However, the clinical significance of these genetic aberrancies in PTCs arising in ectopic locations is not well established.Mutations of the v-Raf murine sarcoma viral oncogene homolog B We describe a patient with a previous history of radioiodine (RAI)-treated hyperthyroidism and a surgically resected right-sided follicular thyroid adenoma. In 2013, a 6 mm follicular variant papillary thyroid carcinoma was diagnosed following a left-sided thyroid lobectomy. The central compartment displayed 9 tumor-free lymph nodes, and no adjuvant treatment was planned.BRAF mutation and a C228T TERT promoter mutation.Three years later, a 26 mm pre-tracheal relapse was noted, however, the excised lesion was consistent with a tall cell variant of papillary thyroid carcinoma (TCV-PTC) arising in ectopic thyroid tissue. RAI treatment was commenced. Four years later, a 5 mm subcutaneous lesion in the anterior neck was surgically removed and diagnosed as metastatic TCV-PTC with a codon 600 BRAF mutation and a TERT promoter wildtype sequence, while the primary TCV-PTC was positive for mutations in both codon 600 of BRAF as well as the TERT promoter.RAI treatment was re-initiated. Molecular re-examination of the primary follicular variant papillary thyroid carcinoma demonstrated a codon 600 The patient is alive and well without signs of relapse 7 months after the latest round of RAI.BRAF and TERT promoter mutations in the primary lesion from 2016 was associated to the manifestation of distant metastases 4 years later, strengthening the benefit of mutational screening of these genes in clinical routine for thyroid carcinomas arising in aberrant locations.We conclude that the occurrence of combined  As a consequence, treatment options and follow-up schemes for these lesions probably vary across different institutions. The most common sites of ectopic cervical thyroid tissue are lingual, sublingual, thyroglossal, laryngo-tracheal and lateral cervical compartments, and believed to be a result of aberrant embryonic migration from the pharyngeal pouches.,4Ectopic cervical thyroid tissue is an uncommon finding in the clinical setting with an estimated prevalence of 1:10,000 individuals.,5 Interestingly, different ectopic locations seem to confer a predilection for certain tumor types; for example, lingual thyroid tissue most often harbor follicular thyroid carcinomas, whereas thyroid carcinoma arising in a thyroglossal duct cyst most often are PTCs, and rarely squamous cell carcinoma or anaplastic thyroid carcinoma.Although rare, the possibility of tumor development in an ectopic location is frequently discussed in the clinical setting, as the distinction between a primary thyroid carcinoma arising in ectopic thyroid tissue and a cervical lymph node metastasis is not always easily determined. This is especially true in the preoperative setting using fine-needle aspiration biopsy (FNAB) examination of lesions separated from the thyroid gland, when the thyroid itself is free of visible tumor masses from a radiological perspective. Although rare, whenever a thyroid tumor develop in ectopic thyroid tissue, findings of papillary thyroid carcinomas (PTCs) dominate, though follicular thyroid carcinomas and occasional reports of medullary thyroid carcinoma and poorly differentiated thyroid carcinoma also exist.,6 While surgical removal seems to be the most appropriate treatment option for ectopic primary thyroid cancer of the cervical compartment, the beneficial use of postoperative radioiodine (RAI) is not established. Moreover, little is known regarding the risk of metastatic spread and what factors that predict aggressive biological features.Thyroid carcinomas arising in ectopic thyroid tissue cannot be risk stratified as conventional tumors, and predictive factors such as extrathyroidal extension and the pathological tumor node and metastasis (TNM) staging are redundant for these cases. Therefore, practicing clinicians are recommended to make assessment based on individual risk stratifications.BRAF) and telomerase reverse transcriptase (TERT) promoter analyses of PTCs arising in ectopic thyroid tissue might be a beneficial strategy to pinpoint cases at risk for recurrences similar to the established risk stratification used for tumors arising within the thyroid gland itself.In this report, we describe the clinical, histological, and molecular features of a PTC arising in pre-laryngeal, ectopic thyroid tissue, and the subsequent development of subcutaneous metastatic deposits. More specifically, we highlight how v-Raf murine sarcoma viral oncogene homolog B , paired box gene 8, and thyroglobulin. The diagnosis was a PTC (Bethesda VI). FNAB of the 10 mm lymph node in level 2 could not pinpoint metastatic disease.BRAF, and the Ki-67 proliferation index was 1%. For the former staining, we employed the Ventana anti-BRAF V600E mouse monoclonal antibody (clone VE1) that specifically targets the V600E mutation, and thus fails to recognize wildtype protein at this codon. To obtain a Ki-67 index, we count at least 2000 tumor cells in hot spot regions (defined as areas with the highest proportion of Ki-67 positive cells), dividing the number of positive nuclei with the total amount of tumor nuclei manually using an ocular with a counting grid. Nine tumor-free lymph nodes were found in the central compartment. The pTNM was pT1aN0, and the patient was discharged from further surveillance without adjuvant RAI treatment.The patient underwent left-sided hemithyroidectomy and central neck dissection 2 weeks later. The thyroid lobe weighted 1.6 g. and measured 28 × 18 × 9 mm. By gross pathology investigation, a 6 mm large lesion was evident in the central aspects of the lobe and submitted entirely for microscopy. The histopathological investigation displayed a poorly circumscribed, follicular-patterned tumor growing infiltrative in the surrounding thyroid parenchyma. Tumor nuclei were enlarged and displayed crowding, nuclear grooves, loose chromatin, and occasional pseudo-inclusion bodies . The pre-laryngeal lesion was resected shortly thereafter. The specimen displayed a weight of 3 g, measured 26 × 16 × 13 mm, and the lesion was submitted entirely for microscopy. The ensuing investigation displayed a well-demarcated tumor with a papillary growth pattern against a background of a sclerotic stroma . This assay screens for NRAS proto-oncogene GTPase (NRAS) gene mutations in exon 2, 3, and 4 as well as mutations in exon 15 of the BRAF gene  using a real-time PCR based methodology. The methodology regarding Sanger sequencing of the TERT promoter has been previously described in detail. The 6 mm FV-PTC diagnosed in the left thyroid lobe was found to display a codon 600 BRAF mutation but negative for TERT promoter mutations as well as hotspot NRAS mutation of exons 2, 3, and 4. Both the pre-laryngeal TCV-PTC and the ensuing subcutaneous metastasis were positive for mutations in both BRAF codon 600 and the C228T TERT promoter mutation. Moreover, both these lesions were NRAS wildtype . For the The study was approved by the Swedish Ethical Review Authority , and informed consent was obtained from the patient.3BRAF and the TERT promoter.