Liquid biopsy is likely to play an increasing role in identifying patients with colorectal cancer (CRC) who are likely to relapse after surgery, and has potential for optimising treatment for individual patients, according to new research.
Of 805 patients in the phase III IDEA-FRANCE trial who had liquid biopsy prior to adjuvant chemotherapy for stage III CRC, 109 (13.5%) had circulating tumour DNA (ctDNA) in their blood.
In this group, two-year disease-free survival (DFS) was 64%, compared to 82% in those who were ctDNA negative.
“In this large prospective trial, we confirmed that ctDNA is an independent prognostic factor in colorectal cancer and that approximately six out of 10 patients who are ctDNA positive will remain disease-free two years after standard adjuvant chemotherapy, compared to eight out of 10 of those who are ctDNA negative,” said study author Prof Julien Taieb, Hôpital European Georges Pompidou, Paris, France.
IDEA-FRANCE also showed that six months of adjuvant treatment was superior to three months in both ctDNA positive and negative patients, and that ctDNA positive patients treated for six months had a similar prognosis to ctDNA negative patients treated for three months.
Adjuvant therapy was FOLFOX (folinic acid, fluorouracil and oxaliplatin) in 90% of cases.
“ctDNA testing did not predict which patients should have three or six months of adjuvant chemotherapy and there is continuing debate over the optimal type and duration of treatment for patients who are ctDNA positive, but we do now know that ctDNA is a major prognostic factor which will be very useful in stratifying patients and driving future trials of colorectal cancer,” said Taieb.
“In all subgroups, ctDNA positive patients who only had three months of adjuvant therapy had the worst prognosis,” he added.
Thirty to 50% of patients with localised CRC relapse despite primary optimal therapy, and a second study reported at the ESMO Congress 2019 investigated whether ctDNA can be used to detect minimal residual disease and identify those at risk of recurrence.
The results showed that post-surgical plasma ctDNA predicted metastatic relapse a median of 10 months before recurrence was visible on radiological scans (hazard ratio 11.33; p=0.0001).
The researchers concluded that plasma ctDNA testing opens up an opportunity for precision treatment of patients with localised CRC.
Commenting on the results of the CRC presentations, Prof Alberto Bardelli, University of Turin, Italy, said: “When patients have surgery for early stage colorectal cancer, doubts remain as to whether the disease has been completely eradicated and, as a result, patients often receive adjuvant chemotherapy. However, the IDEA-FRANCE results have shown we can now use a blood test to say whether the patient is clear or not.”
ecancerecancer.org/en/news/16682-esmo-2019-liquid-biopsy-has-prognostic-role-in-colorectal-cancer-and-potential-for-guiding-therapy
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by Prof. Michael Vogeser, Dr Judy Stone and Prof. Alan Rockwood While analytical standardization and metrological traceability are well-defined terms, ‘methodological standardization’ in clinical mass spectrometry is still in a developing stage. We propose a framework that facilitates the widespread implementation of this highly complex and very powerful technology and is based on two pillars – standardization of the description of LC-MS/MS methods and standardization of the release of clinical test results as a three-step sequence of method validation, batch validation and validation of individual measurements. Mass spectrometry in the clinical laboratory Mass spectrometry (MS)-based methods now play an important role in many clinical laboratories worldwide. To date, areas of application have focused especially on screening for hereditary metabolic diseases, therapeutic drug monitoring, clinical toxicology and endocrinology. In fact, these techniques offer significant advantages over immunoassays and photometry as basic standard technologies in clinical chemistry: high analytical selectivity through true molecular detection; wide range of applications without the need for specific molecular features (as in UV detection or specific epitopes); high multiplexing capacity and information-rich detection; and, in many cases, matrixindependent analyses, thanks to the principle of isotope dilution [1].
