Diagnosing peripheral lung cancer with the bronchoscope is challenging with near miss of the target lesion as major obstacle. Needle-based confocal laser endomicroscopy (nCLE) enables real-time microscopic tumour visualisation at the needle tip (smart needle).To investigate feasibility and safety of bronchoscopic nCLE imaging of suspected peripheral lung cancer and to assess whether nCLE imaging allows real-time discrimination between malignancy and airway/lung parenchyma.Patients with suspected peripheral lung cancer based on (positron emission tomography-)CT scan underwent radial endobronchial ultrasound (rEBUS) and fluoroscopy-guided flexible bronchoscopy. After rEBUS lesion detection, an 18G needle loaded with the CLE probe was inserted in the selected airway under fluoroscopic guidance. The nCLE videos were obtained at the needle tip, followed by aspirates and biopsies. The nCLE videos were reviewed and compared with the cytopathology of the corresponding puncture and final diagnosis. Five blinded raters validated nCLE videos of lung tumours and airway/lung parenchyma twice.The nCLE imaging was performed in 26 patients. No adverse events occurred. In 24 patients (92%) good to high quality videos were obtained . The nCLE imaging detected malignancy in 22 out of 23 patients with lung cancer. Blinded raters differentiated nCLE videos of malignancy from airway/lung parenchyma (280 ratings) with a 95% accuracy. The inter-observer agreement was substantial  and intra-observer reliability excellent (mean±SD κ=0.81±0.05).Bronchoscopic nCLE imaging of peripheral lung lesions is feasible, safe and allows real-time lung cancer detection. Blinded raters accurately distinguished nCLE videos of lung cancer from airway/lung parenchyma, showing the potential of nCLE imaging as real-time guidance tool. Does bronchoscopic needle-based confocal laser endomicroscopy (nCLE) imaging allow real-time peripheral lung cancer detection?In this prospective proof of principle study, we demonstrated for the first time that bronchoscopic nCLE imaging of peripheral lung lesions suspected of lung cancer is feasible, safe and enables real-time malignancy detection at the needle tip. Blinded raters were able to consistently distinguish nCLE videos of lung cancer from airway/lung parenchyma with high accuracy (95%) after a short training. The results of this study demonstrate the potential of nCLE imaging as a real-time bronchoscopic guidance tool to reduce the current near miss rate of sampling peripheral lung lesions.Our study provides novel information on how to improve peripheral lung cancer diagnostics by reducing the current substantial near-miss rate of the target lesion.Lung cancer is the leading cause of cancer related deaths worldwide with annual 2 million incident cases and 1.7 million deaths.Over 70% of the lesions suspected of lung cancer develop in the periphery of the lung.Confocal laser endomicroscopy (CLE) is a high resolution laser-based imaging technique that is performed with intravenous administration of the contrast agent fluorescein to visualise individual cells and structures. Recently, the CLE probe has become small enough to fit through a biopsy needle (needle-based CLE (nCLE)), enabling real-time in-vivo microscopic analysis at the needle tip (smart needle principle).Currently, feasibility, safety and interpretability of nCLE imaging of peripheral lung lesions is not known. In this proof of principle study, we hypothesise that bronchoscopic nCLE imaging of peripheral lung lesions is feasible, safe and allows real-time malignancy detection. Additionally, we aim to evaluate nCLE imaging as a real-time guidance tool by identifying nCLE criteria for airway/lung parenchyma and assessing whether blinded raters can differentiate malignancy from airway/lung parenchyma on nCLE imaging.This is a prospective proof of principle study, conducted between May 2019 and July 2020 in the Amsterdam University Medical Centers, the Netherlands. In the initial design of the study we aimed to perform nCLE imaging both in conjunction with flexible bronchoscopy for peripheral lung lesion analysis and linear EBUS for mediastinal lymph nodes analysis. In the absence of EBUS needles that could accommodate the CLE probe, we only performed nCLE imaging in conjunction with flexible bronchoscopy for peripheral lung lesion analysis. Patients with a peripheral lung lesion suspected of lung cancer based on (positron emission tomography-)CT scan were considered eligible for study inclusion. Exclusion criteria were: <18 years of age, known allergy to fluorescein, pregnancy or lactating. Since beta-blockers are known to increase the anaphylaxis risk of intravenous fluorescein use,Feasibility of bronchoscopic nCLE-imaging was defined as acquiring adequate real-time CLE video footage of the target lesion in >80% of cases. Safety was defined as the absence of adverse events related to the nCLE measurements and fluorescein use. To evaluate the nCLE malignancy detection rate, in-vivo nCLE videos were compared with the final diagnosis using published pre-defined nCLE malignancy criteria.In order to assess nCLE malignancy and airway/lung parenchyma discrimination accuracy, this study was divided into three steps . Step 1 Patients with a suspected malignant peripheral lung tumour and referred for bronchoscopic analysis, were screened and enrolled in the study provided that the bronchoscopist had sufficient confidence on reaching the lesion to attempt a needle puncture pass. Two bronchoscopists (JTA and PB) performed the procedures with the patient under deep sedation. The nCLE videos were obtained using the Cellvizio system and the AQ-Flex 19 miniprobe with an external diameter of 0.85 mm and a resolution of 3.5 μm . The diameter of the probe is compatible with the diameter of the 18G-Flex needle . Prior to bronchoscopy, an 18G needle was preloaded with the CLE miniprobe using a locking device . The proRight before nCLE imaging, fluorescein (2.5 mL of 10% fluorescein dinatrium solution) was administered intravenously. After lesion puncture and fluorescein administration, the CLE miniprobe was advanced in a forward direction, securely positioning the miniprobe past the needle tip with the locking device. Based on real-time nCLE imaging, the optimal TBNA/biopsy location was identified and localised on the fluoroscopy. After nCLE imaging, the CLE miniprobe was retracted, followed by aspiration of tumour cells at the same spot using suction. Every nCLE video was followed by a separate lesion puncture, enabling direct correlation between the nCLE videos and the cytology results of the corresponding needle aspirate. The cytology specimen was prepared on glass-slides with H&E staining and cell blocks were obtained. Rapid on site evaluation was available and was part of the clinical decision-making how many TBNA-nCLE passes were performed. Following TBNA, forceps biopsies were taken under fluoroscopic control.All nCLE videos were reviewed on the presence of malignant criteria and compared with the final diagnosis and corresponding cytopathology. At least one of the three nCLE lung cancer criteria  had to During a training of approximately 15 min, five raters  were trained in published nCLE lung cancerDirectly after training, the first validation session took place. Raters, blinded for the cytopathology results, independently scored the videos on the individual presence of the three malignancy criteria and four airway/lung parenchyma criteria (yes/no). The rater’s final nCLE diagnosis was established by assigning the dominant pattern (tumour or airway/lung parenchyma) and level of confidence (good/moderate/poor). Videos used for the training session were excluded. The second validation session was performed after a 2-week wash-out period with the nCLE videos in a different order to assess the intra-observer reliability (IOR).The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and the IOR were calculated using the standard definitions and software from the SPSS statistical package V.25.0 (IBM Corporation). The accuracy of the performance of the raters was calculated using the scores of the five raters in both validation sessions .The inter-observer agreement (IOA) was calculated with MATLAB R2020b  using multi-rater Fleiss’ κ.Bronchoscopic nCLE imaging was performed in 26 patients with suspected peripheral lung cancer. In 24 out of 26 patients  adequate nCLE videos were obtained. Two patients  were excluded from analysis because of inadequate nCLE imaging due to staining of the probe or improper contact between the probe and the tissue. No adverse events related to the nCLE imaging or fluorescein administration occurred.Patient characteristics are displayed in nCLE imaging detected a malignant nCLE pattern  in 22 out of 23 patients with lung cancer, including two patients with a false-negative tumour sampling. In one patient, nCLE imaging did not detect malignancy and an alveolar pattern was visualised.The three predefined malignant nCLE criteria were observed in all different subtypes of lung cancer . The mal10.1136/thoraxjnl-2021-216885.supp1Supplementary videoIn 14 out of 67 (21%) TBNA samples of patients with lung cancer, no malignancy was detected in both the nCLE imaging and the cytopathology of the corresponding puncture. Based on these videos, four nCLE criteria for guiding airways and lung parenchyma were proposed: (1) ‘elastin fibre bundle’ and (2) ‘still image without cellular structures’ representing conducting airway, (3) ‘small, homogeneous cells’ representing the bronchial epithelium and (4) ‘autofluorescent alveoli networks’ representing the lung parenchyma .After identification of novel airway/lung parenchyma criteria, nCLE videos with a malignant diagnosis were reviewed and in 12 nCLE videos a combination of malignancy and airway/lung parenchyma criteria was present, resulting in a total of 26 nCLE videos with airway and lung parenchyma visualisation. Of the airway and lung parenchyma criteria, the criterium ‘elastin fibre bundle’ was most present  followed by ‘bronchial epithelium’ , ‘still image’  and ‘alveoli’ .The blinded raters scored the nCLE videos on the presence of malignancy and airway/lung parenchyma criteria with an overall sensitivity and specificity of 0.96 (95% CI 0.89 to 1.0) and 0.94 (95% CI 0.85 to 1.0), respectively . Of the In this prospective trial, we demonstrated that bronchoscopic nCLE imaging of peripheral lung lesions suspected of lung cancer is feasible and safe in selected patients. Importantly, smart needle CLE imaging enables real-time malignancy detection at the needle tip during bronchoscopy. Blinded raters were able to consistently distinguish nCLE videos of lung cancer from airway/lung parenchyma with high accuracy (95%) after a short training. The results of this study demonstrate the potential of nCLE imaging as a real-time bronchoscopic guidance tool to reduce the current near miss rate of sampling peripheral lung lesions.et al used a similar nCLE approach, however without intravenous fluorescein administration.To the best of our knowledge, this is the first systematic study analysing nCLE imaging in peripheral lung tumours. We demonstrated that nCLE imaging in a peripheral lung tumour allows real-time visualisation of individual malignant cells at the needle tip, enabling tissue acquisition at the optimal position. In a single case, Su Initial experience with CLE imaging for the diagnosis of lung cancer was gained by advancing the CLE miniprobe trough the working channel of the bronchoscope and scanning the surface of the airway wall and lung tumours (probe-based CLE (pCLE) imaging). Although some indirect abnormal pCLE patterns in patients with lung cancer have been described , pCLE imaging was unable to directly visualise the individual tumour cells despite fluorescein use.et al performed for the first time needle-based CLE-imaging in central lung tumours and metastatic lymph nodes during endosonographic ultrasound procedures and identified three nCLE lung cancer criteria .Wijmans In the present study, we proposed four nCLE criteria for airway and lung parenchyma. We showed that autofluorescent elastin fibre bundles and alveoli can be visualised with nCLE imaging as was reported in previous studies performing pCLE imaging.In this study in 20 out of 24 suspected patients with lung cancer, the pathological analysis of TBNA and biopsies established a lung cancer diagnosis. What can we learn from the nCLE imaging in the four patients without bronchoscopic tissue proof of malignancy? In two patients nCLE imaging revealed a malignant pattern and both were later diagnosed with adenocarcinoma. Probably the needle had slightly moved during the retraction of CLE miniprobe before the actual aspirate, despite the fluoroscopic guidance indicating the needle was in the right position. This might have resulted in a slightly different sampling area. For future studies, the systematic use of a guide sheath might prevent the needle to change position during the retraction of the probe. In a single patient, nCLE imaging revealed an alveolar pattern, indicating the needle had not reached the lesion. In the fourth patient, nCLE imaging showed an increased density of small cells but no malignant features. The patient underwent lobectomy and was diagnosed with an organising pneumonia. These four cases demonstrate that microscopically visualising the tissue at the tip of the needle with the nCLE technique might provide additional information in case of a non-malignant tissue sampling.This study has several limitations. We performed nCLE imaging in a limited number of selected patients with relatively large lesions and a high prevalence of lung cancer. Although nCLE imaging provides high-quality microscopic images in a real-time fashion, its use is inevitably dependent on the guidance tool, such as rEBUS, electromagnetic navigation and robotic bronchoscopy, to bring the needle to the target area. Whether feasibility and outcomes of nCLE imaging in larger, multicentre cohorts with smaller lesions without a bronchus sign and different (benign) pathologies will be similar, has to be evaluated. Another limitation is the selection of video material used in the validation set. Like most published CLE studies, we had to make a selection to make the validation sessions feasible in time. Consequently, limited videos were included in the validation set to validate each individual airway/lung parenchyma criterium.Strengths of this innovative study are the prospective design, the direct comparison between the nCLE videos and the cytopathology of the corresponding puncture, the strong reference standard and the consistent and highly accurate judgement of the blinded and inexperienced raters. Of every evaluable nCLE-imaged lung lesion at least one video was included in the validation set to make the selection of nCLE videos most representative.In addition to improving the current diagnostic bronchoscopic procedures and pending technological developments, application of nCLE into robotic bronchoscopic navigational technology might result in an optimal real-time peripheral lung cancer detection tool.In conclusion, we have demonstrated that bronchoscopic nCLE imaging in peripheral lung cancer is feasible, safe and allows real-time malignancy detection at the tip of the needle. Blinded raters were able to make a clear distinction between malignant tissue and airway/lung parenchyma, demonstrating the potential of nCLE imaging as a real-time guidance tool to reduce the bronchoscopic near-miss rate of peripheral lung cancer.


Thermo-mechanical loading can occur in numerous engineering geological environments, from both natural and anthropogenic sources. Different minerals and micro-defects in rock cause heterogeneity at a grain scale, affecting the mechanical and thermal properties of the material. Changes in strength and stiffness can occur from exposure to elevated temperatures, with the accumulation of localised stresses resulting in thermally induced micro-cracking within the rock. In this study we investigated thermal micro-cracking at a grain scale through both laboratory experiments and their numerical simulations. We performed laboratory triaxial experiments on specimens of fine-grained sandstone at a confining pressure of 5 MPa and room temperature (20 Coupled thermo-mechanical processes present engineering challenges in a number of geological environments. Climate and weather can induce thermo-mechanical loading in surface rock, with diurnal temperature fluctuations causing strength and stability issues due to progressive damage , in West Yorkshire, UK . Test procedures were designed to replicate thermo-mechanical boundary conditions that may be expected in a deep rock engineering scenario, such as a deep tunnel, mine or generic geological disposal facility with the emplacement of heat generating radioactive waste. The worst case thermal load on the rock mass in any of these cases is likely to be approximately 100The test apparatus consisted of a servo-controlled stiff frame , with a confining pressure vessel (capable of pressure up to 140 MPa). The confining cell was fitted with external heater bands and utilised cascade control from internal and external thermocouples (accurate to ± 0.5k) of 2.3 r) is An initial axial pre-load of 2.3 kN was applied to specimens, to ensure a stable contact and alignment of the platens. In tests undertaken above ambient room temperature a confining pre-load of 0.5 MPa was then applied whilst heating was undertaken. Heating was undertaken at 1.5Micro-structural analysis was also undertaken on laboratory specimens. Polished thin sections were prepared from specimens after thermo-mechanical loading, however the mechanical deformation was seen to overprint any thermal micro-cracking. Therefore two new specimens were prepared. One specimen (# 601) was prepared and oven dried at 40Results of the eight thermo-mechanical triaxial tests undertaken in this study are tabulated in Table entclass1pt{minimaentclass1pt{minima Woodman . HoweverAll specimens subjected to thermal loading under pre-load conditions of 1E)  and Poisson’s ratio  v6.0 Itasca  was usedConventionally in UDEC rock failure is captured either through plastic yielding of the rock matrix, or through sliding on explicitly modelled discontinuities  of Specimens were discretised into Voronoi tessellations with an average grain edge length of 3All GBMs have to make a compromise between grain size and computational efficiency. Hence, it is common for simulations to be run with a grain size considerably greater than the true grain size of the rocks being simulated e.g.  are solved numerically. Heat transfers across Voronoi block contacts without resistance, provided that the blocks are in contact. The thermal calculation procedure is coupled uni-directionally to the mechanical stress calculations through the linear thermal expansion coefficients applied to the Voronoi blocks Itasca . The conWhilst only one set of mechanical properties were applied to all Voronoi blocks and contacts within the mechanical simulations, multiple different thermal properties corresponding to the mineralogy of the Thornhill Rock were applied in the thermo-mechanical Voronoi GBM simulations. This allows for heterogeneous thermal loading to occur within the specimen. Whole rock mineral percentages for the Thornhill Rock were obtained from quantitative X-ray diffraction (XRD) analysis and used to apply mineral percentages to the thermo-mechanical simulations Table . The minNumerical simulations were thermally loaded to the same temperatures as used in laboratory testing , and also the normal load present on those contacts, to deduce shear or tensile failure.Fig. Micro-cracking is seen to initiate between 38ion Fig. . Micro-cmentclasspt{minimaOur laboratory triaxial testing data show that increased thermal loading causes reduced strength in the studied Thornhill Rock Fig. c, in linMicro-structural analysis of specimens subjected to just thermal loading, further validate the development of thermally induced micro-cracking. The specimen not subjected to thermal loading except to drive off moisture (# 601) shows largely planar contacts between quartz grains Fig. a. Minor ing Fig. b & c. Inx-component of the stress tensor (The thermo-mechanical triaxial simulations of the Thornhill Rock show comparable results to laboratory testing, with thermal loading alone causing progressive damage to specimens through the propagation and accumulation of tensile micro-cracks. Micro-cracking initiates at approximately 40Sugawara , this vaWhen assessing the mechanical strength of specimens after thermal loading, simulations of the Thornhill Rock show good agreement with the loss of strength observed in the laboratory, with the existing thermally induced micro-cracks acting as nucleation points for further progressive damage to occur Fig. . Whilst Overall, the thermo-mechanically coupled calibrated GBMs can capture micro-cracking as a mechanism of progressive damage, reproducing the stress-strain behaviour of laboratory specimens. Whilst good agreement is found between laboratory results and numerical simulations, the numerical simulations deform in 2D plane strain and therefore can not be used as predictive tools to quantify crack densities or distributions in the laboratory specimens. It is also accepted that the simulations are simplifications and neglect other potential mechanisms of progressive damage such as pore collapse and grain comminution. The simulations also do not account for specimen porosity, heterogeneous mechanical properties or anisotropic mechanical and thermal grain properties, which as shown by microstructural analysis of laboratory specimens are likely important factors in the progressive damage that occurs in laboratory specimens.In this study we undertook elevated temperature laboratory triaxial testing on specimens of fine-grained sandstone. Increased thermal loading caused reduced strength upon mechanical loading, attributed to progressive damage through thermally induced tensile micro-cracking. Microstructural analysis showed the thermally induced micro-cracking to occur along quartz-quartz grain boundaries, and at boundaries between quartz grains and pore space, with micro-cracking dominantly occurring within authigenic overgrowths of the quartz grains. Thermo-mechanically coupled DEM GBM simulations were undertaken representing the microstructure of the laboratory specimens. Numerical simulations showed the onset and development of tensile thermal intra-granular micro-cracking due to the accumulation of localised thermal stresses from heterogeneous thermal properties of the polymineralic specimens. The thermal micro-cracking caused reduced strength with subsequent mechanical loading, with the strength reduction correlating well with laboratory results, showing the ability of a DEM GBM to help establish the mechanisms of damage and capture representative thermo-mechanical behaviour of brittle granular rock.Interesting questions can be posed for future work regarding up-scaling the results of this study. It is widely recognised that the strength of intact rock decreases with increasing scale due to increased heterogeneity and the presence of critically orientated micro-defects (Hoek and Brown


