Gallium Nitride (GaN) is considered as the second most popular semiconductor material in industry after silicon. This is due to its wide applications encompassing Light Emitting Diode (LED) and power electronics. In addition, its piezoelectric properties are fascinating to be explored as electromechanical material for the development of diverse microelectromechanical systems (MEMS) application. In this article, we conducted a theoretical study concerning surface mode propagation, especially Rayleigh and Sezawa mode in the layered GaN/sapphire structure with the presence of various guiding layers. It is demonstrated that the increase in thickness of guiding layer will decrease the phase velocities of surface mode depending on the material properties of the layer. In addition, the Q-factor value indicating the resonance properties of surface mode appeared to be affected with the presence of fluid domain, particularly in the Rayleigh mode. Meanwhile, the peak for Sezawa mode shows the highest Q factor and is not altered by the presence of fluid. Based on these theoretical results using the finite element method, it could contribute to the development of a GaN-based device to generate surface acoustic wave, especially in Sezawa mode which could be useful in acoustophoresis, lab on-chip and microfluidics applications. The demand for surface acoustic wave (SAW) technology, especially in signal processing and filtering has increased significantly due to its robustness, simplicity, inexpensive cost for mass production and good compatibility with CMOS processes . MoreoveCommercially, SAW devices are mass-produced with bulk piezoelectric material and commonly used to convert AC electrical signals into a mechanical propagation on its surface. Normally, bulk piezoelectric materials such as lithium niobate . In piezoelectric devices, the SAW’s propagation was governed by the mechanical equations of motion and Maxwell’s equations for electrical behaviour. FEM couples the stress, strain, electric field and electric displacement in a stress-charge of a piezoelectric layer. In this model, COMSOL solves both structural and electrical equations in the piezoelectric GaN layer, but only solves for the structural equation in the substrate and guiding layer. Meanwhile, the metallic aluminium electrodes were not solved by the electrical equations since the electrical conductivity of aluminium electrode is a few orders of magnitude higher than the piezoelectric layer of GaN, and act as equipotential regions allowing a slight amount of conduction current through them.h from 100 nm to 2 f is the eigenfrequency obtained from simulation results.In the first stage, a simulation without a sensing layer was investigated while the thickness of GaN, p is the acoustic pressure , C is the speed of sound. We implement the acoustic-structure boundary coupling feature in order to consider solid-fluid interaction at the interface. The boundary condition was set with the boundary load F (force/unit area) on the solid:In order to evaluate its performance under the exposure of various liquids and gases, we added a fluid domain on top of our sensing layer through the coupling between the fluid domain (acoustic pressure module) and the solid (solid mechanics module)—refer to cy shift . NeverthWe calculated the eigenfrequency based on the simulation model described previously. h is the thickness of guiding layer, k = 2In ce (QCM) ,29.The eigenmode of surface waves in GaN/Sapphire with addition of 300 nm SiOIn the literature, various materials such as ZnO, gold , titaniuIn the case of Rayleigh’s mode, the phase velocities curve of SiOIn order to evaluate the resonance properties of surface modes, we calculated the Q factor in the frequency domain ranging from 200 to 600 MHz, which is a useful value to indicate the quality of resonator and amount of loss. Nevertheless, the addition of a fluid domain on the propagation of path of surface mode, particularly for Rayleigh SAW has been shown to affect the amplitude due to the radiation of longitudinal wave into the fluid, which caused an attenuation. Therefore, we investigate also the evolution of Q factor in the presence of fluid on top of the guiding layer and compared with the free surface. We opted for gold since it has a significant effect as a guiding layer to reduce the phase velocities of SAW and Sezawa mode by enhancing the in-plane displacement which could avoid the dissipation of energy into the fluid. Unlike conventional SAW devices which suffer considerable loss due to the coupling of surface mode with the bulk substrate, the presence of a gold guiding layer could also reduce this coupling and, hence, increase the energy confinement at the solid–fluid interface. Based on the graph from The graph of Q-factor for GaN/sapphire with a 600-nm of gold guiding layer was compared under different conditions (with and without fluid domain) for a frequency range from 300 to 600 MHz. In the absence of a fluid domain, the first peak represent the Rayleigh SAW mode with a maximum Q factor of 1000 at 320 MHz. We observed the reduction of Q factor with a slight shift to the left of the peak with the presence of a fluid domain indicated by blue line. The reduction of Q factor is expected to occur due to the radiation into the fluid that could attenuate the wave’s amplitude. Meanwhile, the second peak was located at 444 MHz indicating a shear mode in the presence of the guiding layer shows a low value of Q-factor. However, the third peak with the highest Q factor value indicating Sezawa mode at 534 MHz does not show a significant attenuation of the Q factor for both cases (with and without fluid domain) unlike the previous Rayleigh mode. The advantage of Sezawa mode producing a high Q factor even in the presence of fluid domain could be attributed to the energy confinement dominantly in the piezoelectric GaN layer. In the absence of guiding layer in the structure, the shear mode is not observed as displayed by the black curve where the first (409 MHz) and second (563 MHz) peak represent Rayleigh SAW and Sezawa mode, respectively.In conclusion, we have demonstrated the effect of a guiding layer on the propagation of a surface mode of GaN/sapphire layered structure. The addition of an SiO


Shoulder arthroscopy is a safe and effective procedure with a low complication rate. Although rare, there are potentially life-threatening risks such as fluid extravasation causing airway compromise.We report the case of a 65-year-old female treated with an arthroscopic rotator cuff repair who had significant extravasation of arthroscopic fluid causing severe facial and neck swelling. Overnight intubation was required for respiratory monitoring until the edema had resolved enough to allow safe extubation.This case highlights the risk factors and clinical course of a patient with airway compromise caused by extravasation of fluid during shoulder arthroscopy. Although shoulder arthroscopy is a safe procedure, surgeon familiarity with the risk factors for this complication and close monitoring can aid in its identification and allow for appropriate treatment. Shoulder arthroscopy facilitates a minimally invasive approach to the joint compared to open procedures which are inherently more traumatic to the soft tissues. Arthroscopy has its own unique complications primarily related to the need for distension of the operative joint with pressurized irrigation fluid to aid in visualization. Extravasation of irrigation fluid and even air into the surrounding soft tissues has previously been with the most significant consequence of this extravasation is edema resulting in airway compromise .Most of the earlier published cases involved patients undergoing arthroscopy under regional anesthesia where the patients vocalized symptoms of impending airway compromise –6, but mWhile the glenohumeral joint has a capsule that may provide an anatomic barrier to fluid extravasation, it is has been postulated that the lack of such a capsule in the subacromial space allows for fluid extravasation during arthroscopy , 11. Ana10.A multitude of other risk factors are identified in the literature. Obese patients have multiple tissue planes that may allow fluid to spread more easily into the surrounding soft tissue . PatientWe describe a case where arthroscopic rotator cuff repair in a patient with several risk factors for fluid extravasation resulted in facial and neck edema requiring prolonged intubation and respiratory monitoring overnight.The patient provided written consent for publication.A 65-year-old female with a body mass index of 29 presented with a 7-month history of left shoulder pain and weakness. Physical exam and diagnostic imaging were consistent with a symptomatic full thickness rotator cuff tear of the supraspinatus and a partial tear of the subscapularis tendon Fig. . She wasAnesthesia evaluation on the day of surgery was performed and her neck was recorded as “unremarkable.” She was assigned an American Society of Anesthesiologists (ASA) score of 2. On the day of surgery, she was easily intubated with an endotracheal tube in the supine position and then placed in the standard lateral decubitus position for arthroscopic shoulder surgery. Exam under anesthesia was performed followed by a diagnostic shoulder arthroscopy. Normal saline was fed into a Stryker CrossFlow® Integrated Arthroscopy Pump  set at 25 mmHg initially. Epinephrine was not added to the irrigation fluid. Shortly after beginning the case, the arthroscopic fluid pressure was raised to 35 mmHg to aid in visualization where it remained for the duration of the case. No lavage cycles were utilized. Standard posterior, anterosuperior and anteroinferior portals were placed as well as a lateral working portal. She was found to have a type 1 superior labrum anterior to posterior (SLAP) tear, degenerative changes in the anterior, inferior and posterior labrum, a subscapularis tear in the upper one third which was retracted medially, and a complete supraspinatus tear. A biceps tenotomy was performed followed by rotator cuff repair of the subscapularis and supraspinatus tears utilizing suture anchors. Bone quality was remarkably poor with pull-out of multiple suture anchors during the rotator cuff repair adding to surgical complexity and time. Total operative time was 3 h and 53 min. Upon completion of the case and removal of the surgical drapes, significant unilateral face and neck swelling was noted on the side of the operative shoulder (the non-gravity dependent side). Upon consultation with the anesthesia providers the decision was made to obtain a computed tomography (CT) scan for visualization of the soft tissues surrounding the airway, with a plan to leave the patient intubated overnight. The CT demonstrated diffuse soft tissue edema in the subcutaneous tissues of the neck, chest and face. The airway was deviated at the level of the trachea due to the paratracheal edema. There was no focal collection or extravasated contrast indicative of a hematoma or vascular injury . After resolution of the edema, she met standard ICU extubation criteria, and was extubated on the morning of postoperative day one. Postoperatively, the patient has done well without any airway or pulmonary complications, complete resolution of preoperative symptoms, and return