Various MS technologies – in particular tandem MS (MS/MS-coupling with molecular fragmentation), time-of-flight (TOF) MS and Orbitrap-MS – with front-end fractionation technologies such as HPLC or UPLC potentially allow very reliable analysis, but the technology itself is no guarantee of this: these techniques have a very high complexity and a wide range of potential sources of error [2] which require comprehensive quality assurance [3–5]. Indeed, the high degree of complexity is still the main hurdle for the application of MS in the special environment of clinical laboratories. Specific challenges of this type of laboratory – in contrast to research and development laboratories – include: heterogeneous mix of staff qualifications; requirement for maximum handling safety when operating a large number of analysis platforms; work around the clock; and direct impact on the outcome of the individual patient.
Indeed, after more than two decades of commercial availability of LC-MS/MS instruments, their application in a global perspective has remained very limited. The translation of MS into fully automated ‘black box’ instruments is underway, but still far from being realized on a large scale [6], with laboratory developed tests (LDTs) still dominating the field of clinical MS applications. Kit solutions for specific analytes provided by the in vitro diagnostics (IVD) industry are becoming increasingly available, but their application also requires a very high level of skills and competence from laboratories.
Two main differences of MS-based LDTs as opposed to standard ‘plug-and-play’ analysis systems in today’s clinical laboratories can be identified: first, the high heterogeneity of device configurations and second, the handling of large amounts of data, from sample list structures to technical metadata analysis.
In fact, the random access working mode is now so widespread in all clinical laboratories that the ‘analytical batch’ is no longer standard in laboratories. In the same way, modern analytical instruments no longer challenge the end users with extensive metadata (such as reaction kinetics or calibration diagrams). To achieve the goal of making the extraordinary and disruptive analytical power of MS fully usable for medicine to an appropriate extent, approaches to master the heterogeneity of platform configurations and to regulate the handling of batches and metadata are urgently needed – and standardization efforts seem to be crucial in this context. Standardization of the method description IVD companies manufacture many different instrument platforms, but each of these platforms is very homogeneous worldwide and is produced in large quantities for years. In contrast, MS platforms in clinical laboratories have to be individually assembled from a very large number of components from many manufacturers (sample preparation modules, autosamplers, high performance pumps, switching valves, chromatography columns, ion sources, mass analysers, vacuum systems, software packages, etc). As a result, hardly any two instrument configurations in different laboratories correspond completely with each other. This makes handling very demanding for operators, maintenance personnel, and service engineers.
Methods implemented on these heterogeneous platforms (e.g. instruments from various vendors) are in turn characterized by a very considerable number of variables, e.g. chromatographic gradients, labelling patterns of internal standards, purity of solvents, dead volume of flow paths, etc.
Taken together, the variety of assays referring to an identical analyte (such as tacrolimus or testosterone) is enormous, with an almost astronomical combinatorial complexity.
However, method publications are still traditionally written more or less in a case report approach: the feasibility and performance of a method realization is demonstrated for one individual system configuration. It is usually not clear which features are really essential for the method and which features can be variable between different implementations – and which second implementation can still be considered ‘the same’ method. This means that the question of the true ‘identity’ of a method has not yet been deepened by application notes or publications in scientific journals; thus the level of abstraction required here is missing.
In an attempt to standardize the description of MS/MS-based methods, we selected a set of 35 characteristics that are defined as essential for a method (see Table 1) [7], for example, main approach of sample preparation (e.g. protein precipitation with acetonitrile), main technique of ionization (e.g. electrospray ionization in negative mode); molecular structure of the internal standard; mass transitions; calibration range. In addition, we define 15 characteristics of a method that cannot or should not be realistically standardized in time and space (examples: manufacturer and brand of the MS detector; dead volume of the flow path; lot of analytical columns and solvents). These characteristics – identified as variable – should be documented in the internal report files.
We found it feasible to describe several exemplary MS/MS methods using this scheme and a corresponding matrix. On the basis of this matrix, the method transfer to different platforms and laboratories will be much easier and more reliable. Specifying the identity of a method in the proposed way has the essential advantage that a method revalidation can be transparently triggered by defined criteria, e.g. the use of a novel internal standard with a different labelling pattern.