–12 Mutations in these genes are known to collectively highlight PTC specimen at risk of future recurrences. Indeed, in our case, the detection of synchronous manifestation of mutations in these genes was associated to the occurrence of distant metastases 4 years later.In this report, we provide evidence that ectopically derived PTC could harbor established mutations in genes characteristically associated to worse clinical outcome, namely  Moreover, the possibility that the tumor developed within a thyroglossal duct cyst was excluded, as the lesion was void of cystic features and unassociated to the hyoid bone.The development of a carcinoma in ectopic thyroid tissue mandates several clinically relevant considerations, not least to pinpoint whether the tumor developed de novo or in fact constituted a metastasis from a primary tumor within the thyroid gland itself. In our case, the detection of a histologically normal thyroid remnant at the periphery of the excised lesion strongly argued in favor for the former rather than the latter assumption Fig. . The queRAS-driven tumors, while TCV-PTCs are BRAF-driven cancers with poor prognosis, and therefore no apparent genetic association exist – even though the FV-PTC in our case was BRAF mutated, which is unusual but not entirely uncommon.The patient was diagnosed with a follicular thyroid adenoma 30 years ago, first by cytology using a fine-needle aspiration biopsy and subsequently also by histopathology following a hemithyroidectomy. Unfortunately, as this surgery was performed in another hospital, we were unable to retrieve these slides for review purposes. As of this, the chance of this tumor actually being a misdiagnosed FV-PTC was considered. Even so, a rendered FV-PTC diagnosis should have little impact upon the conclusions of this study - as the metastatic-prone PTC in our case was a TCV-PTC without the morphological characteristics of an FV-PTC. Moreover, we do not believe that the occurrence of an FV-PTC in the thyroid gland and a separate TCV-PTC in ectopic thyroid tissue would indicate a genetic association, but rather a coincidence given the high incidence of PTC in the general population. However, we cannot fully exclude environmental factors as contributors of the disease presentation. In general, FV-PTCs are clinically indolent ,15 As the practicing clinicians cannot rely on standard prognostic parameters such as tumor size and the TNM staging when assessing thyroid carcinomas arising in an aberrant location, the detection of genetic aberrancies with a known coupling to worse outcome might be useful in determining the future surveillance and/or adjuvant treatment options for these rare patients. In fact, genomic testing as a diagnostic and predictive marker in advanced well-differentiated thyroid carcinomas is nowadays recommended for subsets of cases according to The National Comprehensive Cancer Network guidelines. The effect of BRAF and TERT promoter mutations in PTC is well established, with a strong coupling to high-risk clinico-pathological variables such as extrathyroidal extension, TNM stage, lymph node, and distant metastases in addition to worse overall survival in mutated cases.,11 While the V600E BRAF mutation stimulates the mitogen-activated protein kinase signaling cascade and results in increased tumor growth and oncogenic transformation, the C228T and C250T mutations of the TERT promoter stimulates TERT gene expression, thereby presumably immortalizing the cells through the telomerase driven effects on telomere length.,18 Of particular interest, the coexistent occurrence of BRAF and TERT promoter mutations in PTCs seem to exhibit synergistic effects on clinical outcomes.,11 Interestingly, both V600E BRAF mutations as well as TERT promoter mutations are overrepresented in TCV-PTCs compared to many other variants of PTCs, including classical variant PTCs, possibly providing a molecular explanation for the overall poorer prognosis in TCV-PTCs.,19 As both mutations are tightly coupled to patient outcome, sequencing of these variants are often used in single-gene, clinical routine screening programs as well as in comprehensive gene panels for both clinical and research purposes.,20–22 The single-gene analyses are cheap, reproducible, and easily interpretable, providing a possibility to screen thyroid tumors even in laboratories lacking state-of-the-art next generation sequencing equipment. The finding of both a codon 600 BRAF and a C228T TERT promoter mutation in the TCV-PTC arising ectopically in our case mirrors previous observations in high-risk cases of thyroid tumors arising within the thyroid gland, as our patient developed distant metastases 4 years after surgical removal. Therefore, if these genetic events would have been known to the clinician at the time of the surgical excision, adjustments to the administered postoperative RAI dose and/or follow-up scheme would possibly have been adequate – even though our patient exhibited a risk profile irrespectively of molecular data given her age (>55 years at the time of diagnosis) and the occurrence of a histological PTC subtype frequently associated to worse clinical outcome.Previous literature suggest that thyroid carcinomas occurring in ectopic cervical locations not seldom give rise to lymph node metastases, thereby potentially arguing that the malignant potential of these lesions demand careful risk assessments.NRAS-BRAF Assay, although highly sensitive and specific, do not discriminate between BRAF V600E and V600D mutations. Thus we cannot with certainty state that the BRAF mutations found herein were identical. However, in PTCs, the V600E mutation is by far the most common mutation type – and as the BRAF V600E mutation specific immunohistochemistry was also clearly positive for all described tumors, the chance of these BRAF codon V600 mutations being disparate is minimal. Another drawback to our study design is the lack of TERT gene expressional data in the TERT promoter mutated tumors, which was due to the lack of fresh frozen material for mRNA analyses. Although antibodies are available for the immunohistochemical detection of the TERT protein, our previous experiences suggest that the latter method is not yet a reliable tool, at least not for follicular thyroid tumors.A limitation to the current study is the fact that the Idylla BRAF mutation and TERT promoter mutations has been found to predict worse outcome in PTCs in general, these molecular aberrancies could probably be of use to predict the risk of distant metastases also in PTCs arising in aberrant locations. Although these initial observations probably need to be reproduced in larger case series, the inclusion of BRAF and TERT promoter sequencing when assessing thyroid cancer from ectopic thyroid tissue might be a beneficial strategy – not least as the current recommendations are based on individualized risk assessments.We conclude that this TCV-PTC arising in ectopic laryngotracheal thyroid tissue did exhibit similar high-risk molecular features as counterpart tumors primary within the gland itself. As the combination of the codon V600 The authors are indebted to the professional technical service of the clincial pathology team at our department, especially Dr. Kenbugul Jatta, MSc, PhD and Mr. Robin Brolin, MSc, both who have agreed upon being named in this section of the manuscript.Conceptualization: Catharina Ihre-Lundgren, Carl Christofer Juhlin.Data curation: Adam Stenman, Anna Koman, Carl Christofer Juhlin.Funding acquisition: Carl Christofer Juhlin.Investigation: Adam Stenman, Anna Koman, Catharina Ihre-Lundgren, Carl Christofer Juhlin.Writing – original draft: Carl Christofer Juhlin.Writing – review & editing: Adam Stenman, Anna Koman, Catharina Ihre-Lundgren, Carl Christofer Juhlin.