Panax ginseng C. A. Meyer) is a highly cherished traditional Chinese medicine, with several confirmed medical effects and many more asserted health-boosting functions. Somatic chromosomal instability (CIN) is a hallmark of many types of human cancers and also related to other pathogenic conditions such as miscarriages and intellectual disabilities, hence, the study of this phenomenon is of wide scientific and translational medical significance. CIN also ubiquitously occurs in cultured plant cells, and is implicated as a major cause of the rapid decline/loss of totipotency with culture duration, which represents a major hindrance to the application of transgenic technologies in crop improvement. Here, we report two salient features of long-term cultured callus cells of ginseng, i.e., high chromosomal stability and virtually immortalized totipotency. Specifically, we document that our callus of ginseng, which has been subcultured for 12 consecutive years, remained highly stable at the chromosomal level and showed little decline in totipotency. We show that these remarkable features of cultured ginseng cells are likely relevant to the robust homeostasis of the transcriptional expression of specific genes  implicated in the manifestation of these two complex phenotypes. To our knowledge, these two properties of ginseng have not been observed in any animals  and other plants. We posit that further exploration of the molecular mechanisms underlying these unique properties of ginseng, especially somatic chromosomal stability in protracted culture duration, may provide novel clues to the mechanistic understanding of the occurrence of CIN in human disease.Chinese ginseng ( A given combination of chromosome number and structure constitutes a distinct chromosomal complement, i.e., karyotype, which characterizes every eukaryotic organism, and hence is usually very stable. Yet, it is known that the karyotype undergoes dramatic changes under intrinsic or environmental stresses, some of which can be mitotically and/or meiotically heritable. Under natural settings, changes in karyotype can be of major evolutionary significance, such as speciation ,2. HowevThus, understanding the factors influencing, and mechanisms underpinning, somatic CIN is of great scientific importance as well as bearing significant implications for human health.CIN is known to be instigated and sustained by various external stress conditions, of which in vitro cell culture is a typical one. This is especially well documented in plant tissue culture, a phenomenon recognized more than 40 years ago and termed somaclonal variation . Plant tIn contrast to cultured animal cells, those of plants  have the unique capacity to redifferentiate into an intact plant under suitable conditions, a phenomenon termed totipotency, first conceptualized in 1902 by Haberlandt . A trendPanax ginseng C. A. Meyer) is a slow-growing, long-lived perennial species belonging to the Araliaceae family. Like many angiosperms, ginseng has speciated and evolved via multiple rounds of whole genome duplication (WGD) events followed by diploidization [Panax has yet undergone full-fledged diploidization [n = 4x = 48. Ginseng has been the most renowned herb in traditional Chinese medicine for thousands of years, with its medicinal values being documented by numerous modern pharmacological and clinical investigations [Chinese ginseng  or other plant species hitherto studied. We propose that further elucidation of the molecular mechanisms underlying these properties of ginseng, especially somatic chromosomal stability, is not only scientifically intriguing but also may bear important implications for human health with respect to the occurrence of CIN in many human pathogenic conditions.A callus was initiated from one piece of a bud taken from a single ginseng (a landrace cv. Damaya) plant in September 2004. The standard Murashige–Skoog (MS) solid medium containing 2 mg/L 2,4-dichlorophenoxyacetic acid  was used. A Petri dish was kept at 26 °C in darkness in a plant incubator for 1 month. After ca. 2 months of propagation, a typical embryogenic callus appeared, and which was judiciously selected and subcultured on the same medium under the same conditions at 30-day intervals for 12 years (until 2016). Totipotency and regeneration efficacy of the callus was evaluated on a yearly basis by transferring the subcultured callus to a plant regeneration medium of MS basal + 0.3% casamino acid, 0.1% proline, and 1 mg/L 2,4-D, incubated at 26 °C under a 14/10 light/dark regime .Pg167TR [Conventional and florescence in situ hybridization (FISH)-based cytogenetic analyses were performed essentially as reported in . The 45SPg167TR , were ushttp://www.ncbi.nlm.nih.gov/sra, accessed on 1 March 2021) with the BioProject accession number PRJNA718727.Total RNAs were isolated from liquid nitrogen-frozen ginseng callus of three subculture durations  using Trizol (Invitrogen) according to the manufacturer’s instructions. Integrity of the extracted RNA was determined using an Agilent 2100 Bioanalyzer . Transcriptome libraries were constructed and sequenced using the Hiseq 2000  with standard protocols. Two biological replicates were conducted for each sample and sequenced as parallel experiments. Low-quality reads (Phred < 30) were removed from the raw data using FASTX-Toolkit . In totaTo complete the transcriptomic mapping of mRNA-seq reads, we de novo assembled the genome of oriental ginseng and annotated its compositional protein coding genes (our unpublished data). For mRNA sequencing reads of each subculture period of ginseng callus, initial transcriptomic mapping of mRNA reads to annotated protein coding genes (in gff3 file) was completed using hisat2  , respectively [The candidate gene homologs related to chromosomal instability (CIN) and totipotency in ginseng were determined by a blast search against annotated ginseng genes using known CIN- and totipotency-related genes curated in ectively ,28, as qThe identified CIN- and totipotency-related candidate gene homologs in ginseng were clustered in terms of their log2-transformed expression levels, which was completed by the clustering method in the pheatmap R package (default parameter settings). Only the gene clusters with consistently high  or low  expression levels across all three callus subculture stages were selected for further gene ontology (GO) enrichment analyses. The Pearson’s correlation coefficients between these selected candidate genes, related to CIN and totipotency across the three sampled ginseng callus subcultures, were calculated.Further filtration for narrowing down the candidate genes putatively involved in the sustained chromosomal stability and totipotency was carried out by characterizing the in-contrast expression variation of respective gene homologs in one- and three-year-old subcultured rice calli . AccordiA GO enrichment test for these CIN- and totipotency-related candidate genes was performed by using the clusterProfiler R package .The ginseng callus initiated from a single bud of cv. Damaya was of the typical embryogenic type, originally described in maize tissue culture . The embA. thaliana and some other plants [A. thaliana as being involved in totipotency [Totipotency, as a plant-specific complex trait, has been documented as occurring under tightly orchestrated gene regulation and reprograming in the model plant r plants ,30. To gr plants ,30 in thipotency . A compaOryza sativa L.) for which transcriptome datasets of callus cultures that were, respectively, totipotent (one year old) and non-totipotent (three years old) of the same genotype (Nipponbare) were available [A. thaliana [It is of apparent interest to further delineate the most likely relevant genes whose persistent higher or lower expression is more likely underpinning the sustained high totipotency of the ginseng callus. Unfortunately, we did not have a ginseng callus culture that had lost totipotency for comparison. However, because the molecular mechanisms orchestrating totipotency are highly conserved in plants , we condvailable . Of the vailable  indicatevailable  showed tthaliana .n = 48) (n = 47 (Pg167TR repeat (n = 48), with structural chromosomal variations also being rare  ; the rem  phenotype for direct comparison, we also analyzed the expression profiles of this set of CIN-related genes in rice using the RNA-seq dataset from the two batches of rice callus cultures, i.e., one and three years old . Our assOf the 154 genes that were consistently (across the three subculture durations) more highly expressed in the ginseng callus , we idenNotably, different from the situation of totipotency in which probably only the set of consistently more highly expressing genes are important, in CIN, both higher- and lower-expressed genes are likely important. This makes sense given that totipotency is primarily an “activation” event  while CIChinese ginseng has been regarded as the symbolic “king” of traditional Chinese medicine and cherished for thousands of years ,18,36. TThe first property is chromosomal stability in long-term subcultured ginseng callus. It is well established that plant tissue culture wherein differentiated tissues or organs are induced into an undifferentiated and disorganized cell mass disrupts normal cellular control  and causWe are not the first to study the genome stability of ginseng in culture. A previous study compared nucleotide substitution rates of three selected gene families, actins, dammarenediol synthase (DDS) genes, and somatic embryogenesis receptor kinase (SERK) genes, in a 20-year-old ginseng cell culture and collected wild ginseng plants . This stThe second unique property of ginseng we unrevealed in this study is its nearly immortalized totipotency as a cultured callus. We have shown that both the plant regeneration frequency and efficacy of the ginseng cultures remained unabated for 12 years. This is distinct compared to all other plants studied, in which cultures usually show a rapid decline and loss of regeneration potential within one or two years. We further demonstrate that compared with rice cultures in which genes related to the maintenance of totipotency showed rapid expression level fluctuations within two years of subculture, these genes exhibited highly stable expression across three disparate subculture stages. Further studies are needed to confirm the causal relationships between expression homeostasis of the candidate genes we identified and the persistent totipotency of ginseng cultures. In addition, based on the findings of the present study, we are tempted to suggest that one unheeded potential benefit of consuming ginseng products is to strengthen genetic stability in our genomes.We report two striking biological properties characterizing long-term callus cultures of Chinese ginseng: high chromosomal stability and virtually immortalized totipotency. Our transcriptome-based analyses show that these properties are probably related to the high expression homeostasis of genes, related to these two complex phenotypes in ginseng. In addition, our results also suggest that these two phenomena are likely mechanistically linked, that is, the karyotypic stability may have enabled the sustainable totipotency, for example, by means of enhanced repair efficacy and fidelity of DNA double-strand break repair in Chinese ginseng.We propose that both features of ginseng, chromosomal stability and totipotency, merit further investigations whereby novel insights might emerge that are pertinent to the pressing issue of understanding the occurrence of CIN in human cancers and the improvement of tissue cultured-based transgenic technology in crop improvement. Although we currently do not know if all ginseng plants will show these features or if what we found is genotype dependent, the phenomena per se are truly remarkable.