to baseline shoulder function Fig. .Fig. 3ClIn this case, the patient was intubated for the duration of the procedure, which ensured airway protection and obviated the need for intubation upon recognition of the condition. Had the procedure been performed under regional anesthesia with sedation or with a laryngeal mask airway (LMA) a conversion to an endotracheal airway would have been required. Fortunately, inspection of the neck and surrounding soft tissues prior to extubation identified this complication and the patient remained intubated in a monitored setting until extubation was safe.A CT of the neck and chest was requested by the anesthesia team to assess for laryngeal edema. Additional strategies exist to assess for a potentially compromised airway. If the edema is noted before extubation of the patient, a cuff-leak test can be performed. This involves deflating the cuff of the endotracheal tube and then digitally occluding it. If the patient is able to breath around the deflated cuff, it is hypothesized they are at lower risk for post-extubation stridor and need for reintubation. Results of this test have varied widely depending on patient population and study so results of the test should be utilized in conjunction with other clinical tools , 13. FurEstablished risk factors for fluid extravasation present in this case included lateral decubitus positioning, prolonged operative time, and performing a subacromial decompression. These represent modifiable risk factors and are important preoperative and intraoperative considerations for the treating surgeon. Adequate visualization of the surgical field during shoulder arthroscopy is a necessity, but the surgeon should have an understanding of the risks of elevated pump pressures and liberal use of lavage cycles. Visualization of the subacromial space can be aided by narrowing the pressure differential between the arthroscopic pressure in this space and the systolic blood pressure. Morrison et al. demonstrated that the visual clarity of the subacromial space in the lateral decubitus position was excellent as long as there was a pressure difference of less than 49 mmHg. This pressure differential can be maintained through a combination relative hypotensive anesthesia and higher pump pressures , 15. TheAlthough the risk of fluid extravasation may be decreased with the beach chair position, this positioning is also not without risk. Beach chair positioning carries a higher risk of cerebral hypoperfusion especially with relative hypotensive anesthesia. It also carries higher rates of venous and arterial air embolism, and even quadriplegia from incorrect head positioning during surgery , 15.Memon et al. conducted a systematic review of the published literature concerning fluid extravasation as result of shoulder arthroscopy. Approximately 65% of the patients who experienced fluid extravasation were positioned in the lateral decubitus position compared to 30% in the beach chair position. The authors also suggest limiting the pump pressures to less than 150 mmHg. Utilizing a gravity feed irrigation setup may be a safer alternative to pump systems which result in higher intra-articular pressures –18. TheiTotal operative time is directly proportional to the volume of irrigation fluid used and subsequent weight gain by the patient, presumably from absorption of the irrigation fluid . AlthougFluid extravasation not only affects the local soft tissues but may have other systemic complications such as electrolyte disturbances. Ichai et al. demonstrated that there is systemic absorption of glycine irrigation fluid during shoulder arthroscsopy. Although the absorption of glycine may be different than that of other irrigation fluids including normal saline or Ringer’s lactate, the authors suggest that the risk of significant systemic absorption can be mitigated with lower pump pressures and a lower volume of irrigation fluid used during arthroscopy , 21.Shoulder arthroscopy is a generally safe and effective procedure, but surgeons and anesthesia providers must be aware of the rare and potentially life-threatening complication of fluid extravasation and airway compromise. Risk factors such as lateral decubitus positioning, obesity, high pump pressures, and long operative times may increase this risk. Surgeons and anesthesia providers should consider routine endotracheal intubation in arthroscopic shoulder procedures with multiple risk factors for dangerous fluid extravasation.


Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of the molecular heterogeneity of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 ‘trios’ of matched primary tumour, PDX, and PDO. We developed a pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Tumour-model comparisons of mutations displayed single-gene concordance across major PDAC driver genes, but relatively poor agreement across the greater mutational load. Genome-wide and chromosome-centric analysis of structural variation (SV) events highlights previously unrecognized concordance across chromosomes that demonstrate clustered SV events. We found that polyploidy presented a major challenge when assessing copy number changes; however, ploidy-corrected copy number states suggest good agreement between donor-model pairs. Collectively, our investigations highlight that while PDXs and PDOs may serve as tractable and transplantable systems for probing the molecular properties of PDAC, these models may best serve  in vitro and in vivo preclinical platforms to study PDAC at the molecular level. However, current investigations of these models provide limited snapshots of donor-model comparisons. To date, comprehensive assessment of PDAC disease models using whole-genome sequencing (WGS) has not been performed. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumors and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 ‘trios’ of matched primary tumor, PDX, and PDO. Globally, our findings indicate that PDX and PDO successfully recapitulate primary and metastatic disease at the level of simple somatic mutations mainly in driver genes. Disease model fidelity is not fully retained when assessing structural variation (SV) events, based on heterogeneity we observed across donor-model pairs. Strikingly, we found that clustering of large SV events across particular chromosomes is retained when the tumors are implanted into their respective disease models. Polyploidy presents a major challenge when assessing copy number changes; however, ploidy-corrected copy number states suggest good agreement between donor-model pairs. Our work provides biological insights into PDAC disease heterogeneity and presents a tractable pipeline for future WGS research that compares donor samples with matched PDX and PDO.PDAC is a highly lethal, therapy-resistant malignancy, whose etiology is poorly understood. PDAC PDX and PDO have emerged as important  Multet al. demonstrated that PDAC PDO maintain differentiation status, recreate histo-architectural heterogeneity, and retain patient-specific physiological changes  recapitulated much of the somatic mutational landscape observed in the primary tumours. Across 5 liver metastasis samples, we observed averages of ~5,300 mutations per tumour, and ~5,500 mutations per PDX, with a range of 66–86% overlap of mutation calls in metastasis-PDX pairs  revealed that the majority of variants were intra-chromosomal, including deletions (DEL), inversions (INV), and duplications (DUP)  to identify distinct chromosomal instability profiles observed in primary tumours that could also extend to liver metastasis pairs. Metastasis-PDX pairs exhibited variable counts of SV events, ranging between 16–172 events in the samples [c = 0.6). In contrast to the primary-PDX pairs however, the majority of the pairs (4/6) demonstrated greater agreement across the genome . This agreement was also reflected when assessing different categories of SV events across the genome [We conducted a comparative analysis of SV events in paired metastasis and matched PDX samples [ samples . Not sur samples , and scoe genome .Chromosome-centric analyses of metastasis-PDX pairs indicated consistency of SV events across the majority of the chromosomes (Jaccard index ≥ 0.6)  Tables. c <0.6), and across many chromosomes (Jaccard scores ≤0.4) for the remaining 4 trios [c scores of PDX-PDO comparisons scored higher than tumour-PDX and tumour-PDO comparisons for these pairs [c ≥ 0.8) was observed across all tumour-PDX, tumour-PDO, and PDX-PDO pairs for several chromosomes, including PCSI_0592 (chr19), PCSI_0624 , PCSI_0642 (chr8) [Analysis of the primary-PDX-PDO trios highlighted the extent to which structural variation of primary-PDX pairs were captured in organoid models . The dis 4 trios . Interesse pairs . Despite2 (chr8) .c scores) also demonstrates that PCSI_0590 and PCSI _0624 were the lowest scoring among the trios; all of the pairs of those trios demonstrated overall concordance Sc < 0.6.Comparison of tumour-PDX, tumour-PDO, and PDX-PDO pairs highlighted particular cases that were discordant at both chromosome and genome-wide levels. We observed the most discordance in all specimens of the trio for chromosome 17 of PCSI_0624. Further investigation into this chromosome revealed a very high count of SV events in the primary tumour, with reduced events in the matching PDX, and almost no events in the PDO . CompariGenome-wide copy number state was assessed for primary-PDX pairs, trios, and metastasis-PDX pairs Figs  Tables. Jaccard indices were computed to quantify overall similarity between the ploidy-adjusted copy number states of primary tumours and matching PDX, across all bases of the genome, and for individual chromosomes. Using ploidy-normalized scores, all tumour-PDX pairs demonstrated concordance ≥ 0.8 across the entire genome . ChromosAssessment of copy number variation in the liver metastasis set demonstrated comparable ploidy states across the 6 metastasis-PDX pairs, and none of those pairs had to be excluded from the analysis , S7 Fig.Copy number state was compared within tumour-PDX, tumour-PDO, and PDX-PDO matched pairs for trios  Figs. AsWe tested for clonality of primary tumours, metastases, and matching models using PyClone . PyCloneCritical evaluation of disease models is gaining importance across several cancer types that utilize these surrogates as preclinical tools for exploring tumourigenesis and drug testing. Previous investigations of PDAC provide a limited snapshot of donor-model comparisons, in terms of morphological, pathological, and mutational correlates . Our hisIn our PDAC cohort of paired tumour-PDX-PDO trios, the organoid samples are derived from the PDX, not the original patient donors. Given this experimental setup, the overall expectation is that the PDO would demonstrate greater fidelity with the PDXs. An assessment of structural variation changes across the trios demonstrated stronger concordance between PDX-PDO pairs than patient-PDX or patient-PDO pairs . Our analysis of copy number variation across 3 trios also indicated that PDX-PDO pairs scored marginally higher in terms of overall concordance, with fewer copy number changes in individual chromosomes. Collectively, these findings suggest that true PDO representation of the donor tumour would benefit from direct growth from the patient