Fluorescence polarization microscopy (FPM) analyzes both intensity and orientation of fluorescence dipole, and reflects the structural specificity of target molecules. It has become an important tool for studying protein organization, orientational order, and structural changes in cells. However, suffering from optical diffraction limit, conventional FPM has low orientation resolution and observation accuracy, as the polarization information is averaged by multiple fluorescent molecules within a diffraction-limited volume. Recently, novel super-resolution FPMs have been developed to break the diffraction barrier. In this review, we will introduce the recent progress to achieve sub-diffraction determination of dipole orientation. Biological applications, based on polarization analysis of fluorescence dipole, are also summarized, with focus on chromophore-target molecule interaction and molecular organization. For example, the orientation of protein structure determines the dipole orientation of the fluorophore molecule if the linker between the fluorescent molecule and target protein is rigid enough. Therefore, the spatial direction of the target protein can be reflected by the dipole orientation of fluorescent molecules.Utilizing the polarization and intensity characteristics of fluorescence molecules, fluorescence polarization microscopy (FPM), also called label-based polarization microscopy, can simultaneously measure dipole intensity and orientation information to study the underlying biological structure organization. For example, FPM is used to monitor the dynamic activation process of membrane proteins Because of the optical diffraction limit, conventional FPM technologies resolve the dipole orientation averaged by multiple fluorescent molecules within diffraction-limited volume. Enabling high-resolution polarization analysis has gradually attracted more and more attention in recent years and some strategies have been developed, such as combining traditional FPM technology with super-resolution microscopy In this paper, the optical setups of FPM based on polarization detection (PD) and polarization excitation (PE) are reviewed. The super-resolution FPMs, including polarization single-molecule localization, polarization demodulation and polarized structured illumination, are introduced thereafter. The applications of super-resolution FPM in life sciences, especially about chromophore-target molecule interaction and molecular organization, are summarized.2The core principle of FPM is to extract dipole moment orientation and higher resolution orientation accesses to more detailed observation. Three issues, containing description of dipole orientation, optical setups of FPM and super-resolution FPM, are exhibited in this section.2.1A fluorescent molecule is constituted by atoms, whose spatial arrangement interacts with light and accounts for a defined transition dipole moment. The specific transition dipole moment interacts with target proteins and is an important indicator of potential subcellular organelle organization. The dipole moment orientation is parameterized by azimuth e system A. In twoe signal B [23]. F2) of the angle between the direction of the polarized excitation light and the direction of the absorption dipole. The electromagnetic radiation from a molecular chromophore is proportional to sine-squared (sin2) of the intersecting angle between emission dipole orientation and direction of observation. Mostly the absorption dipole moment orientation and emission dipole moment orientation of fluorophores can be regarded as parallel The absorption probability of the dipole is proportional to cosine-squared , defined as OUF = A/B, is purposed to describe the local dipole orientation distribution and wobbling. OUF is represented by the length of stick or double-sided arrows, whose direction corresponds to dipole orientation. OUF is equivalent and proportional to polarization factor (p) 2.2Both absorption and emission dipole moment orientation are anisotropic. Polarization detection could be used to analyze the emission dipole while polarization excitation to resolve absorption dipole. Combinations of polarization excitation and polarization emission are required to simultaneously analyze both dipole orientations.The optical setup of wide-field fluorescence microscopy is presented in In this review, all optical setups of FPM are categorized into four strategies G-1 J. InBased on polarization detection scheme, classical FPM has been developed to measure polarization anisotropy 2.3Super-resolution microscopy is achieved by three typical methods, stimulated emission depletion (STED) Traditional FPM, such as LC-PolScope which uses liquid crystal modulation device to achieve polarization excitation Instantaneous FluoPolScope, based on polarization detection setup and single molecule tracking, achieves instantaneous tracking direction and position of fluorophores in live cells In conventional SIM setup Since the fluorescent molecules has polarization dependent response to the excitation beam, Hafi et al. developed SPoD based on polarization excitation setup and sparsity penalty-enhanced estimation by demodulation (SPEED) algorithm A comparison of different super-resolution FPMs is carried out in 3FPM detects both fluorescence and orientation information, which is essential for a multitude of biological observations and further understanding of cellular functions and processes. Super-resolution FPM is developed recently to bring the conventional diffraction limited FPM to a whole new scale, so that the ensemble dipole number can be reduced, and the orientation can be detected at a higher precision. Two categories of biological applications are demonstrated here: chromophore-target molecule interaction, and molecular organization.3.1In life sciences, fluorescent protein and chemical dye are the most commonly used chromophores. FPM imaging can reveal the polarization interaction between the chromophore and target molecule.S. cerevisiae), dipole orientation of Cdc12-conGFP4 construct orients perpendicular to septin filament while Cdc12-conGFP3 orients parallel to the filament (To create a rigid linker between green fluorescence protein (GFP) and septin, one part of the α-helix from N terminus of truncated GFP can be fused to another part of the α-helix from C terminus truncated CDC3, to form a complete α-helix with enough rigidity for PFM imaging. In the FPM imaging of budding yeast  and single particle dynamics, where FPM can provide a better insight view.Via fluorescence anisotropy analysis, FPM unveils a relatively parallel arrangement of human nucleoporin Nup133–Nup107 and yeast nucleoporin Nic96 to the nuclear envelope plane. This observation supports the “head-to-tail ring” model that Y-shaped subcomplexes form an octameric ring through interaction between the end and the short arms of subcomplexes A [10].FiNanoscale structural imaging of fundamental cytoskeleton and DNA fiber is exhibited by super-resolution FPM and more detailed structures could be detected. During budding, yeast septin proteins are arranged in an orderly manner to form short filaments and rotate 90° along the plane of the membrane, from hourglass structure to double ring structure B [11], [Wang et al. synthesized SERS nanoparticles with sensitive polarization effect and without photobleaching. SERS-SDOM effectively realizes nanoscale particle tracking to provide trajectory and rotational changes with nanometer spatial resolution in living cells F and G [4FPM excavates the polarization property of fluorescent probe to supplement orientation information for the study of target protein