The objectives of this paper were to identify and compare clinical prediction models used to assess the risk of venous thromboembolism (VTE) in ambulatory patients with cancer, as well as review the rationale and implementation of a pharmacist-led VTE screening program using the Khorana Risk Score model in an ambulatory oncology centre in Sault Ste. Marie, Ontario, Canada.PubMed was used to identify clinical practice guidelines and review articles discussing risk prediction models used to assess VTE risk in ambulatory patients with cancer.Three commonly used VTE risk prediction models in ambulatory patients with cancer: the Khorana Risk Score, Vienna Cancer and Thrombosis Study (CATS) and Protecht Score, were identified via literature review. After considering guideline recommendations, site-specific factors  and evidence from the CASSINI and AVERT trials, a novel pharmacist-led VTE risk assessment program using the Khorana Risk Score was developed during a fourth-year PharmD clinical rotation at the Algoma District Cancer Program (ADCP) [ambulatory cancer care centre]. ADCP patients with a Khorana Risk Score of The Khorana Risk Score was the chosen model used for the pharmacist-led VTE risk assessment program due to its user-friendly scoring algorithm, evidence from validation studies and clinical trials, as well as ease of integration into pharmacy workflow. More research is needed to determine if pharmacist-led VTE risk assessment programs will impact patient outcomes, such as morbidity and mortality, secondary to cancer-associated thrombosis. Cancer induces a hypercoagulable state in affected patients via several pathophysiologic mechanisms beyond the scope of this practice tool.3 Additionally, patient risk factors including comorbid medical conditions  and low mobility may further exacerbate VTE risk. Cancer-associated thrombosis also confer increased morbidity  and mortality risks. In a prospective observational study of 4,466 patients with cancer receiving chemotherapy, thrombosis was the leading contributor of non-cancer causes of death in addition to infection (9.2% for each). Additionally, a staggering 47-fold increase in VTE death rate was noted in this study compared to the general population .7 Furthermore, cancer-associated thrombosis place added stress on government healthcare spending secondary to thrombotic complications that require urgent intervention and patient hospitalization (i.e. pulmonary embolism).Patients with cancer are estimated to have a four to seven-fold increased risk of experiencing venous thromboembolism (VTE) compared to patients without cancer.911 Each risk prediction model uses a combination of laboratory parameters , patient-specific factors (i.e. body mass index [BMI]), as well as chemotherapy-specific factors to calculate the percentage risk of developing VTE over a three to six-month period. The aims of this work are to summarize the intricacies of each risk prediction model mentioned above, as well as review the rationale and implementation of a pharmacist-led VTE screening program using the Khorana Risk Score model in an ambulatory oncology centre in Sault Ste. Marie, Ontario, Canada.Several risk prediction models have been developed to help clinicians assess VTE risk in ambulatory patients with cancer. Among the most widely recognized and used risk prediction models include the Khorana Score, Vienna Cancer and Thrombosis Study (CATS) Score and the Protecht Score.1217 review articles and author knowledge. A PubMed search  was conducted to identify systematic reviews discussing venous thromboembolism risk models used in ambulatory patients with cancer (search strategy: (“venous thromboembolism”[MeSH Terms]) AND (“risk”[MeSH Terms] OR “risk”[All Fields]) AND (“ambulatory care facilities”[MeSH Terms] OR “ambulatory”[All Fields]) AND (“neoplasms”[MeSH Terms] OR “cancer”[All Fields]) AND Review[ptyp]). Qualitative and quantitative data were extracted from review articles and primary literature identified via hand-searching reference lists. Qualitative data regarding the specific variables of each risk prediction model were extracted, including very high-risk tumor types, high-risk tumor types, presence of platinum chemotherapy and presence of gemcitabine chemotherapy. Patient inclusion and exclusion criteria were also extracted. Furthermore, quantitative data including reported three and six-month VTE rates, risk prediction model scoring algorithms and threshold laboratory values associated with each risk model scoring system were extracted.A pharmacist-led VTE risk assessment program was developed during a fourth-year PharmD clinical rotation at ADCP in Sault Ste. Marie, Ontario, Canada. In the summer of 2020, ADCP pharmacists and a fourth-year PharmD clinical rotation student approached the program oncologists and hematologist with the idea of a pharmacist-led referral program for VTE risk assessment. The pharmacists, oncologists and hematologist agreed that a literature review be conducted to identify prediction models used to assess VTE risk in ambulatory patients with cancer. Risk prediction models were identified based on models reported in clinical practice guidelines,Upon completion of the literature review and identification of relevant VTE risk prediction models, the pharmacists and clinical rotation student reviewed site-specific factors that may impact the implementation of identified models into ADCP pharmacy workflow. These site-specific factors included access to laboratory values required to calculate a score, as well as estimated time to complete a VTE risk assessment with each identified model. Once site-specific factors had been considered, ADCP pharmacists and clinical rotation student presented the oncologists and hematologist with a preferred VTE risk assessment model to be trialed in a pharmacist-led assessment program. The pharmacist-led assessment program implemented at ADCP consists of four steps: (1) calculation of the patient’s VTE risk score, (2) documentation of the score, as well as corresponding percentage risk and risk category in the patient’s chart, (3) referral of the patient to the clinical oncologist or hematologist for a full VTE workup, and (4) assessment of potential drug interactions and cost issues to optimize pharmacotherapy if an anticoagulant was initiated for the patient. VTE rates at ADCP were not available prior to program implementation.911 Six clinical practice guidelines identified via the search strategy were used to select the three risk prediction models: (1) National Comprehensive Cancer Network (NCCN), (2) American Society of Clinical Oncology (ASCO), (3) British Committee for Standards in Haematology, (4) International Initiative on Thrombosis and Cancer (ITAC), (5) European Society for Medical Oncology (ESMO), and (6) The Scientific Standards Committee (SSC) of the International Society of Thrombosis and Haemostasis (ISTH).1217 Extracted data including the specific variables for each risk prediction model, risk prediction model scoring algorithms and threshold laboratory values associated with each risk model scoring system can be found in Review of the literature consistently identified three commonly used risk prediction models that assess venous thromboembolism risk in ambulatory patients with cancer: (1) Khorana Risk Score, (2) Vienna CATS Score, and (3) Protecht Score. It may be used to calculate VTE risk for patients with several solid tumor types and lymphomas; however, patients with myelomas and brain tumors were excluded from validation studies. Early recommendations suggested considering thromboprophylaxis for patients with a high-risk Khorana Score of  Randomized controlled trials have used the Khorana Risk Score as the VTE risk prediction model of choice in ambulatory patients with cancer. For example, both the CASSINI and AVERT randomized controlled trials used a Khorana Risk Score threshold of 21 Furthermore, the AVERT trial included patients with myelomas (2%) and brain tumors (5%), both of which were excluded from Khorana Score validation studies. The AVERT trial also reported a significant increase in major bleeding, while results from the CASSINI trial did not support this association.21 Although convenient, published literature has identified poor discriminatory ability between low and high-VTE risk patients for specific cancers using the Khorana Risk Score, such as pancreatic and lung.23The Khorana Risk Score uses five readily available parameters to assign patients to low, intermediate, or high risk of VTE  and 2.9 The positive predictive value (probability of VTE in those deemed high-risk) of the Khorana Risk Score was found to be 7.1% and negative predative value (probability of not having VTE in those deemed low-risk) was found to be 98.5%. Sensitivity and specificity in the derivation cohort were found to be 40% and 88%, respectively. The Khorana Risk Score has also demonstrated benefit in identifying early VTE and predicting inpatient VTE-both of which are unknown with the Vienna CATS and Protecht Score models.The Khorana Risk Score is the only independently validated VTE prediction