The proposed scheme for method description may also be the basis of a comprehensive traceability report for any result obtained by an MS-based method in the clinical laboratory. Standardization of batch release (Table 2) While today’s routine analyser platforms essentially provide unambiguous final results for each sample, the process of generating quantitative results from primary data in MS is open and transparent. Primary data in MS are the peak areas of the target analyte observed in diagnostic samples. In addition to these primary data, a range of metadata is provided (e.g. internal standard area, peak height-to-area, peak skewness, qualifier peak area; metadata related to analytical batches, e.g. coefficient of variation (CV) of internal standard areas). This transparency and abundance of data is a cornerstone of the high potential reliability of MS-based assays and therefore their interpretation is very important [8, 9].
However, the evaluation of this metadata – related to individual samples and batches – is nowadays done very heterogeneously from laboratory to laboratory [10]; this applies to LDTs as well as to commercially available kit products. The structure of analytical batches is also very variable and there is no generally accepted standard (number and sequence of analysis of calibration samples in relation to patient and quality control samples, blank injections, zero samples, etc).
While the validation of methods – which is performed before a method is introduced into the diagnostic routine – is discussed in detail in the literature (and in practice), the procedures applied to primary data before release for laboratory reporting have not yet been standardized. Validation is generally defined as the process of testing whether predefined performance specifications are met. Therefore, quality control and release of analytical batches and patient results should also be considered a process of validation, and criteria for the acceptance or rejection of results should be predefined.
A three-step approach to validation, covering the entire life cycle of methods in the clinical laboratory, can be conceptualized: dynamic validation should integrate validation of methods, validation of analytical batches and validation of individual test readings. We believe that standardization of this process of batch and sample result validation and release is needed as a guide for developers of methods, medical directors, and technicians.
In a recent article published in Clinical Mass Spectrometry [11], we propose a list of characteristics that should be considered for batch and sample release. In this article we only mention figures for merits and issues to be addressed and do not claim to have specific numerical acceptance criteria. Therefore, this generic list of items is intended as a framework for the development of an individual series and batch validation plan in a laboratory. Furthermore, we consider this list to be a living document, subject to further development and standardization as the field matures.
We believe that it is essential to include basic batch and sample release requirements as essential characteristics in the description of a method [7]. Therefore, we believe that efforts to standardize method description and batch/sample release should be synergistically linked to facilitate the use of MS in routine laboratories.
The approach proposed to clinical MS in these two companion articles [7, 11] can be the basis for discussion and eventually for the development of official standards for these areas by the Clinical and Laboratory Standards Institute (CLSI) and/or International Organization for Standardization (ISO). We believe that these documents can provide a solid basis for internal and external audits of LC-MS/MS-based Quality Control April/May 2020 9 | LDTs, which will become particularly relevant in the context of the IVD Regulation 746 in the European Union [12].
Both approaches – standardized description of MS methods and standardization of batch release – aim at implementing methodological traceability. This corresponds to the analytical standardization and metrological traceability of measurements to higher order reference materials [13, 14]. Future perspectives In the future, a commercialization of MS-based black-box instruments on a larger scale is expected. However, LC-MS/MS will remain a critical technique for LDTs, and the flexibility of MS to develop tests on demand – independent of the IVD industry on fully open LC-MS/MS platforms – will remain a key pillar of laboratory medicine.
Both publications, which this article puts into context [7, 11], have been published in Clinical Mass Spectrometry, the first and only international journal dedicated to the application of MS methods in diagnostic tests including publications on best practice documents. Both articles are freely available.
Clinical Mass Spectrometry is the official journal of MSACL (The Association for Mass Spectrometry: Applications to the Clinical Laboratory; www.msacl.org). MSACL organizes state-of-the-art congresses that focus on translating MS from clinical research to diagnostic tests (i.e. bench to clinic).
In summary, we advocate innovative approaches to methodological standardization of LC-MS/MS methods to master the complexity of this powerful technology and to facilitate and promote its safe application in clinical laboratories worldwide. The authors Michael Vogeser*1 MD, Judy Stone2 PhD, Alan Rockwood3 PhD 1 Hospital of the University of Munich (LMU), Institute of Laboratory Medicine, Munich, Germany University of California, San Francisco Medical Center, Laboratory Medicine, Parnassus Chemistry, San Francisco, CA, USA 3 Rockwood Scientific Consulting, Salt Lake City, UT, USA
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New insights into how the colon functions and actually expels its contents have been revealed for the first time following decades of study by Flinders University researchers.