structure. However, suffering from optical diffraction, conventional FPM is difficult to resolve orientation with high resolution. Recently super-resolution FPMs have been achieved to zap nanoscale orientation information, which is essential for a more detailed study of the polarity, orientational order of intracellular proteins, and thus understanding of life processes. In this review, we focus on the novel advances of technology and application in super-resolution FPM. There are three strategies to accomplish super-resolution FPM, including polarization single molecule localization, polarization demodulation and polarized structured illumination. Biological applications, with the special focus on chromophore-target molecule interaction and molecular organization, are summarized herein.From perspective of signal processing, polarization modulation can contribute to information enhancement with two more components – phase and period, in addition to the common amplitude which corresponds to intensity. Dipole orientation refers to the phase of polarization modulation and structural information at nanoscale could be accessed by pushing FPM into super-resolution, which brings a new dimension for super-resolution imaging and greatly enriches the texture details of fluorescence microscopic imaging. If over two periods of polarization data are acquired, there is great potential to improve signal-to-noise ratio (SNR) of polarization modulation images with optical lock-in detection to separate the periodical signal from optical intensity with different frequencies, such as auto-fluorescence, stochastic noise, sampling noise and so on Accurate determination of fluorescence dipole orientation/anisotropy is vitally important for development of FPM. However, the anisotropy would be affected by the energy transfer between fluorescent molecule and its environment. To discern the contribution of energy transfer to measured anisotropy is an important issue. Control measurements, such as red-edge excitation etc, could be applied to isolate the effect of fluorescence resonance energy transfer (FRET) from total anisotropy Due to the lack of axial information, the out-of-focus dipole will cause significant averaging error and affect the 2D measurement. Therefore, 3D dipole orientation determination is very necessary. Several aforementioned FPM techniques can measure 3D dipole orientation. For defocused pattern analysis, the PSF library of 3D orientations can be calculated. Then the 3D orientation of the dipole can be fitted with a matching algorithm Novel biological applications are expected to boost the development of FPM. On the other hand, with more and more advanced super-resolution FPM developed, wider and more elegant biological applications could be investigated. For example, super-resolution FPM with high temporal resolution, fast imaging speed and being highly compatible with live cell imaging could access more elaborate structural dynamics study. However, much more progress is expected. For example, the detailed septin organization during budding yeast, between hourglass and double ring structure, is still not observed and interpreted yet. In immunofluorescence microscopy, since there are multiple linkers between fluorescent molecule and secondary antibody, primary antibody and secondary antibody, anti-body and the target to label the target protein, the distribution of dipole orientations is usually in random order and is not able to represent structure information. To introduce FPM into immunostaining, it is vitally important to further develop more molecular probes that bind target with strong polarization fluorescence.Incorporating multiple modalities into FPM is promising. Built upon confocal, FPM is used in studying conformation change by measuring fluorescence anisotropy The authors declare that they have no competing interest.


Cancer cells depend upon a continuous supply of nutrition and interrupting that supply curtails tumor growth. One-carbon metabolism pathway is central to tumor sustenance and, through its role in DNA methylation and nucleotide synthesis and repair, several chemo-radiation strategies have exploited the cycle. Solid tumors (including cancer stem cells) are auxotrophic—essential for tumor growth but cancer cells are unable to synthesize it indigenously—for methionine, an essential amino acid involved in one-carbon metabolism. At the cellular level, methionine is needed for the reduction of oxidative stress and repair of DNA damage. Exploiting this cancer vulnerability could be a druggable target to achieve effective and sustained cancer kill, in conjunction with conventional DNA-damaging therapies such as chemotherapy and radiation.Pre-clinical and clinical studies have shown that methionine restriction ceases tumor growth and sensitizes cancers to chemotherapy, due to an S/G2 cell-cycle arrest –4. The sGao et al. extended these findings and established a direct link between methionine restriction and cancer outcomes. In a concurrent murine and controlled human study, they demonstrated that methionine restriction impeded tumor growth in conjunction with anti-metabolite chemotherapy and radiation—cancer therapeutic modalities that exploit the same cancer metabolism pathway as methionine depletion .Kras (activating mutation) and tumor-suppressor p53 (inactivating mutation). They showed that methionine restriction did not inhibit tumor by itself but did so in combination with radiation. Metabolomic analysis further revealed that methionine restriction disrupted one-carbon metabolism cycle, perturbed nucleotide synthesis and markedly reduced the levels of methionine-related metabolites.The group first unveiled the link between methionine restriction and tumor growth retardation in two patient-derived xenograft mice models of colorectal cancer with RAS mutation. The findings were validated via metabolomic profiling. The investigators then showed that methionine restriction synergizes with 5-FU to markedly reduce tumor growth in one of the 5-FU-resistant cell lines. The group then examined the impact on a radiation-resistant soft-tissue sarcoma mouse model with compound conditional mutations in oncogenic These interesting, pre-clinical findings were reproduced in a proof-of-concept clinical study. When six healthy individuals were subjected to a possibly safe methionine restricted diet, it fundamentally disrupted one-carbon metabolism and plasma methionine-related metabolites highly correlated with those found in mouse models. This study provided evidence that strategic dietary modifications could be used to inhibit tumor growth by disrupting cancer metabolism, and potentially make relapsed/refractory cancers sensitive to frontline treatments again. In another murine study, Wang et al. showed that cancer stem cells exhibit overactive methionine metabolism and an overexpression of methionine adenosyltransferase II α (MAT2A) enzyme .Evolving data around tumor metabolism may potentially disentangle the controversy related to the use of TPN in terminally ill cancer patients. Albeit earlier studies showed some benefit of TPN in patients undergoing bone marrow transplantation (BMT) , more reEven though metabolism-based diagnostic strategies are standard in cancer care, the safety and efficacy of metabolism-based therapeutics still need to be established. In contrast to other non-communicable illnesses, such as cardiovascular diseases and diabetes mellitus, where the role of metabolic interventions is a fundamental part of treatment, the role of dietary interventions in cancer still needs to be validated. Depletion of essential amino acids may have detrimental toxicities on normal