tumour itself.At the single-base and gene-centric level, our findings emphasize that PDAC disease models capture many of the mutational patterns and driving events involved in PDAC tumourigenesis, up to an extent. About ~1/3 of the observed SNVs are not shared by the tumour of origin and its matching PDX, rendering an intermediate scoring of tumour-PDX agreement across the majority of the PDAC pairs, and a rather low concordance in 30% of the models. However, there is greater concordance between tumours (primary or metastases) and matched PDX for functional mutation types (missense and nonsense mutations). A more detailed evaluation also demonstrated retention of genetic features for 9 driver genes involved in PDAC tumourigenesis. As such, our study confirms and extends prior findings that the genetic mutations of key driver genes are maintained between tumour-PDX pairs , 8, 20, Investigations into copy number variation emphasize that ploidy changes are a substantial factor that must be considered when conducting tumour-model investigations. Two of our 16 samples had undergone severe ploidy changes that resulted in their exclusion from the analysis. While many of the remaining tumour-PDX pairs and trios demonstrated comparable ploidy, our analysis also captured conspicuous cases of whole genome polyploidization events that gave rise to tetraploid genomes in matched disease models (notably across PCSI_0590 and PCSI_0633). Accordingly, measuring consistency had to be conducted on ploidy-scaled copy number events. With these corrected measurements, we were able to conclude that there is agreement of copy number events at genome-wide scales  and chromosome-centric levels. However, we are cognizant that these normalized measures potentially mask discrepancies that are more readily apparent when comparing raw (uncorrected) copy number measurements. This is of import when assessing copy number changes in chromosomes that exhibit large-scale, complex genetic events, such as chromothripsis.et al, based on observations from breast and hematopoietic cancers, suggest that clonal evolution of PDX occurs through directional selection of pre-existing clones [in vivo PDX passages, such that CNAs acquired through PDX passaging differ substantially from that in their parental tumours [Deviation in ploidy underscores potential changes that arise when transferring portions of primary tumours to other disease model mediums, particularly across multiple passages. In our work, WGS profiling of PDX and PDOs was undertaken following third passage (P3) engraftment of tumours into mice. While the lack of profiling of earlier PDX passages prevents us from drawing conclusions about copy number aberrations (CNAs) in PDAC across multiple passages, one limitation includes the selection of subclones when different portions of the tissue are grown in future passages within PDXs and PDOs. Subclones may gain survival advantage growth in the disease models, and subsequently, tumour engraftment in PDXs may be confounded by underlying biological mechanisms that promote adaptation and growth of these tumours in a new environment . Indeed,g clones . Interes tumours . These rc score) showed poor agreement between tumours and PDX, which promoted further investigation into chromosome-specific scoring methods to efficiently depict this heterogeneity. At this deeper level, many chromosomes still scored poorly in tumour-PDX comparisons. Strikingly however, we identified cases of genomic consistency across chromosomes with clustered SV events that suggest chromothripsis. In that context, chromosomes with chromothripsis in the primary tumour exhibited similar behavior in matching PDX and matching PDO. For these particular chromosomes, in resected patients, the actual rearrangements of the SV clusters appear to be quite different between tumour and PDX. However, these rearrangements are almost identical when comparing metastases-PDX pairs. Despite evidence of overall structural heterogeneity between tumours and their matched models, these findings argue that major structural changes that occur in a tumour sample can be largely ‘transmitted’ to matched PDXs and PDOs. There remain, however, other anomalous cases where clustered SV events in specific chromosomes within tumour samples have been ‘lost in translation’ in matched PDX and PDO . Equally difficult to rationalize are cases where clustering of SV events have arisen in the matched PDX, but not in the source tumour . While these events may once again be explained as a result of subclonal events or tumour selection, our limited sample size hinders investigations as to whether those events could be recurrent events in a larger PDAC series.Assessment of structural variation (SV) events sheds light on biological phenomena that have not been previously described in PDAC disease models. Genome-wide scoring of SV concordance  in comparison with the primary series (and their matched PDX). Across all types of genomic events studied , we observe that metastases demonstrate higher concordance with their matched PDX compared with primary-PDX pairs. All of the pairs demonstrate higher concordance levels (Jaccard index ≥ 0.6) for both SSM and CNV. SV heterogeneity across these pairs is less pronounced than those of primary tumours, producing improved concordance scores for SV events. Concordance at the SV level is also higher in metastases than primary tumours when considering chromosome-centric analyses, or focusing on categories of SV events . The rearrangement of SV events for chromosomes with chromothripsis is almost identical between metastases-PDX pairs, and there are fewer discrepancies in copy number states. One possible explanation for this behavior may reflect upon the tumour microenvironment of metastatic samples compared to primary tumours, as metastatic samples represent more stable, aggressive, and proliferative derivatives of the primary tumour. Interestingly, our cohort of metastasis-PDX pairs also includes a sample demonstrating DNA mismatch-repair (MMR) deficiency (PCSI_0489) due to a germline MLH1 mutation. Alterations in MMR genes lead to microsatellite instability (MSI), a genotype found infrequently in PDAC , 24, 25.Our approach presents quantifiable parameters of model fidelity; assessing molecular similarity between tumour-model pairs is necessary towards utilization of these models in future preclinical testing. For example, our analysis highlighted a discordant tumour-PDX pair (PCSI_0169) that was consistently difficult to decipher across both SSM and SV comparisons. One possible explanation for this discordance may be a mutational process that is only present in the PDX, due to a clone from the primary tumour growing as the main clone in the PDX. Further investigations would be needed to explore this phenomenon; however, this is a clear case where the PDX may not a good surrogate for preclinical testing of the donor tumour. Deciphering the extent of discordance for that PDX sample would not have been possible without scoring donor-model comparisons across genome-wide, single-base, and chromosome-centric scales.in-vitro models [This study has several potential limitations. Our limited sample size hampers investigations into subclonal events within individual samples. Due to a lack of sufficient technical replicates for each disease model studied, it is difficult to measure within-line variability in the models. We have assessed potential clonality changes between tumours and their matched models for each patient, and present findings that suggest stable clonality in tumour-model comparisons, with notable exceptions. These however, are preliminary findings, as our limited sample size precludes explanations as to the prevalence of different clones or their statistical significance. Our findings demonstrate that larger-scale investigations to assess transcriptional and clonal evolution across these disease models will be warranted in the future, similar to previous analyses highlighting genomic evolution across o models . CurrentGenomic comparisons between primary tissues and disease models are necessary for future studies that focus on gene-drug associations across model systems. While we have highlighted in this manuscript examples of common and discrepant genomic aberrations between tumour-model pairs, the phenotypic relevance of these aberrations remains to be investigated. Previous work on PDAC disease models, using whole-exome sequencing, demonstrated that drug sensitivities remained stable across multiple passages of PDAC cell lines, suggesting a conservation of dominant genetic drivers and related therapeutic sensitivities . HoweverIn summary, we have conducted a detailed molecular dissection of WGS data to quantify concordance of genomic events between model systems and matched human PDAC. We present the first extensive genomic evaluation of consistency and in-consistency across pre-clinical models, for different levels of genomic complexity . Broadly, our findings shed light on consistency between disease models for mutations in PDAC driver genes, but highlight relatively poor agreement between tumours and their matching models across the greater spectrum of mutational load. Disease model fidelity is not retained well when assessing structural variation events, but there are striking cases of clustering of SV events across particular chromosomes that are retained when tumours are implanted into their respective disease models. Copy number comparisons suggest good agreement across tumour-model comparisons, conditional of reducing potential changes in ploidy. Across all of these platforms, our systematic comparison of tumours, PDX, and PDO also highlights several genetic aberrations that are sample-specific, and which may not be shared across donor samples and matched models. Collectively, our investigations highlight that while PDXs and PDOs may serve as tractable and transplantable systems for probing the molecular properties of PDAC, these models may best serve selective analyses across different levels of genomic complexity. We expect that our analytic pipeline may serve as a framework for future WGS research that compares donor samples and matched PDX and PDO.A schematic overview of the biospecimens and analytic design is presented in PDX were established by subcutaneous implantation of fresh surgically resected primary tumour tissue into immunodeficient mice . All ani™ TH . Dissociated cells were collected and embedded in growth factor-reduced Matrigel , which is overlaid with growth medium [PDO models were generated by the Princess Margaret Living Biobank core facility using previously described protocols . Brieflyh medium 3 or larger were obtained for each case and stored at -80°C. Each tissue was serially cryosectioned (10um thickness) at -20°C, fixed with 100% ethanol for up to 30 min and mounted onto PEN membrane 1.0 slides . All but one section was stained to visualize structures using a cresyl violet protocol that stains Nissl granules purple. Sections were rinsed in deionized water and stained in 1% cresyl violet solution  for 1 minute, dipped in 70% ethanol and dehydrated by dipping in absolute ethanol. Slides were left to dry for approximately 15 minutes before being stored at -80°C in air tight, aluminum foil wrapped slide boxes. The