-quality viral proteins. We’re delighted that by collaborating with The Native Antigen Company, we can take advantage of our technology to support the needs of researchers racing to develop much-needed diagnostics and vaccines against COVID-19.”
The Native Antigen Company’s recombinant SARS-CoV-2 antigens are produced in mammalian cells to ensure full glycosylation and proper protein folding, both of which are essential for full biological and antigenic activity. The rapid scale up production of SARS-CoV-2 antigens is critical for the development of widely available diagnostic tests.
Dr Andy Lane, Commercial Director, The Native Antigen Company, said: “We are committed to developing the highest-quality reagents in rapid response to emerging epidemic diseases. Since the start of the crisis, the demand for our COVID-19 antigens has increased significantly, and by scaling up production of these vital reagents in collaboration with OXGENE, we hope to be able to support more researchers in their critical work developing diagnostics and vaccines.”
This collaboration builds on a long-standing collegiate relationship between the two Oxford-based businesses as they work towards developing more scalable technologies for the diagnosis of disease, and the cost-effective manufacture of high-quality diagnostics and vaccines.
OXGENE and The Native Antigen Company aim to complete the first validation of this new paradigm in protein expression by May 2020, which could have a demonstrable impact on the race to develop diagnostic kits and vaccines against this virus.
For further information about The Native Antigen Company’s Coronavirus Antigens, visit: https://thenativeantigencompany.com/coronavirus-dashboard/
GC-MS discovery of biomarkers will allow non-invasive early disease detection by breath biopsy
, /in E-News /by 3wmediaOwlstone Medical and Thermo Fisher Scientific recently announced a collaborative partnership to advance the early diagnosis of cancer and other diseases. This will involve the integration of Orbitrap gas chromatography mass spectrometry (GC-MS) instrumentation into Owlstone Medical’s Breath Biopsy platform, aiding metabolomics studies of breath samples for unique biomarkers that could translate into non-invasive, routine screening solutions for improved early diagnosis of cancer and other disease. CLI caught up with Dr Max Allsworth, Owlstone Medical, and Dominic Roberts, Thermo Fisher Scientific, to discuss how MS has benefited clinical lab diagnostics.
Mass spectrometry is an incredibly powerful technique, used increasingly in clinical lab diagnostics. How has it been of benefit in this application?
Clinical laboratories involved in both routine and research applications are under ever-increasing pressure to deliver fast results, while maintaining the highest levels of accuracy and confidence. The majority of these laboratories currently rely on targeted analytical approaches, using both gas chromatography (GC) and liquid chromatography coupled to triple quadrupole mass spectrometry (MS) instrumentation. These techniques cover the wide range of chemical classes to be monitored at the required levels of sensitivity and selectivity. However, they are limited to those compounds in the target list and they require careful optimization of acquisition parameters for each compound. High-resolution, full-scan MS using Orbitrap technology provides a solution to meet:
While MS adoption in clinical settings has been somewhat limited to date, that is rapidly changing. A small number of MS-based assays have received United States Food & Drug Administration (U.S. FDA) clearance over the past few years in areas including microbiology pathogen identification, vitamin D quantitation, newborn screening and genetic analyses. One of the key benefits of MS adoption in clinical settings is its flexibility. The same instrumentation platform can be deployed into a wide variety of applications, being able to detect and measure protein, lipid, genomic, and the area with perhaps most clinical promise, metabolites. As a result, a broad range of laboratorydeveloped tests now exist in Clinical Laboratory Improvement Amendments (CLIA)-facilities with more being developed all the time.
One of the areas of greatest promise of MS in clinical settings is through the deployment of Breath Biopsy®. Metabolites, being the furthest downstream in biological processes, represent the most phenotypically relevant biomarkers that take into account both endogenous and external drivers of disease. Breath represents an extremely exciting approach to capturing these chemicals at very low levels with powerful implications for the early detection of disease and the effective delivery of precision medicine.
What current work is underway for developing the use of MS in the clinical lab?
GC-MS is Owlstone Medical’s core discovery technology, enabling us to explore volatile organic compounds in breath, seeking to link specific chemicals, and the changes in their levels, to specific diseases. In many metabolomics studies samples have to undergo a complex sample preparation protocol that can lead to complexity and variation if not controlled adequately. This is particularly true of liquid samples. However, as Owlstone Medical is identifying breath-based volatile biomarkers directly, sample preparation is relatively simple. By using thermal desorption to release the chemicals found in breath, which we have captured on a sorbent matrix in cartridges as part of our ReCIVA® Breath Sampler, the outflow can be directly introduced into a GC-MS system.
Owlstone Medical is focused on developing diagnostic and screening solutions in oncology (for example through LuCID, the world’s largest breath-based clinical trial for the discovery of breath-based biomarkers of early-stage lung cancer), liver disease (with whom they have partnered with the Cleveland Clinic), respiratory disease (working with AstraZeneca and GSK on asthma and COPD), and environmental exposure. In the future, once tests have been developed and launched into the market, sample analysis for a substantial portion of these tests will also be via GC-MS.
DPD identification is key in avoiding serious reaction to 5-FU cancer drug
, /in E-News /by 3wmediaBefore starting cancer treatment with fluoropyrimidine-based chemotherapies, it is highly recommended to check for dihydropyrimidine dehydrogenase (DPD) deficiency by measuring uracilemia (or calculating the dihydrouracil:uracil ratio). This article discusses some of the ways of doing this.
Background
Approved for treatment of humans 60 years ago, fluoropyrimidinebased chemotherapies remain important antineoplastic agents. They are widely used in Europe, for example in France 100¦000 patients are medicated with this group of anticancer drugs.