cells as well. Toxicity and feasibility studies in larger clinical cohorts are awaited before any targeted metabolism therapeutics could be employed in clinical practice.Another metabolomic-based intervention strategy that is finding its role in cancer therapeutics is the gut microbiome. Strategies are being evaluated to transform the resident gut taxa from a dysbiotic  state to a symbiotic state and re-direct the adaptive immune system toward cancer cells. Resident gut taxa and its diversity impact the adaptive immune system and differentially activate either the effector T-cells or regulatory T-cells, via production of metabolites. This distinctive T-cell differentiation then either enhances or dampens responses to immunotherapy and, secondarily, survival .Firmicutes and Verrucomicrobia phyla have specifically been shown to enhance responses to immunotherapy  proteins and sophisticated immune-engaging therapy, simple dietary interventions to achieve tumor control certainly holds similar promise. Strategic restriction of essential amino acids, in conjunction with chemotherapy, is being re-explored. The safety profile and optimal dosing regimen still need to be determined in large prospective trials. The intriguing question of whether methionine restriction could impart a similar metabolite-induced impact on immune-based cancer treatment also needs to be explored. Yet again, the key will be to bridge the gap between the bench and the bedside collaboratively.MBA and MAA conceived of the idea and wrote the manuscript. MAA drew the figure. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The serratus anterior plane block (SAPB) has been shown to effectively treat pain following breast surgery, thoracotomies, and rib fractures. We present the first reported case of a bilateral ultrasound-guided SAPB in a multimodal analgesic regimen after an acute large, thoracic, deep partial-thickness burn.A 72-year-old male presented in severe pain two days after sustaining a deep partial- thickness burn to his anterior chest wall after his shirt caught on fire while cooking. The area of injury was on bilateral chest walls, and the patient was consented for bilateral SAPBs at the level of the third thoracic ribs (T3). With ultrasound guidance, a mixture of ropivacaine and lidocaine with epinephrine was injected into the fascial plane overlying bilateral serratus muscles at T3. The patient reported complete resolution of pain for approximately 15 hours and required minimal additional intravenous analgesia.The ultrasound-guided SAPB may be an excellent addition to the multimodal analgesic regimen in superficial and partial-thickness burns of the anterior chest wall. Chest wall burns are relatively common, and often do not require specialized treatment at a burn center.2The ultrasound-guided serratus anterior plane block (SAPB), first described by Blanco et al in 2013, is an effective method of achieving analgesia of the hemithorax from the second thoracic (T2) to ninth thoracic T9) dermatomes. dermatomA 72-year-old male with diabetic polyneuropathy presented to the ED with a chief complaint of chest pain two days after sustaining a flame burn while cooking. His vital signs were stable and his physical exam revealed a deep partial-thickness burn over the majority of his right pectoralis extending across midline to the left sternal border . The patAfter obtaining informed consent, bilateral SABPs were performed in the manner described by Blanco et al.10What do we already know about this clinical entity?Serratus anterior plane blocks (SAPB) effectively treat pain following breast surgery, thoracotomies, and rib fractures.What makes this presentation of disease reportable?This is the first description of an ultrasound-guided SAPB performed in the emergency department (ED) for the management of pain from an extensive partial-thickness thoracic burn.What is the major learning point?Ultrasound-guided SAPBs can provide safe and rapid analgesia for patients with superficial and partial-thickness chest wall burns in the ED.How might this improve emergency medicine practice?Ultrasound-guided SAPBs can facilitate chest wall burn treatments such as dressing changes and wound debridement while reducing reliance on opioid medications.The patient was positioned in the left lateral decubitus position with right arm overhead for the right-sided block. A high-frequency 10-5 megahertz linear transducer was used to locate the third rib in cross section in the mid-axillary line. Superficial to the rib lies the SA, which is deep to the latissimus dorsi (LD) . The tarAfter sterilizing and anesthetizing the skin, an in-plane technique was used to inject the 15-mL mixture of ropivacaine and lidocaine with epinephrine between SA and LD muscles  followinFive minutes after the initial injection, the patient reported significant analgesia over his right chest, and 20 minutes after the termination of the procedure he was able to sleep comfortably. The patient reported no discomfort during the injection process and experienced no side effects. A local burn center recommended admission for treatment with silver sulfadiazine and fluid resuscitation; when the patient was interviewed on hospital day two, he reported the SABP took approximately 20–30 minutes to take full effect and lasted for an estimated 15–18 hours. He ultimately required two doses of 2 mg intravenous morphine at approximately 30 hours and 35 hours after his SABP. His only other medications were acetaminophen 500 mg orally every six hours, ibuprofen 600 mg orally every six hours, and home medications gabapentin and trazodone. The patient was discharged in stable condition on hospital day two.The SAPB, originally used to control pain following breast surgery, is now becoming increasingly popular among emergency physicians to control pain from thoracic injuries in the ED.Burns are a leading cause of accidental injury, many of which will not require management at a burn center. Aggressive pain control is necessary in the treatment of these injuries. A multimodal regimen including opioids and ketamine is often required to achieve adequate analgesia for large burns. However, opioids carry risks and side effects of respiratory depression, nausea/vomiting, constipation, and addiction; and ketamine may cause dizziness, dysphoria, and altered mental status.With regard to anesthetic choice, ropivacaine was selected for its long duration of anesthesia and good safety profile. When compared to lidocaine, ropivacaine has a slightly longer mean onset time but provides a much longer mean duration of anesthesia .17We recognize that the ultrasound-guided SAPB may not be ideal for burns posterior to the mid-axillary line. We have noted minimal success of this planar block in patients with posterior rib fractures and are unclear of the efficacy of this block for these injuries. We also recognize that clinicians performing high-volume SAPB blocks should be aware of maximal anesthetic dosing to prevent administering a toxic dose. The patient’s weight should be measured and a dosing calculator should be used to ensure safety.Ultrasound-guided serratus anterior plane blocks can potentially provide a safe and rapid method of analgesia for patients with superficial and partial-thickness chest wall burns in the ED. They may be easily performed at the bedside with minimal risk of affecting respiration, mental status, or motor function. As such, they may be a valuable addition to a multimodal pain regimen used in the evaluation and treatment of chest wall burns, and may facilitate treatments such as dressing changes and wound debridement while reducing reliance on opioid medications.