last section of each series was mounted onto an uncharged slide and stained using a modified haematoxylin and eosin (H&E) protocol. Sections were rinsed in deionized water, stained in Mayer’s haematoxylin (10 minutes), rinsed with deionized water, blued in Scott’s tap water, rinsed in deionized water, stained in aqueous eosin Y (1 minute), dipped in 3 changes of absolute ethanol, dehydrated in three changes of xylene (2 minutes each), prior to coverslipping. Additional details on the histochemistry and laser capture microdissection of xenograft samples have been previously described [Snap frozen tumour tissue 5mmescribed .LCM was performed on the PDX as previously described . BrieflyLCM of fresh frozen tissue samples from PDAC was performed on a Leica LMD 7000 instrument. Frozen tissue for tumour samples was maintained in vapor-phase liquid nitrogen and embedded in OCT cutting medium and sectioned in a cryotome into 8-μm thick sections. These sections were then mounted on PEN membrane slides (Leica) and lightly stained with hematoxylin to distinguish tumour epithelium from stroma. A pathologist (SF) marked tumour sections and LCM was performed on the same day according to manufacturer’s protocol on the Leica LMD7000 system. Microdissected tumour cells were collected by gravity into the caps of sterile, RNAse-free microcentrifuge tubes. Approximately 150,000–200,000 tumour cells were collected for each DNA extraction and stored at –80°C in Arcturus PicoPure Extraction Buffer.http://broadinstitute.github.io/picard) was used to sort, merge, and mark duplicates from multiple lanes of the same sample, followed by the Genome Analysis Toolkit  [Paired-end cluster generation and sequencing was carried out using the Illumina HiSeq 2500 platform, on DNA isolated from fresh frozen tissue following LCM. Whole genome sequencing (WGS) of tumours, PDX, and PDO was performed with a minimum depth of ~30X per sample. Xenome (version 1.0.1)  was used 1.3.16)  to improGermline single nucleotide variants (SNV) were called using the Genome Analysis Tool Kit , using best practice guidelines made available by the Broad Institute. Briefly, data were locally realigned around indels and the base quality values were recalibrated prior to variant calling using the Unified Genotyper. This was followed by filtering using the VariantFiltration module, and subsequent classification of germline variants as those mutations which have a QUAL score greater than 50 in the normal sample. Both tumour and matched normal samples were processed simultaneously.Strelka (version 1.0.7)  and MuTevcfR (version 1.4.0) [VariantAnnotation (version 1.18.7) [GenVisR package (version 1.0.4) [Parsing of VCF files containing the filtered calls was conducted using the n 1.4.0)  and Vari 1.18.7)  packagesn 1.0.4) .М) was calculated as follows:Concordance of mutation calls between a tumour and its matching PDX was calculated using the Jaccard Index. For each mutation category annotated by Oncotator, the Jaccard Index for that mutation type , annotated with mutation мCStructural variations (SV) were called using CREST   and DELLRCircos library (version 1.2.0) [RCircos (version 1.2.0) [GenomicRanges (version 1.24.3) [rtracklayer (version 1.32.2) [PharmacoGx (version 1.1.6) [Genome-wide structural changes across all the four categories of structural variation  in tumour-PDX pairs were rendered using the n 1.2.0) . Quantifn 1.2.0) , Genomic 1.24.3) , rtrackl 1.32.2)  and Pharn 1.1.6)  packagesAssessment of concordance and discordance among SV events was conducted for each chromosome individually across tumour-PDX pairs. We first tabulated the number of SV events observed across tumour, PDX, and PDO, to determine the overall distribution of SV events across tumours and their matched disease models. We identified chromosomes with ≥ 5 SV events in both tumours and their matching PDX. To count SV events per chromosome, intra-chromosomal events  were assigned a score of ‘1’ for their respective chromosomes, while a score of 0.5 was assigned to each of the chromosomes involved in a translocation event. Instances of discordance (where one sample of a pair had ≥ 10 SV events different from the other sample) were also noted.Ѵ), across all SV events as follows:Ѵ = chromosome (autosomes in addition to chrX and chrY)Chromosome-specific concordance of structural variation events for a tumour and its matching PDX was quantified using the Jaccard Index, taking into account the genomic size (number of the genomic bases) of the SV events. For deletion (DEL), Inversion (INV), and Duplication (DUP) events, the genomic size of the event was calculated from the annotated breakpoints. For translocation (TRA) events, the total number of bases affected was set to 1. Jaccard indices of a tumour-PDX pair were then calculated individually for each chromosome  score to summarize the agreement between tumour-PDX pairs. The score determines the ratio of ‘positive’ chromosomes across a tumour-PDX pair that have a Jaccard index ≥ 0.6. The score is calculated as follows:We also generated an overall concordance , inversion (INV), duplication (DUP), and translocation (TRA) events. Concordance of SV events, per category, was determined as follows:К = Sum of genomic bases in the tumour sample, across all SV calls annotated as category КTК = Sum of genomic bases in the PDX sample, across all SV calls annotated as category КPК = Sum of genomic bases for SV events that are identical in the tumour and the PDX, across all SV calls annotated as category КCJaccard scores by SV category were calculated genome-wide , and calculated individually for each chromosome (autosomes in addition to chrX and chrY).Ѵ, JК, and Sc, as described previously.In the case of trios (tumour-PDX-PDO), SV concordance was quantified by splitting the trio into pairwise calculations of tumour-PDX, tumour-PDO, and PDX-PDO, and then calculating JCopy number segments were obtained using CELLULOID (version 0.11.2) to estimate gene copy number and tumour ploidy from WGS . Unless imean scores generated by CELLULOID (which represents average integer copy-number) for the genomic loci were rescaled by the ploidy of the respective samples. Genomic loci in tumour-PDX pairs were considered identical if they shared a copy number state with a difference ≤ 0.25. Genome-wide concordance scores (G) were calculated across all bases of a tumour-PDX pair as follows:Concordance of copy number state was calculated by first identifying overlapping genomic loci in tumour-PDX pairs using bedtools (version 2.24.0) . To consX = Number of genomic basis in the PDX with a defined copy number C = Number of genomic basis in either the tumour or PDX with a defined copy number I = Subset of C, where the absolute value of the difference between the imean score in the tumour and PDX is ≤ 0.25Ѵ) were also calculated individually for each chromosome as follows:Chromosome-specific concordance scores (G Ѵ = chromosome (autosomes in addition to chrX and chrY)ѴT = Number of genomic bases in the tumour with a defined copy number  in chromosome ѴX = Number of genomic basis in the PDX with a defined copy number  in chromosome ѴC = Number of genomic basis in either the tumour or PDX with a defined copy number  in chromosome Ѵ, where the absolute value of the difference between the imean score in the tumour and PDX is ≤ 0.25I = Subset of C, in chromosome Ѵ scores, as described previously. Plots of overlapping copy number states were drawn using the copynumber package (version 1.12.0) [In the case of trios (tumour-PDX-PDO), CN concordance was quantified by splitting the trio into pairwise calculations of tumour-PDX, tumour-PDO, and PDX-PDO, and calculating G and G  1.12.0) .М, JѴ, JК) or concordance scores , as described previously. These scores follow a scale from 0 to 1. A score of 0 indicates complete discordance between a tumour and its matching model, and signifies that none of the genomic aberrations identified in the tumour could be identified in the matching model. A score of 1 indicates complete concordance, such that all genomic aberrations identified in the tumour are successfully recapitulated in the matching disease model.Quantitative comparison of SSM, SV, and CNV events between tumours and their corresponding PDX and PDO samples were developed by calculating Jaccard indices , accession code EGAS00001002597. Comparison of SSM, SV, and CNV events between tumours and their corresponding PDX and PDO sample was conducted using R (version 3.3.1) . All sofS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(A) Frequency of reads carrying the variant allele for primary-PDX pairs. (B) Frequency of reads carrying the variant allele for metastasis-PDX pairs. (C) Frequency of reads carrying the variant allele for the trios. The trio is split into primary-PDX (top), primary-PDO, and PDX-PDO (bottom) pairs.Oncogenes, tumour suppressors, and genes involved pathways of PDAC tumourigenesis are plotted. (PDF)Click here for additional data file.S4 Fig(A) Circos plots of SV events across the genomes of 10 primary-PDX pairs (B) Circos plots of SV events across the genomes of 6 metastasis-PDX pairs.Each type of SV event is color-coded with a similar color between tumours and matching PDX. For each SV type, tumours are annotated on the outer rings of the circos plot and the matching PDX on the inner rings. SV events are colored as follows: deletions (red), inversions (green), and duplications (blue). Translocation events between chromosomes are also depicted (center). (PDF)Click here for additional data file.S5 Fig(A) Genome-wide jaccard scores, by SV category, for primary resected tumours and matched PDX. (B) Chromosome-specific jaccard scores, per SV category, for chromosomes exhibiting clustered SV events in primary resected tumours and matched PDX (C) Genome-wide jaccard scores, by SV category, for metastases and matched PDX (D) Chromosome-specific jaccard scores, per SV category, for chromosomes exhibiting clustered SV events in metastases and matched PDX.(PDF)Click here for additional data file.S6 FigEach type of SV  is represented as one circos plot, with 3 rings indicating tumour (outer), PDX (middle), and PDO (inner). SV events are colored as follows: deletions (red), inversions (green), and duplications (blue). Translocation events between chromosomes are also depicted (center).(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 FigFor each pair, the copy number state of the primary tumour (magenta) and matching PDX (black) is plotted across all chromosomes. A detailed panel also shows the copy number of the tumour and PDX across each chromosome. Values presented are raw (uncorrected) copy number values.(PDF)Click here for additional data file.S9 FigComplete CELLULOID solutions (solutions 1–5) are also provided for each of the samples which have been excluded from the analysis (PCSI_0592 and PCSI_0602).(PDF)Click here for additional data file.S10 FigFor each trio, the copy number of the tumour (black), matched PDX (red), and matching PDO (blue) is plotted across the genome. A detailed panel also shows the copy number of the tumour and PDX across each chromosome. Values presented are raw (uncorrected) copy number values.