Indeed, 5-fluorouracil (5-FU) and its oral pre-prodrug capecitabine are the backbone in the treatment of colorectal, pancreatic, gastric, breast, head and neck cancers. They work by interfering with enzymes (principally thymidylate synthase) involved in producing new DNA, thereby blocking the growth of cancer cells. They are administered by injection or by mouth. However, the use of fluoropyrimidines is associated with an important risk of toxicity, mainly due to deficiency of the enzyme involved in its catabolism, dihydropyrimidine dehydrogenase (DPD).
In France, health authorities recommend the determination of uracil concentration to guide dosing of fluoropyrimidines. Numerous liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been proposed but they include complex liquid–liquid or solid-phase extraction procedures.
Prescribers may be unaware that their patients lack functional DPD (encoded by the DPYD gene) and hence cannot break down fluorouracil, resulting in its build-up. This can lead to severe and life-threatening side effects such as neutropenia, neurotoxicity, severe diarrhea and stomatitis.
Up to 15% of patients exhibit a partial deficiency, whereas 0.1–0.5% may have a complete deficiency. Consequently, a 5-FU dose can lead to severe or lethal toxicity, and it is therefore highly recommended to screen for DPD status to determine a safe dose for the patient.
This deficiency may be detected either by genotyping (an approach that explores the polymorphisms of the DPYD gene) or by phenotyping, which consists of measuring uracilemia or calculating the 5,6-dihydrouracil:uracil (UH2:U) ratio.
Brief methodological overview
In genotyping, genes causing the deficiency are focused on, whereas with LC-MS/MS, the activity of DPD is estimated by measuring the ratio of the compounds UH2 and U. The first method looks only at the cause, whereas the second, safer method, looks at the result considering all deficiency cases while reducing toxic risks.
Need for accuracy, reliability and robustness
Proposed threshold values of 16 and 150 ng/mL for uracilemia characterize a partial or complete DPD deficiency, respectively. Inaccurate quantification of these threshold values may totally influence patient care and medical decisions. Analytical methods must therefore be accurate, reliable and robust. Automation is undoubtedly the best solution for reduction of errors while ensuring best reproducibility, robustness and reliability.
In this context, Shimadzu has developed a fully-automated procedure for the measurement of U and UH2 in human plasma. It is known as indirect phenotyping and provides faster testing as well as greater accuracy, safety and standardization. It is a method where the extraction is carried out by a programmable liquid handler directly coupled to a LC-MS/MS system.
The Centre Hospitalier Universitaire de Limoges (CHU Limoges), France, has been involved in proposing a method combining accuracy and time-efficiency. They suggested a new solution based on a novel sample preparation system, coupling an HPLC instrument and a triplequadrupole mass spectrometer.
Extraction is performed by an automated sample preparation system, the Clinical Laboratory Automation Module (CLAM)-2030 (Shimadzu Corporation) coupled to an LC-MS/MS system. Responding to the needs of clinical research sites, the CLAM-2030 provides stable data acquisition, lower running costs and improved work efficiency. It can be connected to four models of triple-quadrupole liquid chromatography mass spectrometers. Once the primary (or secondary) tube is loaded onto the automated system, no further human intervention is required as the CLAM-2030 resulting in high standardization.
The system was used in positive electrospray ionization mode. Acquisition method targeted multiple reaction monitoring (MRM) transitions for uracil, dihydrouracil, uracil-13C, 15N2 and dihydrouracil-13C, 15N2. The workflow procedure is summarized in Figure 1.
The CLAM-2030 targets pharmaceutical and medical departments as well as biological analysis labs. It is a technological key system applied in Shimadzu’s European Innovation Center (EuIC) programme. The EuIC merges the cutting-edge analytical technologies of Shimadzu with game-changing topics and expertise in markets and science covered by opinion leaders, strategic thinkers and scientific experts in order to create new solutions for tomorrow. In France, the CHU University Hospital is a cooperation partner of the EuIC.
The CLAM-2030 module automates everything from the preparation of urine, blood, and other biological samples to measurement via liquid chromatography mass spectrometry (LC-MS). Within a few minutes, the CLAM-2030 preparation module completes the blood-sample preparation process including the addition of reagents, mixing of the solution and the addition of a deproteinization liquid, compared to the 15–20 minutes that this process conventionally takes. Further, if the samples and reagents are placed and positioned in special containers for automatic conveyance to the LC-MS by an autosampler, the module can perform all of the processes automatically, on weekends and overnight.
Quick results
By overlapping sample treatment, a result is obtained every 14 minutes after the first sample. This method is fully validated according to ISO 15189 requirements. The result of the validation study are summarized in Table 1. A 5 ng/mL limit of quantification is obtained for both U and UH2 with good linearity (R² >0.995). At 16 ng/mL (threshold value) the inaccuracy and coefficients of variation were less than 5% for intra- and inter-assay tests, clearly sufficient to avoid misdiagnosing the level of DPD activity.
The method has been applied successfully in 64 consecutive patients tested at the CHU Limoges, and its results were similar to those of a classic LC-MS method (LLE for sample preparation) used routinely until then. For each patient, the same diagnosis (absence or presence of DPD deficiency) was given and the Bland–Altman plot (Fig. 2) shows good agreement between the two methods.
Conclusion
As DPD deficiency screening in patients given fluoropyrimidine-based chemotherapy is now highly recommended, most labs in charge of the measurement of U (and UH2) will or are already facing an increase in this activity. Shimadzu therefore proposes a fully-automated solution ensuring an accurate and robust measurement without requiring precious laboratory staff time. The simplicity of operation and the minimization of user involvement in the sample preparation process will help obtain high throughput for the monitoring of 5-FU and capecitabine treatments.