tool that provides clinicians with a quick means of identifying ambulatory oncology patients that may benefit from the addition of thromboprophylaxis. Patients with brain tumors were included in Vienna CATS model development and placed in the ‘very high risk’ cancer site category (i.e. 2 points). Furthermore, a patient’s VTE risk score would increase by 1 point if the D-Dimer was elevated  The positive predictive value of the Vienna CATS Score was 22.1%, which was an improvement versus the Khorana Risk Score. The negative predictive value of the Vienna CATS score was reported as 94.9%, while the sensitivity was reported as 31.9% and specificity as 91.9%.The Vienna CATS Score introduced two biomarkers predictive of VTE risk in patients with cancer, D-Dimer and soluble P-selectin, in addition to the variables derived from the Khorana Risk Score  and 2.1 From a practical standpoint, having to manually request these biomarkers may introduce disruptions to the pharmacist workflow via additional wait times for biomarker results to be reported. However, if D-Dimer and soluble P-selectin values are readily available as part of routine testing at a specific oncology centre, the Vienna CATS Score may be the preferred risk model as it has demonstrated superior positive predictive value of VTE compared to the Khorana Risk Score. The clinician should consider that evidence for thromboprophylaxis using the Vienna CATS Score employs injectable LMWH as the therapeutic intervention, while evidence for more convenient agents  exists from the CASSINI and AVERT trials using the Khorana Score.21A significant drawback of this model is the potential difficulty for laboratories to report D-dimer and soluble P-selectin as part of routine testing. This VTE risk prediction model employs the same variables included in the Khorana Risk Score, with the exception of BMI. Additionally, the Protecht Score adds an additional 1 point to the patient’s VTE risk score if the patient is on a platinum or gemcitabine-based chemotherapy , as well as time taken to complete the VTE risk assessment calculation. Via interview, ADCP pharmacists stated that the Khorana Risk Score calculation added approximately five minutes of working time for each patient assessed. The pharmacists did not feel that the addition of this task negatively impacted daily workflow. Evidence for the literature review also clearly indicated the Khorana Risk Score was the most validated tool among the three options, so this further influenced the decision to use the Khorana Risk Score model in practice versus the Protecht Score. ASCO guidelines recommend that high-risk outpatients with cancer  from the study population. Furthermore, the SCC and ISTH guidelines suggest the use of DOACs (i.e. apixaban or rivaroxaban) as primary thromboprophylaxis for ambulatory patients with cancer if they have a Khorana Score The ITAC guidelines state that the Khorana Risk Score is the most widely used model to identify ambulatory patients with cancer at high risk of thrombosis. These guidelines recommend prophylaxis with apixaban or rivaroxaban if the patient’s Khorana Risk Score is As some laboratories may have difficulty obtaining specific biomarkers , the Khorana Risk Score is a convenient tool that uses readily accessible laboratory parameters available across most oncology practice sites. Therefore, pharmacists working in ambulatory clinical oncology centres may be appropriately positioned to incorporate the Khorana Risk Score into daily workflow. The pharmacist-led VTE assessment program implemented at ADCP is summarized in 21 However, one must consider the limitations of these trials when deciding to recommend initiation of rivaroxaban or apixaban for VTE prophylaxis in ambulatory patients with cancer. In the CASSINI trial, nearly 47% of enrolled patients discontinued rivaroxaban early in the treatment group. Additionally, a significant VTE benefit was only observed in a secondary prespecified intervention-period analysis , which is subject to bias. The primary outcome of VTE benefit did not reach statistical significance . In the AVERT trial, only 5.9% of patients had a creatinine clearance of 50 ml/min or less. Therefore, applicability of AVERT trial results to patients with a creatinine clearance of 50 ml/min or less is questioned. After consideration of trial limitations, pharmacists should also notify the oncologist or hematologist if the patient is already receiving anticoagulation or antiplatelet therapy for another indication. Navigating drug coverage options for patients that start thromboprophylaxis is another area in which both hospital and community pharmacists may significantly contribute to optimize patient care. Although pharmacists may not always make the final decision on initiating thromboprophylaxis for a referred patient, identifying those that may benefit from the addition of an anticoagulant is a crucial step in the process of minimizing preventable cancer-associated thrombosis.Using evidence from the CASSINI and AVERT randomized controlled trials, pharmacists may refer patients with a Khorana Risk Score A pharmacist-led model of VTE risk assessment using the Khorana Risk Score was implemented in the summer of 2020 during a fourth-year PharmD clinical rotation at the Algoma District Cancer Centre (ADCP) [Sault Area Hospital] in Sault Ste. Marie, Ontario, Canada. All new ambulatory patients with cancer, as well as patients with cancer recurrence, receiving treatment at ADCP were screened for VTE risk by the pharmacist while therapeutically assessing the first cycle of chemotherapy. If the patient’s Khorana Risk Score was  Risk prediction models are not a replacement for clinician judgement, and patient factors outside of the risk model variables  must be considered when deciding to initiate, or not to initiate, thromboprophylaxis. Furthermore, limitations to implementing a pharmacist-led VTE risk assessment program at an ambulatory cancer centre exist. Although a Khorana Risk Score only takes minutes to calculate, some pharmacists may be reluctant to add this task to an already demanding workday. Additionally, patient drug coverage will vary substantially by geographic region, and the DOACs  supported by evidence from the CASSINI and AVERT trials are significantly more expensive than alternative medications with demonstrated therapeutic benefit in preventing cancer-associated thrombosis (i.e. warfarin). However, it can be argued that patients may make up for this medication cost difference in both time and money saved not having to monitor the INR. Furthermore, warfarin is not considered a first-line agent in VTE prophylaxis for ambulatory patients with cancer. Pharmacists, other clinicians and patients must continue to advocate for government or other third-party drug coverage that will allow greater accessibility to convenient medications, such as DOACs, with increasing evidence for this indication.Despite the conveniences of using risk prediction models to assess VTE risk in ambulatory patients with cancer, limitations to these tools are evident. A patient’s risk of VTE is dynamic and changes over time; however, risk prediction models use parameters from a single laboratory panel to calculate a three to six-month risk of VTE. Furthermore, a notable limitation of this study is the lack of pre and post-intervention VTE rate, as well as patient-specific outcome data for the ADCP pharmacist-led VTE risk assessment program that is available at this current time. ADCP pharmacists have slowly incorporated this model into their workflow since the summer of 2020, and an original research investigation assessing patient outcomes using the pharmacist-led program is currently in development.Limitations of this study are also evident. The three models identified in this study  are not exhaustive of all prediction models used to assess VTE risk in ambulatory patients with cancer. However, these three models were the most consistently discussed in clinical practice guidelines and review articles identified in our search strategy. Although other models exist, the Khorana Risk Score still has the most robust external validation evidence to date.Three risk prediction models used to assess VTE risk in ambulatory patients with cancer, the Khorana Risk Score, Vienna CATS Score, and the Protecht score were identified in this study. Given the Khorana Risk Score’s user-friendly algorithm and potential for integration into pharmacy workflow, a pharmacist-led VTE risk assessment program using the Khorana Risk Score was introduced at an ambulatory oncology centre in Sault Ste. Marie, Ontario to aid in identifying ambulatory oncology patients at risk of developing VTE that may benefit from thromboprophylaxis. Incorporating VTE risk assessment into pharmacist practice may foster interprofessional communication within the oncology care team, especially when deciding to initiate thromboprophylaxis in eligible patients. Further work is needed to determine if pharmacist-led VTE risk assessment will impact patient outcomes, such as morbidity and mortality, secondary to cancer-associated thrombosis.