(PDF)Click here for additional data file.S11 Fig(A) 10 resected primary samples and (B) 6 liver metastasis samples. Each graph represents one sample, with matching PDX and PDO (where applicable) labelled. SNVs are clustered based on their common clonality, and colored by cluster. Points are drawn at the cellular prevalence of the SNV in each sample.PyClone output for (PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(A) primary tumours and matched PDX, (B) metastatic tumours and matched PDX, and (C) primary-PDX-PDO trios. Jaccard index for each tumour-PDX pair for mutation types are also shown for (D) primary tumours and matched PDX and (E) metastatic tumours and matched PDX.SSM calls across (XLSX)Click here for additional data file.S4 Table(A) primary tumours and matched PDX, (B) metastatic tumours and matched PDX, and (C) primary-PDX-PDO trios. For each sample, the total number of deletions (DEL), duplications (DUP), inversions (INV), and translocations (TRA) is indicated.SV across (XLSX)Click here for additional data file.S5 Table(A) primary tumours and matched PDX, (B) metastatic tumours and matched PDX, and (C) primary-PDX-PDO trios. Deletions, duplications, and inversion events that occur in a chromosome were assigned a value of 1 prior to their summation, and translocation events assigned a value of 0.5.Total events are indicated across (XLSX)Click here for additional data file.S6 Table(A) primary tumours and matched PDX, (B) metastatic tumours and matched PDX, and (C) primary-PDX-PDO trios.The indices are indicated across (XLSX)Click here for additional data file.S7 Table(A) Genome-wide jaccard scores, by SV category, for primary resected tumours and matched PDX. (B) Chromosome-specific jaccard scores, per SV category, for primary-PDX pairs (C) Genome-wide jaccard scores, by SV category, for metastases and matched PDX. (D) Chromosome-specific jaccard scores, per SV category, for metastasis-PDX pairs.(XLSX)Click here for additional data file.S8 Table(A) primary tumours and matched PDX, (B) metastatic tumours and matched PDX, and (C) primary-PDX-PDO trios. For each sample, the percentage of normal content (N), percentages of tumour content (T1), and the ploidy of the sample is indicated.Metrics are shown across (XLSX)Click here for additional data file.S9 Table(A) 8 primary-PDX pairs from resected tumour, (B) 6 metastasis-PDX pairs, and (C) 3 primary-PDX-PDO trios from resected tumour.Concordance is computed across (XLSX)Click here for additional data file.


To determine whether magnetic resonance imaging (MRI)-detected extramural vascular invasion (mrEMVI) status is associated with quantitative perfusion parameters derived from dynamic contrast-enhanced MRI (DCE-MRI) in rectal cancer.Ktrans, kep and ve) derived from DCE-MRI were calculated from all sections containing tumours. DCE-MRI parameters and clinicopathological variables in patients with different mrEMVI statuses were compared.Seventy-two patients with rectal adenocarcinoma who underwent curative surgery alone within 2 weeks following rectal MRI were enrolled in this retrospective study. mrEMVI status was determined based on high-resolution MRI. The quantitative perfusion parameters  and higher ve values (P = 0.021) than tumours of patients who were mrEMVI negative, while the Ktrans value displayed no significant difference (P = 0.390). The patients who were mrEMVI positive had larger tumour size, higher pathological tumour stage and increased regional nodal metastases compared to those who were mrEMVI negative .For patients who were mrEMVI positive, the tumours demonstrated significantly lower kep and higher ve values. In addition, these patients were more likely to have a larger tumour size, a higher pathological tumour stage and regional nodal metastases than mrEMVI-negative patients.This study demonstrated for the first time that tumour microcirculation is altered in mrEMVI-positive patients with rectal adenocarcinoma, as evidenced by significantly lower  Extramural vascular invasion (EMVI), defined histologically as the presence of tumour cells within blood vessels outside the muscularis propria of the rectal wall, is associated with a higher risk of local and distant recurrence and poorer survival , 2. AlthKtrans, min− 1), the rate constant between EES and blood plasma  and the volume of EES per unit volume of tissue  [Tumour angiogenesis, which is regulated by numerous factors, is a fundamental process in the growth and invasion of rectal cancer . Among t ve < 1) .Ktrans, kep and ve), with the ultimate aim of identifying a potential relationship between mrEMVI status and tumour microcirculation.Neovascularization usually has an immature vascular structure that can be easily infiltrated by tumour cells , 17. HowThis retrospective study was conducted from November 2014 to December 2015 after approval by our institutional review board. Written informed consent was obtained from all participants included in the study.n = 3) and signet ring cell carcinoma (n = 3); receiving preoperative radiotherapy, chemotherapy or chemoradiotherapy (n = 26); did not undergo surgery (n = 6); no rectal DCE-MRI (n = 6); and poor quality MR images (n = 4). Ultimately, 72 patients  were included  for rectal cancer  within 2 weeks after rectal high-resolution MRI and DCE-MRI were enrolled. A total of 120 consecutive patients with proven rectal cancer had preoperative rectal MRI. Among them, 48 patients were excluded for the following reasons: proven special histopathological type, including mucous adenocarcinoma (An appropriate amount (20–80 mL) of ultrasonic gel was administered in the rectum, except for patients with low or large rectal tumours. To reduce bowel peristalsis, 20 mg of raceanisodamine hydrochloride was injected intramuscularly approximately 10 min before the MRI. In addition, the patients were asked to urinate to prevent the effects of urine inside the bladder on DCE-MRI data postprocessing.Imaging was performed using a 3.0-Tesla MR scanner  with a 6-element body phased-array coil. The coil centre was placed at the level of the pubic symphysis and adjusted according to the tumour location. All of the patients were placed in the supine position with a feet-first orientation.The MRI protocols included the following: (a) sagittal, coronal and oblique axial high-resolution T2-weighted imaging (T2WI) using a turbo spin-echo sequence with the oblique axial plane perpendicular to the tumour basement and (b) oblique axial DCE-MRI using a three-dimensional dynamic T1-weighted sequence based on a time-resolved technique with interleaved stochastic trajectories (TWIST). After five precontrast phases, DCE-MRI was simultaneously initiated at the time of an intravenous bolus injection of 0.1 mmol/kg gadopentetate dimeglumine at a rate of 3.0 mL/s with a power injector, followed by a 25 mL saline flush. A total of 75 consecutive dynamic images with a temporal resolution of 4.25 s were acquired. In addition, a volume-interpolated body examination (VIBE) sequence with a dual-flip-angle was acquired to perform T1 mapping in an oblique axial plane before contrast agent administration. The detailed parameters of the protocols are listed in Table Two radiologists specialized in rectal MRI with 6 and 8 years of experience independently reviewed high-resolution T2WI for the presence of mrEMVI. Both radiologists were blinded to the DCE-MRI results and histopathological findings. Imaging findings suggestive of mrEMVI were serpiginous extension of tumour signal into extramural vessels contiguous with or separated from the primary rectal tumour that resulted in expansion and/or irregularity of these vessels , 18. DisKtrans in the VOI, an arterial input function (AIF) with the smallest chi-square value was selected. Third, regions of interest (ROIs) were manually contoured along the edges of the tumours on all slices containing tumour on DCE-MRI guided by corresponding oblique axial high-resolution T2WI. Lastly, the ROIs were copied to Ktrans map, and the mean quantitative perfusion parameters  were generated from a Tofts two-compartment model [At one-month intervals, DCE-MRI data were independently postprocessed by the same two radiologists  on an MRI workstation  using Tissue 4D software. First, the T1 mapping was registered to the motion-corrected DCE-MRI images. Second, a volume of interest (VOI) was manually drawn on DCE-MRI, and the VOI was kept away from bladder. To calculate the parametric map of nt model  , contrast-enhanced liver MRI or fludeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) were used for more precise evaluation. The distant metastatic lesions were confirmed by histopathology or by sequential 6-month radiologic follow-up.t-test, while non-normally distributed data were analysed using Mann-Whitney U test. Categorical data were compared with χ2 or Fisher’s exact tests. A two-sided P value < 0.05 was considered statistically significant. The above statistical analyses were performed using SPSS software (version 20.0).A Kolmogorov-Smirnov test was conducted to evaluate the data distribution. Normally distributed data were expressed as the means with standard deviations (SDs), while non-normally distributed data were reported as medians with ranges. Normally distributed data were analysed using independent samples Interobserver agreement for mrEMVI status evaluation between the two radiologists was analysed using Kappa statistics  with a 95% confidence interval (CI) [The median duration between MRI and curative resection was 5 days (range: 1–14 days). Table P = 0.011). Higher pathological tumour stage (T3–4) was significantly more frequent in the mrEMVI-positive group . mrEMVI-positive status was associated with increased regional nodal metastases when compared with mrEMVI-negative status .The tumour size was significantly larger in mrEMVI-positive group . Among 56 patients with at least 6-month follow-up after the initial diagnosis, distant metastases to liver (3 patients) and lung (1 patient) were noted at the initial diagnostic workup, and one patient newly developed distant lymph node metastases within the 6-month follow-up.There was no significant difference in tumour location, differentiation or synchronous distant metastasis between different mrEMVI statuses . However, the kep values in the mrEMVI-positive group (0.710 ± 0.337 min− 1) were significantly lower than those in the mrEMVI-negative group (0.938 ± 0.364 min− 1) (P = 0.012). The ve values in the mrEMVI-positive group (0.344 ± 0.101) were significantly higher than those in the mrEMVI-negative group (0.288 ± 0.094) (P = 0.021) .There were nine discrepant cases for mrEMVI status evaluation between the two radiologists. The interobserver agreement for mrEMVI status evaluation between the two radiologists was good . Figure Interobserver reproducibility was excellent for Ktrans between mrEMVI-negative and mrEMVI-positive patients, whereas the DCE-MRI parameters kep and ve were revealed to be associated with mrEMVI status. It is interesting to note that mrEMVI-positive patients with rectal adenocarcinoma had significantly lower kep and higher ve values than mrEMVI-negative patients.In the present study, we evaluated the association between mrEMVI status and tumour microcirculation in rectal adenocarcinoma. No significant difference was found in the DCE-MRI parameter kep values. The kep value equals the flux rate of contrast agent from the EES to plasma, which depends