It promises new diagnostics tools and treatments for gastrointestinal disorders to address problems with bowel movements leading to constipation, diarrhoea and pain, affecting hundreds of millions of people worldwide.
Propulsion of intestinal contents is controlled by millions of neurons within the wall of the gut, known as the enteric nervous system. Capable of operating independently of the brain, a functioning enteric nervous system is essential for life – but exactly how it functions has been a mystery.
By unravelling the neural circuits of the enteric nervous system in guinea pigs and humans Professor Marcello Costa and colleagues are able to understand how the enteric nervous system ensures that food is slowly mixed and propelled along the digestive tube, allowing for absorption of nutrients and excretion of waste.
“For the first time we have combined video recording intestinal movements with a pressure-measuring manometric probe, enabling movements, pressures and electrical activities to be recorded all at the same time within the colon.
“This powerful combination of techniques applied to a guinea pig colon identified several distinct neural mechanisms involved in the propulsion of colonic contents.
“This answers the deceptively simple question of how neural mechanisms within the colon manage the propulsion of bowel contents” Professor Costa says.
“The findings also show how studies in human and animals can be complementary, identifying fundamental mechanisms that are shared across species – in this case guinea pigs and humans. “Currently we treat intestinal disorders by addressing the symptoms, such a stopping-up diarrhoea or softening stools to ease constipation, but as a result of this new understanding of the neural networks of the enteric system, clinicians may be able to develop treatments that treat the cause of the problems” Professor Costa says.
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Japanese scientists report a new method to construct 3D models from 2D images. The approach, which involves non-rigid registration with a blending of rigid transforms, overcomes several of the limitations in current methods. The researchers validate their method by applying it to the Kyoto Collection of Human Embryos and Fetuses, the largest collection of human embryos in the world, with over 45,000 specimens.
MRI and CT scans are standard techniques for acquiring 3D images of the body. These modalities can trace with unprecedented precision the location of an injury or stroke. They can even reveal the microscopic protein deposits seen in brain pathologies like Alzheimer’s disease. However, for the best resolution, scientists still depend on slices of the specimen, which is why cancer and other biopsies are taken. Once the information desired is acquired, scientists use algorithms that can put together the 2D slices to recreate a simulated 3D image. In this way, they can reconstruct an entire organ or even organism.
Stacking slices together to create a 3D image is akin to putting a cake together after it has been cut. Yes, the general shape is there, but the knife will cause certain slices to break so that the reconstructed cake never looks as beautiful as the original. While this might not upset the party of five-year olds who want to indulge, the party of surgeons looking for the precise location of a tumour are harder to appease.
“The sectioning process stretches, bends and tears the tissue. The staining process varies between samples. And the fixation process causes tissue destruction,” explains Nara Institute of Science and Technology (NAIST), Nara, Japan, Associate Professor Takuya Funatomi, who led the project. Fundamentally, there are three challenges that emerge with the 3D reconstruction. First is non-rigid deformation, in which the position and orientation of various points in the original specimen have changed. Second is tissue discontinuity, where gaps may appear in the reconstruction if the registration fails. Finally, there is a scale change, where portions of the reconstruction are disproportional to their real size due to non-rigid registration.
For each of these problems, Associate Professor Takuya Funatomi and his research team proposed a solution that when combined resulted in a reconstruction that minimizes all three factors using less computational cost than standard methods.
“First, we represent non-rigid deformation using a small number of control points by blending rigid transforms,” says Funatomi. The small number of control points can be estimated robustly against the staining variation.
“Then we select the target images according to the non-rigid registration results and apply scale adjustment,” he continues.
The new method mainly focuses on a number of serial section images of human embryos from the Kyoto Collection of Human Embryos and Fetuses and could reconstruct 3D embryos with extraordinary success.
Notably, there are no MRI or CT scans of the samples, meaning no 3D models could be used as a reference for the 3D reconstruction. Further, wide variability in tissue damage and staining complicated the reconstruction.
Nara Institute of Science and Technology
https://tinyurl.com/y4ealgow
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