Dystonia is one of the most common forms of movement disorder, involving repetitive or sustained contractions of antagonistic muscle groups causing pain and abnormal posturing. The motor phenotypes observed in dystonia are varied with single muscle group involvement , multiple adjacent  and more generalised forms involving multiple body regions. Some dystonia’s are task specific, being induced by undertaking a particular task or movement, whereas with others the involuntary movements and posturing are independent of activity. Dystonia is increasingly considered to be a network-based disorder involving multiple motor pathways in the brain, with the cerebellum, basal ganglia and sensorimotor cortex particularly implicated. Injected botulinum toxin represents the mainstay of treatment for many patients with focal and segmental forms and Deep Brain Stimulation (DBS) is used in the treatment of more severe forms.TorsinA) genetic form of generalised dystonia.While in excess of 30 disease-causing genes for dystonia have been identified, understanding of the pathophysiological mechanisms underpinning dystonia remains poor. Neuroimaging, whilst providing only indirect measures, is our most accessible tool in assessing in vivo brain structure and functioning in humans. The papers discussed below make use of a range of neuroimaging techniques to further investigate dystonia pathophysiology, with these including (i) structural imaging techniques to evaluate grey matter volume and white matter diffusion properties, (ii) resting-state functional MRI to compare regional and global motor network changes in isolated focal dystonia, (iii) positron emission tomography (PET) to glean insight into acetylcholine release in the DYT1  compared to those with non-task-specific dystonia (NTSD). Three techniques were used to assess for subtle grey matter volumetric differences: (1) whole-brain cortical thickness analysis, an automated process which divides the cortex into subsections and measures for average cortical thickness; (2) Voxel-based morphometry (VBM), taking a voxel-by-voxel approach to compare localised brain volume changes; (3) Regional grey matter volume analysis. Changes to white matter structures were also assessed using Tract based Spatial Statistics (TBSS) of diffusion MR images, with this approach allowing for measurement of the diffusion properties within individual voxels.A total of 97 patients with isolated focal dystonia and 83 age- and gender-matched controls were recruited. Amongst the dystonia cohort, 36 had TSD  and 61 NTSD . Each patient was scanned at least 3 months after their last botulinum toxin injection, and in addition to MR scanning an extensive clinical assessment included dystonia severity rating, determination of dystonia triggers, pain severity and the presence of a sensory trick. Although matched for age and gender the clinical groups did differ with lower clinical severity and higher reported disability in those with TSD, while patients diagnosed with NTSD were more likely to have received botulinum toxin treatment and report pain.p < 0.001) thicker primary somatosensory cortex  amongst those with TSD compared with both those with NTSD and unaffected controls, as well as greater thickening in multiple regions involved in somatosensory processing compared to NTSD, including the right paracentral, precentral gyri and inferior parietal gyri, and left superior parietal, rostral middle frontal, supramarginal, fusiform and inferior temporal gyri. In contrast, the NTSD cohort demonstrated cortical thinning compared to controls in a number of regions including the primary somatosensory cortex and premotor cortex. VBM measurements demonstrated similar changes, with increased grey matter volumes in TSD and decreased in NTSD in several regions involved in sensorimotor processing, with substantial overlap with those regions identified in the cortical thickness analysis. However, application of more stringent post-hoc corrections (Bonferroni Holm step down correction for multiple comparisons) these differences were lost with no between group statistically significant differences observed. The deep grey matter regional analysis demonstrated an increased volume in the left amygdala in TSD compared to both NTSD and healthy controls, and an increased volume of the right globus pallidus compared to NTSD. NTSD showed reduced thalamic volumes compared to healthy controls. White matter analysis showed widespread reductions in FA  and MD  in both groups compared to controls but more so in the TSD group.Overall analysis found a significantly  and lower (NTSD) grey matter volumes in brain regions associated with sensorimotor processing compared to controls. The strengths of this study include the large recruited and matched cohort, as well as the use of multiple complimentary techniques in assessing brain structure. However, it is important to note that these techniques predominantly involved use of whole grey/white matter approaches rather than being driven by focusing on specific regions of interest. As a result, the data generated a large number of multiple comparisons, with statistically significant results only observed in the absence of correction for multiple comparisons.Tomić et al. (2020). Movement Disorders 36(1):196–205n = 18), focal upper limb dystonia (n = 10), cervical dystonia (n = 13), craniofacial dystonia (n = 17)] and 47 age and gender-matched controls. Their intention was to undertake the imaging at least 3 months post the most recent botulinum toxin treatment, although this wasn’t achieved in all participants.Functional MRI makes use of variation in the signal produced by oxygenated and deoxygenated blood due to differences in their magnetic properties  and is considered to be a proxy for neuronal activity. Resting state functional connectivity MRI measures changes in the BOLD signal in the brain at rest, and how that signal change correlates between brain regions. Norris et al. performed resting-state functional connectivity analyses in isolated focal dystonia, looking for shared changes between dystonia subtypes. They undertook two sets of analyses for each participant; (i) assessment of specific regional activity focussing on pre-determined regions in the basal ganglia and sensorimotor cortex, (ii) a more global analysis involving 300 regions across the whole brain. They recruited 58 patients diagnosed with isolated idiopathic focal dystonia [adductor laryngeal dystonia . Neurology 95:e2246–2258TorsinA gene (DYT1 dystonia) were recruited, and compared to cohort of age- and gender-matched controls (n = 20). Three of those in the DYT1 cohort were prescribed routine anticholinergic medication at the time of the study, however, this was withheld for 72 h prior to scanning.Neurotransmitter activity is key in brain functioning, with disruption to acetylcholine neurotransmission having been implicated in animal models of dystonia. Mazere et al. applied PET techniques using a radioligand that binds to the vesicular acetylcholine transporter, involved in presynaptic acetylcholine storage and is a rate-limiting factor in the release of acetylcholine. The study focused on 17 brain regions involved in motor control or containing cholinergic cell bodies, with a particular focus on regions in the cerebellum and basal ganglia. In addition to the PET images, resting-state functional MR images were also captured, as well as volumetric structural analysis to enable adjustment for confounding volume loss that may contribute to changes in measured radioligand binding. Twenty individuals with genetic confirmation of a mutation in the In comparison with controls, those with DYT1 mutations demonstrated significantly lower levels of vesicular acetylcholine transporter binding in the posterior putamen. This difference also appeared to be age dependent, only present in the younger group when the cohort was split into two groups above and below the overall median age of the cohort. Reduced binding was also observed in the cerebellum of the patient cohort, most notably the vermis, with no age effect seen in this region. The functional MR analysis once again demonstrated higher between region connectivity in those diagnosed with dystonia, as well as being positively correlated with ligand binding in the same regions, a finding not observed amongst the control cohort. The volumetric analysis only showed between-group differences in the right caudate and right dorsoventrolateral prefrontal cortex, with higher volumes in patients. There was an age effect on volume, but no age by group interaction.Comment This study implicates disruption to cholinergic neurotransmission in the basal ganglia and cerebellum in DYT1 dystonia. The study benefits from its focussed, region of interest-driven analysis, rather than a more global whole-brain approach. The additional volumetric analysis also considerably strengthened this study, ensuring adjustment for potentially confounds of systematic volumetric differences. Finally, the inclusion of only those with confirmed DYT1 mutations also allowed for greater homogeneity in assumed underlying pathogenesis, in spite of there being considerable clinical phenotypic heterogeneity.Mazere et al. (2021). Brain 144(3):909–923


Earlier detection of cancer recurrence using circulating tumor DNA (ctDNA) to detect molecular residual disease (MRD) has the potential to dramatically affect cancer management. We review evidence supporting the use of ctDNA as a biomarker for detection of MRD and highlight the potential impact that ctDNA testing could have on the conduct of clinical trials.www.clinialtrials.gov was searched to identify trials that incorporate ctDNA testing.We searched the literature using MEDLINE (via PubMed) for articles from January 1, 2000, focusing on studies that assessed ctDNA as a predictor of cancer recurrence. Broadly focused searches on ctDNA and cancer were also performed to provide additional background information. Numerous studies across different cancer types indicate that ctDNA-based MRD detection predicts recurrence with high sensitivity and specificity, and with lead times that precede standard imaging by up to 12 months. Recently, ctDNA testing has started being used to enroll MRD-positive patients at high risk of recurrence into trials, promising gains in statistical power that allow clinical utility to be demonstrated with smaller cohorts. Trials where ctDNA testing based-MRD detection is used to stratify patients into low or high-risk categories for treatment assignment are also ongoing. In addition, there is increasing evidence supporting the use of ctDNA dynamics or clearance as a surrogate end point, which could significantly reduce trial duration.ctDNA-based trial enrichment across many cancers seems likely to become increasingly common for cost- and time-reduction benefits. Trial efficiency could also benefit from using ctDNA as a surrogate end point, leading to accelerated approval of new therapeutics. A clear demonstration of efficacy from trials that use ctDNA-based MRD detection to assign treatment could transform clinical practice. Over the past decade, the advent of next-generation sequencing (NGS) and other advances in methods for ctDNA detection contributed to a surge in research evaluating ctDNA as a cancer biomarker. The association of ctDNA with clinical variables has now been investigated in many