largely on capillary permeability and the permeability surface area [kep value in mrEMVI-positive patients. Clearly, a cluster of invasive tumour cells might cause decreased blood supply in the corresponding area due to a reduced permeability surface area, leading to an increased degree of tissue hypoxia and microscopic necrosis and, thus, an enlarged EES. The ve value represents the fractional volume of the EES, which is affected by the integrated effects of cell proliferation and necrosis [ve value was significantly higher in mrEMVI-positive patients. Yeo et al. [kep value. In addition, it has been shown that the loss of function of cell-cell adhesion molecules, a pivotal step in tumour progression, leads to a larger interstitial space, which is reflected by a higher ve value [It is now well established that angiogenesis is essential for tumour growth, invasion and lymphatic and haematogenous spread of tumour cells . Newly face area . Based onecrosis . Thus, io et al.  also demve value , 27.Ktrans value represents the transfer rate from plasma to the EES, which has a mixed microcirculatory blood flow and permeability weighting [Ktrans value in the mrEMVI-positive group, but the trend was nonsignificant. A possible explanation for this might be that the Ktrans value is also impacted by blood perfusion, including cardiac output, hypertension and the circulatory system of an individual, which thus produces greater individual variation than other quantitative parameters. By comparison, kep is only influenced by the contrast concentration and fractional volumes in the tumour EES and might more accurately mirror the capillary permeability [The eighting , 28. Alteighting . Theoreteability . The resKtrans, kep and ve values. This inconsistency may be because lymphovascular invasion may be a different entity from EMVI. In addition, quantitative perfusion parameters from DCE-MRI primarily characterize angiogenesis rather than lymphangiogenesis. Furthermore, tumour development is a complex and heterogeneous process [A recent study also showed that primary rectal cancer blood flow measured by DCE-MRI was significantly lower in patients with metastatic nodes, which also represents an unfavourable prognosis . However process . In addimrEMVI is more likely to occur with more locally advanced tumours in rectal cancer . Thus, iOur study is hindered by a few limitations. First, mrEMVI was not directly compared with histopathology results. However, the reported incidence of EMVI on histopathology is variable, impairing the prognostic predictive value of EMVI. Although tumour deposits within small vessels are still beyond the spatial resolution of current MRI techniques , patientkep and higher ve values, enabling DCE-MRI parameters to serve as an additional tool for potentially predicting prognosis for mrEMVI-positive patients with rectal adenocarcinoma. In addition, larger tumour size, higher pathological tumour stage and metastatic regional nodes were more common in mrEMVI-positive patients.This preliminary study suggests that the microcirculation of rectal adenocarcinoma in mrEMVI-positive patients is altered, with significantly lower


This paper aims to apply a proposed, based on calorimetric measurements, a reliable numerical model for magnetic fluid hyperthermia (MFH) treatment planning of breast cancer. Furthermore, we perform a comparative analysis of magnetic nanoparticles (MNPs) and tumour tissue interactions by means of the magnetic-field-dependent Néel and Brownian relaxation times. The analysis was based on an anatomically correct breast model (developed in-house) and a modified linear response theory, which was applied to investigate the heat dissipation from the magnetic nanoparticles dispersed in the breast tumour. The calculations of the single-domain magnetic power losses were conducted for a case where the magnetic field value and the applied frequency were known, but also for the different concentrations of the MNPs in the tumour. Two scenarios were considered: The MNPs mobilised and immobilised in the tumour. In parallel, the eddy currents effect, together with the related temperature distributions, were considered in order to analyse safety issues. By changing the MNP concentration in the tumour, the corresponding temperature distributions were calculated. The eddy current effect, together with the related temperature distribution, were considered in order to analyse safety issues. Varying the MNP concentration in the tumour, the corresponding temperature distribution was calculated. Moreover, the cumulative equivalent minutes at  Novel therapy methods without negative impacts are needed to reduce mortality rates in patients with breast cancer. One such method is magnetic fluid hyperthermia (MFH) in which magnetic nanoparticles (MNPs) dissipate heat during radiofrequency (RF) application ,2,3,4,5,TM system [Three dimensional (3-D) models were used in the past to investigate the effects of MNP volume fraction, nanocomposite geometry, and treatment parameters on thermal profiles regarding female breast cancer ,16,17. HM system ,20, i.e.It is clear that the reported SLPs values  do not rOur previous studies presented mathematical models, i.e., the single-domain magnetic power losses model and the eddy current model. Both of them were used later in the modified Pennes equation to evaluate a temperature distribution. The current work details the anatomically correct breast model used in the simulations and its parameters, followed by several analyses of volumetric power losses, which determine spatial temperature distribution as a function of time for various concentrations of magnetic fluid preparations. Furthermore, mobilised and immobilised MNPs in the tumour were considered.The mathematical model described in the previous section was used to investigate the temperature distribution during MFH. Different magnetic fluid concentrations were applied in the tumour to control the temperature in the target tissue, then the tumour position in the breast model was determined by maximum electrical loss. These power density values are referred to as “hot spots”. In addition to the power losses due to the eddy current effect, a power dissipation from magnetic nanoparticles should be considered in parallel when dealing with MFH treatment planning. Moreover, particular interest was paid to the comparative analysis of MNPs–tissue interactions, as in MFH, and the heating rates of MNPs which were determined using Néel and Brownian losses, which can be inappropriate for MFH treatment. For a reliable model of heating, two cases were considered, i.e., the MNPs free to move in the tumour (Néel and Brownian relaxations included in the tumour) and the MNPs immobilised in the tumour (in which only Néel relaxation takes place).f = 171 kHz based on the Information Technologies in Society (IT’IS) database [HTR =In our previous study, the breast model containing only skin, breast fat and tumour tissues was evaluated based on field application by a pancake coil . In the database . The parIn our previous work , the tisTM system. The location of the spherical tumour (2 mL volume) in the breast model was determined by the maximum volumetric eddy power density , i.e., to one of the “hot spots”, which were placed in the model excluding the skin layer to improve visualisation. It is important to note that the presence of “hot spots” should be considered during treatment planning as they are hard to predict and they can limit the amount of energy that can be deposited in a patient’s body. Moreover, one should remember that maximal electrical power losses will occur on the surface of the tumour as the eddy currents have to circulate around the tumour centre where they are the smallest. This sometimes can be used as an advantage because high-conductivity tumours surrounded by low-conductivity tissues will have a local eddy current flowing around the approximate centre of the tumour leading to increased heating of deep-seated tumours.The volumetric power density due to the eddy currents was evaluated for frequency In our case, to investigate the worst-case scenario, the tumour was positioned in one of the “hot spots”, and the power density was calculated see —right. OAs the first step, the single-domain magnetic power losses for both the mobilised and the immobilised MNPs were calculated. The ferrofluid concentration was the same as what is was during the calorimetry experiments  in In the case of the immobilised MNPs in the tumour, it can be seen that both the maximum power dissipation see , as wellAccording to the data in However, in order to reach f, the magnetisation lags behind the external field. According to linear response theory (LRT), the heat dissipation in W/kg can be expressed as the specific loss power (SLP) [Hmax), are given by [When polydisperse magnetic nanoparticles in the superparamagnetic regime are exposed to an AMF with the given magnetic field strength, er (SLP) :(1)SLPLRgiven by :(3)[τN and u) together with a magnetic vector potential (A), one can formulate the issue as:f is the angular frequency. Finally, The magnetic field strength ωtumoc is the tissue-specific heat, ω(T) is the perfusion rate, T is the local temperature, HGR is the metabolic heat generated rate and h is the heat transfer coefficient, and κ is the thermal conductivity of the skin layer.Hence, the temperature distribution in the breast model was investigated using the modified bio-heat transfer equation:The modified Pennes heat equation with Robin’s boundary condition to compute the temperature distribution within the realistic breast phantom was investigated. The single-domain magnetic and eddy current mediated power loss densities were applied as the parallel external heat sources. It was shown that due to the anatomical structure of the breast tissues, the “hot spots” could occur together with high-temperature gradients. That is why this phenomenon should be considered during treatment planning. More importantly, the distribution of the magnetic power losses along with the temperature was not homogeneous in the cancer area. It was also demonstrated that the viscosity played a crucial role, and the magnetic fluid concentration was critical for effective MFH treatment. The behaviour of MNPs within the tumour volume was also analysed as their lack of mobilisation leads to lower power dissipation. Consequently, it is vital to know if the MNPs are free to move in the interstitial fluid or whether they are immobilised in the tumour tissue. Thus, magnetic nanoparticles–tissue interactions hold the equal potential to affect treatment. To conclude, from the MFH-treatment planning point of view, it is crucial to prevent healthy surrounding tissues from overheating, so particular attention should be given to the temperature distribution in the tumour vicinity. The accuracy of magnetic fluid thermal therapy determines its role in prospective treatment planning for the individual patient because inadequate thermal doses to the tumour can cause a failure of treatment. Hence, the proposed numerical model will help to calibrate magnetic fluid temperature measurements when exposed to radiofrequency, and realistic models like the one presented here will enable in silico evaluation of MFH efficiency prior to clinical treatment.