Barriers to prevention varied based on the survey participants’ country oforigin; however, there was consensus across all countries on the three most importantstrategies to improve prevention, namely multidisciplinary interventions, patienteducation and introducing performance measures.1 In the UK, shortcomings in the delivery of secondary prevention programmes havealso been linked with suboptimal outcomes for patients with cardiovascular disease(CVD), and recently a group of multidisciplinary team (MDT) members working in secondaryprevention in primary and secondary care settings has developed a UK consensus onoptimising CVD secondary prevention care.2We read with great interest a recent article entitled ‘Secondary prevention andcardiovascular care across Europe: a survey of European Society of Cardiology members’ views’.3 a nurse-led integrated community-based CVD prevention programme and a GP-ledone-stop heart failure diagnosis and management clinic model for primary care. All ofthese models provide practical examples of ways that different healthcare professionalsinvolved in delivering MDT care can work together to improve patients’ outcomes in CVD,for example through recognising and addressing poor adherence to cardioprotective medication.4,5As co-authors of the UK consensus statement, we were mindful that there is not a uniform,one-size-fits-all model of CVD secondary prevention care, and that within our NationalHealth Service (NHS), the roles undertaken by different MDT members can vary dependingon the skillsets and healthcare resources available locally. Within the typicalmanagement pathway for post-myocardial infarction follow-up, numerous opportunities forMDT members to deliver secondary prevention care from the time of initial presentationand hospitalisation through to long-term post-discharge follow-up were identified andoutlined. In addition, a range of best practice models from across the UK that arecurrently achieving success in reducing cardiovascular risk were described and endorsedwith a view to being adopted or adapted elsewhere. These included a pharmacist-led,hospital-based post-myocardial infarction medicines optimisation programme,1 underlines that secondary prevention is often suboptimal across the Europeancountries included in the European Society of Cardiology (ESC) survey. Given that MDTmembers are central to delivering CVD secondary care, it is understandable that anAssociation of Cardiovascular Nursing and Allied Professions (ACNAP) task force iscurrently examining ways to support greater collaboration between allied professions andto address uncertainties about the roles that different MDT members can play in CVDsecondary prevention. We suggest that the UK consensus statement (which is availableonline as an open-access publication) may provide insights and practical guidance whichare of interest to cardiovascular nurses and MDT members working in CVD secondary careacross Europe.The data analysis from Fitzsimons et al.


In this review article, we discuss advances on personalized HIS models using reprogrammed iPSCs and review the use of HIS mice to study requirements for efficient targeting of human Tregs for personalized cancer immunotherapies.Immunodeficient mice engrafted with a functional human immune system [Human immune system (HIS) mice] have paved the way to major advances for personalized medicine and translation of immune-based therapies. One prerequisite for advancing personalized medicine is modeling the immune system of individuals or disease groups in a preclinical setting. HIS mice engrafted with peripheral blood mononuclear cells have provided fundamental insights in underlying mechanisms guiding immune activation vs. regulation in several diseases including cancer. However, the development of Graft-vs.-host disease restrains relevant long-term studies in HIS mice. Alternatively, engraftment with hematopoietic stem cells (HSCs) enables mimicking different disease stages, however, low frequencies of HSCs in peripheral blood of adults impede engraftment efficacy. One possibility to overcome those limitations is the use of patient-derived induced pluripotent stem cells (iPSCs) reprogrammed into HSCs, a challenging process which has recently seen major advances. Personalized HIS mice bridge research in mice and human diseases thereby facilitating the translation of immunomodulatory therapies. Regulatory T cells (Tregs) are important mediators of immune suppression and thereby contribute to tumor immune evasion, which has made them a central target for cancer immunotherapies. Importantly, studying Tregs in the human immune system  One main obstacle remaining in cancer therapy is intra-tumor heterogeneity. The use of personalized medicine offers a promising approach to tackle this major hurdle and HIS mice engrafted with patient-derived hematopoietic cells are an important tool for the development of personalized medicine.With the discovery of checkpoint inhibitors and more recently, the use of chimeric antigen-receptor T cells, immunotherapies have taken center stage in oncology. The common goal for cancer immunotherapies is to boost the immune system to attack and destroy cancerous cells. The gold-standard for pre-clinical testing of cancer therapies has long been the use of xenograft models, where human cancer cell lines are engrafted into immunodeficient hosts. However, both development and evaluation of immunotherapies requires the presence of a functional immune system closely mirroring the immune reaction in the human disease. Immunodeficient mice engrafted with a functional human immune system [human immune system (HIS) mice] have opened up new ways of evaluating immunotherapies directly Prkcdscid or SCID) mice, engrafted with human hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs), can develop a human immune system  that shows enhanced interaction with human CD47 on hematopoietic cells, thereby preventing their phagocytosis by macrophages -HLA-DQ8 transgenic mice engrafted with fresh human cord blood (CB) HSCs and demonstrated a high engraftment efficacy in peripheral blood of these animals , or patient-derived xenografted tumors (PDXs) have been established  within few weeks upon engraftment. Alternatives for long-term studies are the engraftment with patient-derived HSCs or HSCs originating from fetal liver or CB. Here, engraftment with patient-derived HSCs allows for positive selection of T and B cells based on murine antigens in the thymus and bone marrow, therefore limiting GvHD reactions and further enabling MHC compatibility with the autologous tumor tissue. One drawback of HSC engraftment, however, are low frequencies of HSCs in the peripheral blood of adults without mobilization, which impede an effective reconstitution of the human immune system in HIS mice. Of note, in 2006, a group led by Shinya Yamanaka generated pluripotent stem cells from adult cells. The discovery of these induced pluripotent stem cells (iPSCs) has thenceforward substantially revolutionized the field of translational research and personalized medicine  and enabThe discovery that iPSCs are able to self-renew and differentiate into any cell type by introducing the reprogramming factors Oct4, Sox2, cMyc, and Klf4 , 17 has de novo generation of functional HSCs from iPSCs for efficient in vivo engraftment and multi-lineage potential remains challenging due to the complex nature of hematopoietic ontogeny  syndrome  are likewise present on peripheral Tregs, whereas their expression is increased