cancers and it is well established that ctDNA levels are associated with stage, response to therapy, prognosis, and tumor burden.5The presence of extracellular DNA in blood, referred to as cell-free DNA (cfDNA), was first reported in 1948.Key ObjectiveCirculating tumor DNA (ctDNA) detection indicates the presence of molecular residual disease (MRD), identifying recurrence earlier than standard approaches. Key roles for ctDNA-based MRD detection in the design of clinical trials in adjuvant and neoadjuvant settings are examined.Knowledge GeneratedGlobally, hundreds of clinical trials seek to show the benefit of therapeutic interventions. ctDNA-based MRD detection is increasingly being used to select patients at high risk for recurrence into clinical trials, as it can greatly reduce sample size and trial costs. Moreover, using ctDNA as a surrogate end point can result in substantial reductions in trial duration, expediting the introduction of new therapeutics into the clinic. The results from trials that investigate early therapeutic interventions after MRD detection could substantially affect clinical practice.RelevancectDNA-based MRD detection could have a major impact on the conduct of clinical trials and ultimately on the management of disease in patients with cancer.12A significant new development in clinical cancer research is using ctDNA for detection of molecular residual disease (MRD) and molecular relapse. We use MRD  here to mean any molecular evidence of disease, typically when detected shortly after surgery or definitive treatment, whereas molecular relapse, treated here as a subset of MRD, is used to describe molecular evidence of disease found later, during treatment or surveillance. Numerous studies across different cancer types indicate that ctDNA-based MRD detection predicts recurrence with high sensitivity and specificity, preceding standard imaging by months.Reliable detection of MRD has substantial implications for clinical trial design. Identifying patients at high risk of recurrence through ctDNA testing can lead to substantial reductions in trial sample size, as enriching trials with patients likely to recur increases statistical power. Another potential role for ctDNA is as a surrogate end point for treatment response in settings where conventional response biomarkers are unavailable . This could provide an early indication of treatment efficacy relative to conventional measures such as progression-free survival and overall survival (OS). These gains in trial efficiency can reduce study costs leading to expedited approval of new therapies. ctDNA-based testing also provides the opportunity to conduct trials where MRD status guides treatment. These trials could determine whether MRD-positive patients benefit from early therapeutic interventions.In this review, we elaborate on evidence supporting different approaches to using ctDNA in clinical trial design and discuss the utility of ctDNA-based MRD detection for increasing trial efficiency and guiding treatment across neoadjuvant and adjuvant settings.13 particularly in early-stage cancers, and thus, methods with high analytic sensitivity are required for successful ctDNA detection. Detection methods include digital polymerase chain reaction,16 multiplex polymerase chain reaction–based NGS,8 and hybrid capture–based NGS.17 High costs of whole-genome and exome sequencing have discouraged efforts toward plasma-based detection of many types of somatic variants; however, whole-genome approaches for identifying genomic rearrangements in plasma have been used successfully in clinical research.18 Table 28ctDNA concentrations are often low (< 0.1% of cfDNA),29 A study that tracked multiple ctDNA variants in patients with stage I-III non–small-cell lung cancer following definitive treatment reported that a 94% detection rate with multiple markers dropped to 58% using the same platform with only a single marker.29 Recently, there has been a shift toward using personalized, tumor-informed approaches, where a patient's tumor biopsy results determine variants to be tracked in the plasma. These are suggested to have greater sensitivity than tumor-naive multigene panels, as the latter are reported to only detect an average of 2-5 variants per patient, despite using large panels .29 Although multigene panels provide improved sensitivity relative to a single marker, a tumor-naive panel may not cover variants found in some patients. By contrast, prior knowledge from assessment of variants in tumor tissue allows tracking of a greater number of variants (often with relatively high frequency that permits more reliable detection), enhancing sensitivity.22 Knowledge of the tumor variant profile also ensures assays can be focused on variants present in plasma, whereas tumor-naive approaches assay many regions unlikely to contain a relevant variant, increasing false-positive results. False positives are a major clinical concern and a barrier to adoption if an intervention  is to follow a positive test.Considerable attention has been given to broadly applicable assay strategies to enhance ctDNA detection accuracy. This includes assaying many variants instead of a single variant to increase the probability of finding detectable variants in plasma samples.22 It is as yet unclear how this difference in methodology, and other differences such as error models and assay design, influences specificity (and other accuracy measures).Although tumor-informed approaches can reduce the proportion of false-positive tests, the probability of a false-positive result will still increase with the number of variants assayed. To combat this loss in specificity, some tumor-informed methods require at least two variants to be present in both tumor and plasma, substantially reducing false-positive results. Currently used tumor-informed approaches track different numbers of variants, ranging from 16 to as many as 115.30 A summary of differences between tumor-informed and tumor-naive approaches is provided in Appendix Table ADespite the benefits of using multiple variants and/or tumor-informed methods, costs of assaying multiple markers and availability of tumor tissue are potential barriers to using these approaches in some studies. Furthermore, the need to sequence tumor tissue for designing ctDNA assays can result in longer turnaround times for tumor-informed approaches . Using presurgical material or designing assays promptly upon receipt of tumor material can ameliorate this concern.31 The results from several studies indicate that DNA methylation holds promise for MRD detection.35 Current approaches generally examine methylation at a few cancer-related markers .34 Low costs and efficiency are cited as advantages of these assays.36 However, current results indicate that sensitivity for detecting MRD is lower than observed for mutation-based methods , although results for specificity are similar (80%-92%37v 90%-100%41). These comparisons are hindered by small sample sizes and lack of uniformity in timing and frequency of ctDNA testing across studies, particularly in methylation studies. More research is needed to demonstrate the utility of methylation-based MRD testing.Much of the focus of ctDNA testing involves detection of somatic mutations. However, aberrant changes in DNA methylation are widespread in tumor tissue and also reflected in plasma.42 More research is needed to further evaluate the combination of epigenomic and genomic markers in MRD testing. Of interest is whether the addition of genomic variants will improve the accuracy of tumor-naive approaches to levels comparable to tumor-informed approaches, and the impact on accuracy of adding methylation markers to current tumor-informed methods.Methylation approaches have also been used in tandem with testing of somatic mutations. A recent study, using a tumor-naive approach, reported that adding a predetermined methylation cancer signature to somatic genomic mutations increased sensitivity of detecting recurrence.44 Recent studies have reported 14% of patients with early-stage lung cancer and 25% of patients with late-stage solid tumors harbor CHIP mutations.46 Because of the difference in methodology used to detect and define CHIP variants between these studies, a comparison of results does not permit inferences about CHIP mutation frequency by cancer type and stage. However, the high frequency of CHIP variants observed in both studies underlines how misclassifying CHIP variants as ctDNA variants may reduce specificity for MRD detection. Approaches to address this misclassification include sequencing paired peripheral blood mononuclear cells for in silico filtering of variants common to peripheral blood mononuclear cells and ctDNA, and using tumor-informed methods to identify clonal tumor variants.47A significant challenge to maintaining specificity of ctDNA testing is confounding by clonal hematopoiesis of indeterminate potential (CHIP). CHIP mutations originate from hematopoietic progenitor cells.Many other factors can influence the accuracy of ctDNA detection, including tumor shedding, tumor location, weight, and recent surgery. We summarize these in Table There is substantial evidence that ctDNA-based MRD detection can stratify patients into high-risk and low-risk groups, which allows for more efficient trials through targeting high-risk patients for enrollment. Numerous retrospective studies across multiple cancer types have reported that ctDNA-based MRD detection is sensitive and specific for recurrence in both postoperative and serial testing scenarios 41 and 89%-100% specificity41 for CRC in the adjuvant setting, 79%-100% sensitivity and 100% specificity for breast cancer in the neoadjuvant or adjuvant setting,58 and 90% sensitivity and 88% specificity for pancreatic cancer in the adjuvant setting.59 Additional studies report ctDNA detected recurrence at 71% sensitivity and 100% specificity for esophageal cancer for a single ctDNA test performed after neoadjuvant therapy,9 and 94% sensitivity for non–small-cell lung cancer with a single test after local treatment (specificity was not reported).29 These studies used limited numbers of patients and require confirmation from large well-annotated cohorts. Still, because of the rapidly expanding body of evidence surrounding ctDNA-based MRD detection for CRC, a recently convened National Cancer Institute task force released a consensus statement concluding that the presence of ctDNA was strongly associated with a high risk of disease recurrence in CRC, with the results suggesting ctDNA was a robust marker for MRD.60 Currently, several ongoing trials are using ctDNA status to determine enrollment.ctDNA-based MRD detection using serial testing predicted recurrence with 82%-100% sensitivity61 Enrollment on the basis of ctDNA-positivity ensures that only patients with a high probability of recurrence are included in the trial, whereas low-risk patients who are unlikely to benefit are spared from potential treatment-related side effects. Moreover, from a clinical perspective, most experts would agree on the value of offering systemic therapy for patients with low-risk stage II CRC with initial intent of observation and surveillance, given the 100% recurrence for ctDNA-positive patients noted across multiple studies.62Restricting trial enrollment to those at high risk for recurrence is of clear benefit to patients. For example, patients