We find that NaCl significantly decreases the dihedral angle to below 60° in all investigated conditions at concentrations above 5 wt% and, importantly, even at 1 wt% at 2 GPa. Our results show that slab-released fluid forms an interconnected network at relatively shallow depths of ~80 km and can partly reach the fore-arc crust without causing wet-melting and serpentinization of the mantle. Fluid transport through this permeable window of mantle wedge accounts for the location of the high electrical conductivity anomalies detected in fore-arc regions.Recently, high electrical conductors have been detected beneath some fore-arcs and are believed to store voluminous slab-derived fluids. This implies that the for-arc mantle wedge is permeable for aqueous fluids. Here, we precisely determine the dihedral (wetting) angle in an olivine–NaCl–H 2O and olivine-H2O-NaCl systems. The observed effect of NaCl to decrease dihedral angles allows fluids to percolate through forearc mantle wedge and to accumulate in the overlying crust, accounting for the high electrical conductivity anomalies in forearc regions.The authors here perform experiments to investigate the dihedral angle of olivine-H Water bounded in the hydrous minerals in the altered subducting slab is continuously released into the overriding mantle wedge via metamorphic dehydration reactions with increasing pressure and temperature during subduction7. The released aqueous fluid can control the partial melting of the mantle wedge because the presence of aqueous fluid effectively decreases the peridotite solidus temperature, which leads to the formation of arc magma11. The slab-derived water involved in the arc magma returns to the earth’s surface via volcanic emission, which is assumed to regulate water cycling in the subduction zone12.An understanding of fluid circulation in subduction zones is crucial for determining the origin of arc volcanism and to constrain global material recycling12 calculated the global water mass balance constrained by a phanerozoic sea level decrease of 0–360 m and obtained a global water input flux of 1.03–1.17 × 1012 kg/yr at the trench. They considered that the output flux in subduction zones is limited through arc/back-arc hydrous magma production. Recently, Faccenda13 proposed a higher input flux of 1.19–2.0 × 1012 kg/yr based on the geophysical constraints on the extent of hydration in the oceanic lithosphere, which suggests the missing output fluid flux in subduction zones to balance the larger amount of water subducted. Although they invoked serpentinized forearc mantle as the water reservoir, analysis of global seismic data revealed that extensive forearc serpentinization is limited to the hottest subduction zone, such as Cascadia14. Recent magnetotelluric studies have found moderate-to-high electrical conductivity anomalies at the lower crust to uppermost mantle depths at a nearly constant distance of 20–40 km seaward from the arc volcanoes in the fore-arcs of the Cascadia16, Mexico17, Costa Rica18, North Chile21, North Honshu22, Southern Kyushu23, and Ryukyu–Philippine subduction zones24. Because aqueous fluid effectively increases the bulk electrical conductivity by up to several orders of magnitude25, these anomalies are interpreted to be reservoirs of aqueous fluid supplied from the subducting slab without causing mantle melting and thus may account for some of the missing output flux in subduction zones. This interpretation requires the presence of a permeable mantle wedge at a relatively shallow depth of ~80 km to enable upward fluid migration.Water mass balance and geophysical observations, however, have indicated that a water output flux other than volcanic emission should exist in subduction zones. Parai and Mukhopadhydayθ), which is defined by two intersecting interfaces of a pore at a junction with two solid grains. In the equilibrium state, θ can be expressed as a function of interfacial energy as follows:γss is the solid–solid grain boundary energy per unit area, and γsf is the solid–fluid interfacial energy per unit area28. In the case of a low fluid fraction, as expected in a deep mantle wedge setting29, the intergranular fluid forms an interconnected network if θ < 60°, whereas the fluid is isolated at grain corners and edges if θ > 60°27. The critical θ may increase at high fluid fractions, which might occur in local areas such as shear zones. Nevertheless, the critical angle of 60° can provide a fundamental constraint on the dominant regime of fluid transport in the deep mantle wedge28. Mibe et al.30 examined the pressure and temperature dependence of θ in an olivine–H2O system to constrain the fluid migration in the mantle wedge. They showed that θ is >60° at forearc depths of 80–105 km; thus, the slab-derived fluid is transported downward within the down-dragged mantle matrix at the base of the mantle wedge. After reaching a depth of ~105 km, where θ becomes <60°, the fluid can be released into the hot mantle wedge through the interconnected network to cause hydrous melting just beneath the arc volcano.At high pressure and temperature conditions, fluid migration is controlled by the dihedral (wetting) angle 35. These salinities, particularly those of >5 wt% NaCl equivalent, could be influenced to some degree by the chromatographic fractionation through the hydration reaction in the mantle wedge after the fluid is released from the subducting slab38. The original salinity of slab-derived fluid equilibrated with dehydrated subducting rocks is thought to be smaller than those values (0.5–2.0 wt% NaCl39). Therefore, the influence of NaCl on θ between olivine and aqueous fluid should be clarified in a wide range of NaCl concentrations to correctly understand fluid circulation in the mantle wedge. Although Watson and Brenan28 investigated θ between olivine and aqueous fluid with 27.5 wt% NaCl at 1 GPa and 1000 °C, further systematic studies are required to reveal the pressure and temperature dependence of θ in a wide range of NaCl concentrations under mantle wedge conditions.Although many previous experiments assumed that slab-derived fluid is composed of pure Hθ in both olivine–H2O and olivine–H2O–NaCl systems at 1–4 GPa and 800–1100 °C by using a piston cylinder apparatus. The NaCl concentration in aqueous fluid ranged from 1.0 to 27.5 wt% in our experimental systems. On the basis of our data, we clarify the grain-scale fluid connectivity and fluid pathway for forming the electrical conductivity anomalies at lower-crust and uppermost mantle depths at forearc regions.In this study, we constrain the effect of NaCl on the fluid connectivity in the mantle wedge by experimentally determining 40, whereas the flat interfaces were affected by crystallographically controlled minimum interfacial energy41. We measured only olivine–fluid–olivine θ defined by the curved–curved interfaces to focus on the angle not affected by the facetted planes. Additional examples showing our selection and measurement processes of θ are given in Supplementary Figs. θ42. Although a true θ generally occurs in a range owing to the surface energy anisotropies of minerals, the median value can represent the most frequent true θ in the system. Details on the experimental conditions and results as well as frequency distribution histograms of the measured apparent θ are given in Supplementary Table The run products were composed of sintered aggregates of olivine and interstitial pores with no other solid phases Fig. . The graθ in both H2O and H2O–NaCl (27.5 wt%) systems along with the median angles are shown in Fig. 2O–NaCl systems with 1.0, 3.0, 5.0, 10.0, and 15.0 wt% NaCl are shown in Supplementary Fig. θ, which is consistent with the theoretical prediction for an isotropic system with one true θ. However, in some experiments, the data largely deviated from the theoretical curve at the region of high apparent θ. This discrepancy can be explained by the presence of a few cases of very large θ associated possibly with low-angle grain boundaries, where the misorientation between the two adjacent grains is very small43. Even in this case, however, the median angle is assumed to represent the system because the distribution of the true θ is concentrated mostly at the median angle.The cumulative frequencies of the measured apparent θ in both H2O and H2O–NaCl (27.5 wt% NaCl) systems is shown in Fig. θ decreased with increases in pressure and temperature in both systems. Moreover, θ in the H2O–NaCl system was always <60°, and was also smaller than that in the H2O system at the same pressure and temperature conditions. The θ in the H2O system, however, was <60° only at pressures and temperatures above 2 GPa and 1000 °C, respectively. The difference in θ between these two systems was up to 13° at 1 GPa and 800 °C, but gradually decreased with increases in pressure and temperature, mostly diminishing above 3 GPa and 1000 °C, respectively.The pressure and temperature dependence of the median θ in the olivine–H2O–NaCl system at temperatures of 800 and 1000 °C under pressures of 1–3 GPa are shown in Fig. θ showed a nonlinear dependency on the NaCl concentration in aqueous fluid: The angle steeply deceased at low concentrations and then gradually increased with increasing NaCl concentration. At 2 GPa and 800 °C, for example, θ suddenly dropped from 69.9° to 58.7° by adding 1 wt% NaCl to the pure H2O fluid and continued to decrease down to 51.5° with increasing NaCl concentration up to 10 wt%. Further addition of NaCl, in contrast, slightly increased the angle up to 56.4° at 27.5 wt% NaCl. It is worth noting that these angles are still clearly smaller than those in the pure H2O system  at 3 wt% NaCl at 1 GPa became <60° at pressures above 2 GPa. With an increase in temperature from 800 °C to 1000 °C at 2 GPa, the angles became smaller at all of the experimental concentrations; the extent of the reduction was again larger at lower concentrations  as that obtained at Bayreuth University (Run CDM-04), at θ = 55.8° and 56.1°, respectively. Because the number of measured angles in this study, at 208, is significantly larger than that in Watson and