in tumor-infiltrating Tregs. Other tumor-specific Treg signature genes  are exclusively expressed on tumor-infiltrating Tregs and absent in their peripheral counterparts. However, the functional consequence of this gene signature in tumor-infiltrating Tregs remains to be determined.It is now established, that Tregs take residence in various tissues, where they express tissue specific signature genes and exert key non-canonical functions –49. Nota+CD8+T cells/PD-1+Tregs in the tumor microenvironment can predict the efficacy of PD-1 blocking therapy  and cytotoxic T-lymphocyte associated protein 4 (CTLA4) , 54. Man therapy . While P therapy , 38, whi therapy . These sThe identification of tumor-related Treg signature genes is an important step toward more specific targeting of tumor Tregs without affecting the peripheral Treg pool which could potentially lead to autoimmune reactions. CTLA4 mediated tolerization of antigen-presenting cells is an important mechanism of suppression by Tregs. Importantly, CTLA4 blockade has been studied extensively as checkpoint inhibitor in cancer immunotherapy, since CTLA4 is often upregulated by tumor cells for immune evasion . However+ lymphoma bearing NOG mice reconstituted with human PBMCs was used (Like CTLA4 the chemokine receptor type 4 (CCR4), albeit being expressed on peripheral Tregs, is further upregulated on tumor-infiltrating Tregs, while additionally being expressed on different types of cancer cells. To test anti-CCR4 treatment in a pre-clinical setting, a humanized mouse model for lymphoma based on CCR4was used . The trewas used . The latwas used . Howeverwas used , 62, 63.+Tregs harboring the highest levels of GITR  has been studied extensively as a target for cancer immunotherapies. GITR is a coreceptor, which is expressed at low levels on naïve CD4 and CD8 T cells and is upregulated upon activation with Foxp3 of GITR , 65. Imp of GITR . Accordi of GITR –69. Whil of GITR , 70. Thi of GITR , or to TFoxp3 locus  in the p3 locus . We werep3 locus , 72, 73.p3 locus . Importap3 locus . Regardipancreas , 37, 76.pancreas , 78. Thepancreas ], as miRThe perpetual improvements of HIS mouse models have enabled the design of personalized immunotherapies for various diseases including cancer and the evaluation of their efficacy prior to the translation to the human setting. Using patient-specific HIS mice will enable us to better understand the heterogeneity of immune responses and advance the development of novel personalized immunotherapies for the treatment of cancer. Even though still in its infancy, the use of iPSC-derived HSCs for engraftment could be an important step toward personalized HIS models.in vivo.Additionally, the immunosuppressive tumor microenvironment influences antitumor responses and the efficiency of cancer immunotherapies. Targeting Tregs has therefore become an important tool in cancer immunotherapy and has been studied extensively in murine models. In order to target Tregs most efficiently in human diseases the development of novel models depicting the immune reaction of the human disease is pivotal. HIS mouse models offer the opportunity to study the interaction of human immune cells, including Tregs, with human tumors directly Overall, current advances in establishing a “truly” humanized mouse model for the development of immunotherapies and drug delivery systems will guide personalized medicine approaches further to the translation to the clinic.IS and MK reviewed the literature and wrote the manuscript. MGS reviewed the manuscript and prepared the figure using BioRender. CD reviewed the literature and contributed to the conceptualization of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We aimed to evaluate the potential of radiomics as an imaging biomarker for glioblastoma (GBM) patients and explore the molecular rationale behind radiomics using a radio-genomics approach. A total of 144 primary GBM patients were included in this study (training cohort). Using multi-parametric MR images, radiomics features were extracted from multi-habitats of the tumor. We applied Cox-LASSO algorithm to build a survival prediction model, which we validated using an independent validation cohort. GBM patients were consensus clustered to reveal inherent phenotypic subtypes. GBM patients were successfully stratified by the radiomics risk score, a weighted sum of radiomics features, corroborating the potential of radiomics as a prognostic biomarker. Using consensus clustering, we identified three distinct subtypes which significantly differed in the prognosis . Transcriptomic traits enriched in individual subtypes were in accordance with imaging phenotypes summarized by radiomics. For example, rim-enhancing necrotic subtype was well described by radiomics profiling (T2 autocorrelation and flat shape) and highlighted by the inflammatory genomic signatures, which well correlated to its phenotypic peculiarity (necrosis). This study showed that imaging subtypes derived from radiomics successfully recapitulated the genomic underpinnings of GBMs and thereby confirmed the feasibility of radiomics as an imaging biomarker for GBM patients with comprehensible biologic annotation.  Glioblastoma (GBM) is the most lethal primary brain tumor with the median survival of 12–15 months . MoleculRadiomics converts medical images into high-dimensional quantitative data capable of capturing the intra-tumor heterogeneity of the tumor . PreviouWe reason that phenotypical heterogeneity captured in neuroimaging reflect the underlying biological characteristics . As one In this study, we sought to evaluate the feasibility of radiomics as a potent biomarker for GBM patients and explore the transcriptomic traits associated with imaging phenotypes to understand the biological correlates of radiomics. We evaluated the utility of radiomics as an imaging surrogate for GBM patients and identified distinct subtypes using the clustering of the radiomics features. Next, we explored the genomic correlates of these subtypes to establish the genotype–phenotype association. Collectively, we aimed to explore the radiomics-based imaging biomarker for GBM patients with comprehensible biologic annotations.We retrospectively screened GBM patients who underwent surgical resection at the Department of Neurosurgery at Samsung Medical Center (SMC) between January 2010 and June 2017. The inclusion criteria were as follows: (i) histologically confirmed primary GBM without prior treatment history, (ii) availability of preoperative magnetic resonance (MR) images taken at SMC with all of four sequences  with adequate resolution, (iii) contrast-enhancing tumors with measurable volume (≥1 cc), and (iv) availability of clinical records with one or more follow-up visits. In total, 144 patients were finally included. Written informed consent was obtained from all patients, and the Samsung Medical Centre Institutional Review Board approved  this study. All procedures were carried out following the relevant guidelines. The external validation cohort was collected from the Austrian Brain Tumor Registry network . A totalAll MR images were obtained using a 3.0-T MR imaging system (Philips). We used four MR sequences: T1, T1CE, T2, and FLAIR. The imaging parameters for each sequence were as follows: T1 and T1CE ; T2 ; FLAIR . www.mitk.org, German Cancer Research Center, Heidelberg, Germany). Similarly, the peri-tumoral edema on T2WI was segmented. The peri-tumoral edema was delineated on the T2 images using the same algorithms. Tumor