with stage II CRC do not receive adjuvant chemotherapy, although 20% will experience recurrence.Improvement in clinical trial efficiency using ctDNA-based enrollment is highlighted in Figure 65 However, the modest improvement in invasive DFS, a reported lack of benefit in node-negative patients, treatment toxicities, and the cost of running a lengthy trial with a large sample size underscore the importance of enrolling patients at high risk of recurrence into trials.66Recent phase III adjuvant clinical trials that have enrolled thousands of patients further highlight the benefit of enriching trials with high-risk patients. For example, the PALLAS study enrolled 5,706 patients with early-stage hormone receptor–positive and human epidermal growth factor receptor 2–negative breast cancer in a trial with a planned 10 years of follow-up, to determine whether a CDK4/6 inhibitor added over a 2-year period to a minimum of 5-year standard-of-care (SOC) endocrine therapy improves disease-free survival (DFS). Similarly, the APHINITY study randomly assigned 4,805 patients with human epidermal growth factor receptor 2–positive breast cancer to investigate the addition of pertuzumab to chemotherapy plus trastuzumab. Both trials could have substantially reduced sample size and costs if reliable biomarkers to identify patients at high-risk for recurrence were available at enrollment , whereas ctDNA-negative patients receive SOC. This design permits comparison of the intervention on ctDNA-positive patients while ensuring that the ctDNA-negative group is noninferior to the intervention group  and escalation (ALTAIR) trials and an observational study (GALAXY) that serves to screen patients for MRD, leading to their assignment to one of the two trials. These trials fall under the umbrella of CIRCULATE-IDEA , a collaborative effort conducted by groups in Japan, the United States, Europe, and Australia to perform integrated analysis of data from ongoing randomized phase III studies.The ALTAIR study is evaluating efficacy and safety of preemptive treatment with Trifluridine/tipiracil (FTD/TPI) compared with standard of care (SOC). Patients who test ctDNA-positive after undergoing curative resection in GALAXY will be recruited into ALTAIR and randomly assigned to treatment or control. VEGA tests noninferiority of observation versus adjuvant CAPOX. These trials incorporate a crossover component where VEGA participants who become ctDNA-positive can enter the ALTAIR trial Fig B.Trials such as DYNAMIC II, DYNAMIC III, and TRACC Part C use a marker-based strategy design framework. In these trials, patients are randomly assigned on the basis of MRD testing results, with ctDNA-positive patients assigned to treatment escalation and ctDNA-negative to de-escalation. An active comparator receives SOC in the absence of information regarding ctDNA status Fig C.69The TRACC Part C study further illustrates the use of this design, examining whether postoperative ctDNA guided therapy  is noninferior to SOC chemotherapy in patients not tested for ctDNA. This highlights the possible role of ctDNA testing in reducing unnecessary SOC chemotherapy. Indeed, ctDNA testing may ultimately identify patients who are cured and do not require further therapy, a potential role supported by high DFS for ctDNA-negative patients in the adjuvant setting.73 Likewise, serial carcinoembryonic antigen measurements and imaging (CRC74) and CA-125 assessments (ovarian cancer75) for monitoring patients following definitive treatment did not demonstrate a mortality benefit. Conversely, early salvage radiation on the basis of prostate-specific androgen testing is SOC for patients meeting specific risk-benefit criteria following definitive treatment.76 Furthermore, two recent randomized trials in prostate cancer demonstrated that early systemic therapy for rising prostate-specific androgen levels, in the absence of clinically detectable disease, can improve metastasis-free survival and has led to US Food and Drug Administration (FDA) approval of apalutamide and enzalutamide for this indication.78 MRD-directed therapy has also long been part of the treatment armamentarium in hematologic cancers. A recent example is the FDA-accelerated approval of blinatumomab for treatment of patients with a form of B-cell leukemia who had MRD after initial chemotherapy.79 This single-arm trial showed that those with a complete MRD response after blinatumomab had longer progression-free survival and OS durations. Trials that focus on ctDNA as an intervention are in the early stages, and conclusions regarding the clinical utility of ctDNA-based treatment assignment await their completion.Benefit from early detection is key to a successful intervention involving treatment assignment through disease monitoring. However, previous trials investigating intense monitoring versus SOC have not always shown benefit. Two randomized clinical trials in early-stage breast cancer that compared intense monitoring  to SOC failed to show improved survival.83 These observations provide support for using ctDNA status as a surrogate that could act as an early indicator of clinical benefit, reducing trial length and accelerating approval of new therapeutics.Associations of ctDNA dynamics and clearance with response and survival outcomes are consistently reported across cancers in the neoadjuvant and adjuvant settings.84 A surrogate end point validated against an established end point can provide insight into the benefit of new therapeutics, facilitating accelerated approval, although generally, a confirmatory trial, with potentially a large sample size, must be ongoing at time of approval. Pathologic complete response (pCR), discussed below, is a well-known surrogate end point. The FDA approved use of pCR for accelerated approval in the neoadjuvant setting for high-risk, early breast cancer in 2013.85A role for surrogate end points in oncology trials is widely accepted, although debate exists regarding the employment of many existing surrogates.86 A retrospective analysis of the IMvigor010 trial found that patients with urothelial carcinoma undergoing adjuvant treatment with atezolizumab who cleared ctDNA had improved DFS  and OS  compared with patients who did not clear ctDNA. These results suggest that ctDNA testing provides an early readout that informs treatment decisions.64Initial data indicate that ctDNA holds promise as a surrogate end point. In the immuno-oncology setting, a minority of patients with solid tumors respond to immune checkpoint inhibitors (ICI), although treatment is known to have long-term benefits for responders. Early determination of response would enable patients who derive clinical benefit from immune checkpoint inhibitors to continue therapy while others could be spared from unnecessary toxicities.83 Interestingly, data from this trial also indicated ctDNA could serve as a complement to MRI functional tumor volume as a predictor of treatment response.87A potential role for ctDNA testing is to be used as a complementary measure to pCR. A retrospective analysis of the I-SPY-2 clinical trial, which evaluated neoadjuvant treatment with investigational drugs in patients with high-risk breast cancer using pCR as an end point, provides support for using ctDNA testing in this role. The analysis found that ctDNA status was strongly associated with pCR, and lack of ctDNA clearance was a predictor of poor response and metastatic recurrence. Importantly, ctDNA clearance was associated with improved survival in patients who did not achieve pCR, indicating ctDNA testing might provide information regarding outcome in clinical trials beyond that of pCR.88Further evidence is needed before ctDNA dynamics or clearance can be approved as surrogate end points in trials. Approval by appropriate regulatory agencies depends on accumulation of evidence from observational studies and clinical trials. Meta-analyses of clinical trials will be key in determining whether ctDNA dynamics or clearance robustly predicts treatment effect on the true end point.88 Agreements on standardization around key aspects of trial design, such as ctDNA collection time points, would further streamline analyses and facilitate interpretation of results.60Currently, meta-analyses investigating ctDNA clearance as an end point in the adjuvant setting are planned for CRC. In addition, ongoing trials are collecting ctDNA as secondary measures permitting further evaluation across cancer types and treatment settings. Analysis and interpretation of these data has challenges, because of heterogeneity in study design, ctDNA assays, measurement and metrics, and timing of ctDNA samples. Preliminary work from ctMoniTR, a collaboration of private, government, and academic institutions with the aim of harmonizing data from clinical studies using ctDNA-based treatment response monitoring, indicates that trends in individual trials can be replicated in aggregate.As highlighted, alongside evidence presented for using ctDNA for MRD detection in various malignancies, the clinical question, assay timing, biology of shedding for different cancer types, and number of time points being collected cannot be ignored. Furthermore, reliable knowledge of recurrence rates of ctDNA-negative patients by cancer type is needed to allow for appropriate and ethical de-escalation or discontinuation of therapy. The merits of ctDNA testing would have to be weighed with the risks and long-term quality-of-life outcomes. Additionally, a question that often arises is whether ctDNA-positive patients are treatable or have occult metastatic disease that may not be cured with adjuvant therapy. The latter two concerns underscore the need to consider possible harms from molecular monitoring and early intervention, particularly if clinical utility is less than what is currently anticipated. Costs of ctDNA testing may represent added out-of-pocket expense for patients. False-positive results can lead to additional testing, mental stress for patients, and exposure to toxic and unnecessary therapy. Even true-positive results can lead to deterioration of quality of life because of drug-induced adverse effects at a stage when the cancer is asymptomatic. Early systemic therapy for patients who relapse may also run the risk that an effective treatment option will no longer be available when the cancer becomes symptomatic. These concerns can be settled through well-conducted clinical trials.In conclusion, evidence pointing to ctDNA as a biomarker that can predict cancer recurrence continues to accumulate. ctDNA-based MRD detection is now being incorporated into the design of clinical trials. It seems likely that, for many cancers, ctDNA-based trial enrichment will become increasingly common because of cost-reduction benefits. Trial efficiency could also benefit from using ctDNA as a surrogate end point, leading to accelerated approval of new therapeutics. Further research, however, is needed to validate ctDNA dynamics or clearance as end points. In the next few years, trials using ctDNA-based MRD detection to identify patients who may benefit from early therapeutic interventions will release results. A clear demonstration of efficacy of ctDNA-based MRD detection would transform clinical practice. The importance of enrolling patients into ongoing trials incorporating ctDNA as an integral or exploratory marker cannot be overemphasized.