Brenan28, at 100 ± 15, we are convinced that our precise measurements are more reliable than those in the previous study.The median angle in the Hθ with the addition of NaCl is indicative of reduced interfacial energy between the olivine and aqueous fluid in the H2O–NaCl system under the assumption that the grain boundary energy between two olivine grains is constant. Takei and Shimizu46 theoretically demonstrated that the interfacial energy between the solid and fluid phases decreases with an increase in solubility of the solid component into the fluid phase in binary eutectic systems, such as silicate+ fluid, binary alloys, and binary organic systems. In an olivine–H2O system, the experiments conducted by Yoshino et al.47 showed that θ decreased down to 0° with an increase in pressure up to ~7 GPa owing to the enhanced solubility of olivine into the aqueous fluid. Because NaCl is known to increase the forsterite solubility into the aqueous fluid at 1 GPa48, we consider that high olivine solubility effectively reduces the interfacial energy and thus θ between olivine and aqueous fluid in an H2O–NaCl system. As shown in Fig. θ between the H2O and H2O–NaCl systems at pressures and temperatures >3 GPa and 1000 °C, respectively. This fact suggests that the NaCl effect on olivine solubility became less remarkable at such high pressure and temperature conditions owing possibly to the elevated solubility of olivine in the pure H2O system. At low NaCl concentrations, θ significantly decreased, reached its minimum values at 5–10 wt%, and then gradually increased. This is consistent with the data showing that olivine solubility steeply increases at low NaCl concentrations, and is suppressed at very high concentrations48. The interfacial energy between minerals and fluids decreases by positive adsorption of the solute on the mineral surface49. This could also contribute to the nonlinear dependence of θ on the NaCl concentration . Details on the isopleths of θ other than 60° are provided in Supplementary Figs. 51 and van Keken et al.4), where high electrical conductivity has been detected in deep forearc regions of Costa Rica18, North Chile21, North Honshu22, Southern Kyushu23, and Ryukyu-Philippines24. In these thermal models, rheological coupling between the subducting slabs and hot mantle wedges is assumed to occur at a nearly constant depth of 80 km, which leads to dramatic changes in the thermal gradient around the coupling depth. The intersection depth between the 60° isopleth and the pressure and temperature paths of the basal layer of the down-dragged mantle wedges in the H2O–NaCl system (~80 km) is obviously much shallower than that in the H2O system (>100 km) even at a low NaCl concentration of 1.0 wt%  owing to the steep thermal gradient at the coupling depth of 80 km, which is inconsistent with the depth of the volcanic front at ~100 km. Strikingly, the depth of the intersection is strongly consistent with the position of the high electrical conductor in the deep forearc crust. The coupling depth is constrained within a narrow range of 70 to 80 km by observations of surface heat flow and seismic attenuation in the forearc region57. Therefore, the relationship between the coupling depth  and the slab-surface depths beneath the forearc electrical conductors is robust.To infer the consequences of shallow infiltration of NaCl-bearing aqueous fluid, the slab-surface depths beneath the forearc electrical conductors and the arc volcanic fronts are shown in Fig. 60. Although partial melting and interconnection of conductive minerals are also invoked to explain the high conductivity64, the thermal gradient in the forearc region is too low to cause melting, and the volume of such conductive minerals is generally too small to form interconnected network. Recent high-resolution magnetotelluric studies have found a continuous and moderately conductive anomaly in the mantle wedge rooted from the top of the subducting slab to the forearc crust65. These observations can support the steady-state fluid transport via intergranular fluid networks in the mantle wedge with a caveat of a smoothing artifact. While such a continuous conductive anomaly was not always observed in other studies, the interconnection at small fluid fraction in the mantle wedge is still not denied due to the fluid fraction dependence of the electrical conductivity25 and the electrical static effect of the shallow high electrical conductor on electrical amplitude response of deep electrical field66. Electrical conductivity in the deep forearc crust could be enhanced by the high fluid fraction due to the fluid accumulation over the geological time scale and the high fluid salinity resulted from the hydration of the mantle wedge during fluid  transport.The above relationship can be explained if we assume that the NaCl-bearing fluid released at the beginning of the dehydration reaction in subducting slab percolates upward through the interconnected network in the mantle wedge and accumulates at crustal depths as a fluid reservoir Figs. , 7, beca11 conducted melting experiments in a hydrous fertile peridotite system, and proposed that the incipient melting of mantle wedge occurs at 890–870 °C in the chlorite-bearing water-undersaturated system and 850–820 °C in the water-saturated system at pressures of 2–3 GPa. However, these solidi were questioned by the recent research which has found that solidus of water-saturated fertile peridotite is ~1000 °C at 1–3 GPa67. Although no precise experimental data are available for depleted peridotite, which is more applicable to common mantle wedges, it should be higher than that for the fertile peridotite. At lower temperatures, however, serpentine is formed by water infiltration; thus, the slab fluid may be sequestered until the peridotites are dominantly transformed to serpentinites54. It should be emphasized that the temperature gap exists between the hydrous peridotite solidus and serpentine stability, which serves as a window for slab-derived fluid to percolate completely through the mantle wedge and accumulate to form the fluid reservoir near the Moho  angle (θ) between olivine and aqueous fluid. The starting material was San Carlos olivine (Fo91Fa9) handpicked from a gently crushed San Carlos lherzolite xenolith. These grains were finely ground with a tungsten carbide mortar, and were sieved to obtain size fractions of 38–53 µm. Impurities included in the powder were carefully removed under an optical microscope. To prevent absorption of moisture, the olivine powder was placed in an oven, and was held at 100 °C for one night prior to the experiment. Deionized and distilled water was used for the source of pure H2O. We dissolved reagent-grade NaCl (99.99% NaCl) into the deionized and distilled water at room temperature (~25 °C) and atmospheric pressure to obtain solutions with various NaCl concentrations of 1.0, 3.0, 5.0, 10.0, 15.0, and 27.5 wt% NaCl.Annealing experiments were conducted in olivine–H72. The olivine powder along with ~3–5 wt% fluid was loaded in an end-welded noble metal capsule that was sealed by arc welding. Capsules constructed of Au and Au80Pd20 alloy with outer diameters of 2.2 mm and 2.0 mm, respectively, were used for the experiments at temperatures of 800–1000 °C and 1100 °C, respectively. The cells were first pressurized to 90% of the target pressure; the remaining 10% pressure was applied after heating to the target temperature. During the experiments, the temperature was monitored and controlled by using an S-type thermocouple (Pt–Pt90Rh10). The run durations varied from 72 to 210 h, depending on the target temperature. Quenching to room temperature was completed within 1 min, and the pressure was slowly released until atmospheric pressure was reached.End-loaded 3/4- and 1/2-inch piston cylinder apparatuses at Bayerisches Geoinstitut, University of Bayreuth, were utilized for the experiments at pressures of 1–2 GPa and 3–4 GPa, respectively, with a standard talc–Pyrex assembly2O–NaCl system for 120 h by using the end-loaded 3/4-inch piston cylinder apparatus at Tohoku University. A conventional NaCl–Pyrex pressure assembly was utilized for the experiment. We used the same starting materials as those used in the experiments at Bayerisches Geoinstitut. The starting materials were packed into an Au-lined machinable ceramics cylinder , which was also used in the experiments by Jégo et al.73. A thin Au disk was then placed on top of the Au capsule, and the Au capsule inside the cylinder was shut following the initial pressurization of the run. The temperature gradient along the capsule was estimated to be <10 °C at 1200 °C, according to Nakamura and Watson74. The detailed procedures for these high-pressure experiments are similar to those described in Ohuchi and Nakamura75.To check the reproducibility of our measurement, we conducted one annealing experiment at 1 GPa and 1000 °C in the Hθ measurements were performed by using Image-J software for >200 angles on the acquired SE images for each sample. Errors of each measurement were <3°. The details for discussing error are provided in Supplementary Note θ was estimated by calculating the median of the distribution of apparent angles76. This number of measurements was necessary to obtain a steady median value, particularly when the sample incurred severe plucking, which can locally reduce the randomness of the apparent angle distribution. Additional examples showing the selection and measurement of θ are given in Supplementary Note The recovered capsules were cut with a wire saw to expose the run products. The run products were then impregnated with epoxy under a vacuum and were polished down to 1.0 μm with alumina powder and subsequently to 0.06 μm by colloidal silica suspension. The polished sections of the run products were observed by using a field emission-type scanning electron microscope. More than 200 backscattered electron (BSE) or secondary electron (SE) images of 1280 × 960 pixels were obtained for each run product at magnifications of × 3000 to × 150,000, depending on the pore size. The Supplementary InformationPeer Review File