segmentation was performed on multi-parametric MR images (T1CE and T2WI) by two neurosurgical experts in a semiautomatic manner . Multi-habitat regions of the tumors were specified. The contrast-enhancing solid portion was segmented on the axial T1CE images by using the region-growing and thresholding algorithms implemented within the MITK software (http://stnava.github.io/ANTs/) was conducted [To transfer the segmented ROI to other image spaces, image registration using Advanced Normalization Tools  ,17,18 wajβ is the j-th weighted coefficient of the selected feature, and ijX is the j-th selected radiomic features for i-th patient. This method has been widely used in many radiomics studies encompassing various tumor types [Radiomics risk score was defined as the output at the initial time point of Cox’s model  model by fitting a nearest shrunken centroid classifier on the centroids generated from the clusters of the training cohort . We fittRNA sequencing libraries were prepared using the Illumina TruSeq RNA Library Preparation Kit v2. The sequenced reads were trimmed and mapped onto hg19 using GSNAP . The reshttp://software.broadinstitute.org/gsea). Next, to remove the redundancy of the GSEA results, we applied Cytoscape for visualizing the enriched genomic signatures [p-value < 0.01 were finally selected to construct the network visualization. Node size inversely correlates with the nominal p-values and the node color implies the normalized enrichment score (NES); red color signifies up-regulation, while blue signifies the down-regulation of the corresponding node. Single-sample GSEA (ssGSEA) score was computed for every case using the genesets identified as being significantly highlighted in radiomics subtypes. Z-normalized ssGSEA score was utilized to illustrate a heatmap representing the transcriptomic landscape of GBM patients. We performed gene set enrichment analysis (GSEA) to identify the significantly enriched genomic signatures per each radiomics subtypeAll GSEAs were performed using GenePattern from the Broad Institute (gnatures . Genesethttp://www.R-project.org) [t-test or the two-sided Wilcox test, and categorical variables were tested using the chi-square test or Fisher’s exact test. When comparing the continuous variables with multiple categories (more than three categories), ANOVA or the Kruskal–Wallis test was used where appropriate. Survival analysis was performed using a Kaplan–Meier plot, and the log-rank test was used to show statistical differences between survival curves. All statistical analyses were performed using R version 3.6.3 (ect.org)  and Statp < 0.05 was used as a threshold for statistical significance. p values derived from multiple comparisons were appropriately corrected by Bonferroni and false discovery rate (FDR) methods [ methods .We calculated radiomics features from the contrast-enhancing tumor and peri-tumoral edema regions. A total of 478 radiomics features were extracted from multi-parametric MR images . The overall study flow is summarized in a schematic illustration A. A totaWe applied the Lasso-Cox proportional hazard model to the training cohort and seven radiomics features were finally selected as the most relevant features to survival outcome. Among them, one feature was the flatness of the enhancing tumor core, which signified the degree of tumor elongation. Two features were histogram-derived features, which depicted the distribution pattern of voxels of distinct gray-levels. The other four features were texture features reflecting intra-tumor heterogeneity. Of note, no features were selected from the peri-tumoral edema. Detailed information on selected features is shown in p value < 0.001). This finding was also confirmed in the external validation cohort with a hazard ratio of 19.17 .To validate the potential of radiomics as a prognostic biomarker, we defined the radiomics risk score and stratified the GBM patients according to the median value of the score. In the training cohort, the high-risk group demonstrated significantly poor outcomes compared to the low-risk group, and the hazard ratio was 3.56 . To validate the clustering subgroups identified in our training cohort, we applied the prediction analysis for microarrays (PAM) to assign each sample in the validation cohort to one of the clusters [p-value = 0.003, log-rank test).Accordingly, these three GBM subtypes showed distinct survival outcomes as well as radiomics profiling  features. This texture feature measures the variability of gray-level intensity values in the image, with a higher value indicating the massive heterogeneity in signal intensity. Gross morphology of these tumors (contrast-enhancing solid mass with a mixture of necrosis within the core) were in accordance with high values of T1CE GLNN feature, and we named this cluster as the “heterogeneous enhancing” subtype. Tumors in Cluster 2 (“rim-enhancing necrotic” subtype) were characterized by high values of flatness (shape) and T2 autocorrelation , indicating the spherical shape with homogenous T2 signal intensity within the tumor. These tumors showed rim-enhancement with a necrotic core, a typical phenotype observed in GBMs. Autocorrelation of GLCM measures the linear dependency of gray-levels in neighboring pixels and thus reflects the intensity heterogeneity. A larger value of T2 autocorrelation implies a tumor with less heterogeneity in T2 signal intensity, which correlated well with the considerable portion of necrotic core observed in Cluster 2 tumors.Cluster 3 tumors exhibited the most favorable prognosis and were characterized by low values of T1CE skewness and high values of FLAIR kurtosis. Interestingly, this subset also exhibited high values of T2 maximal correlation coefficient (MCC), which indicates the complexity of the texture. Grossly, tumors in Cluster 3 were cystic tumors with thin rim-enhancement on T1CE image and thus were named as the “cystic” subtype. Next, we compared the known confounding factors associated with the prognoses of GBM between different radiomics subtypes to validate the potential of radiomics as a prognostic biomarker ,32,33,34To comprehend the biologic rationale behind the radiomics, we explored the genomic characteristics of radiomics-defined GBM subtypes. Geneset enrichment analysis (GSEA) was performed to identify the genomic signatures enriched in each individual radiomics subtype. We also searched the correlative gene sets to each radiomics feature to account for the biologic implication of radiomics feature. Cluster 1 (the “heterogenous enhancing” subtype) was highlighted by several genomic signatures associated with lysosomal activity and autophagy, as illustrated in Herein, we demonstrated the efficacy of utilizing radiomics as a prognostic biomarker for GBM patients. Radiomics risk score successfully stratified the GBM patients according to survival outcome and clustering analysis using radiomics features revealed that GBMs were composed of three image-defined subtypes which differed in prognosis. To comprehend the biologic rationale of radiomics as an imaging biomarker, we explored the molecular characteristics of these radiomics subtypes. This approach enabled us to discover potential connections between radiomics and specific genomic traits with relevant clinical translation. In the radiomics risk score building step, seven features were identified to be significantly associated with GBM prognosis . There wCluster 1 tumors (the “heterogeneous enhancing” subtype) were characterized by high values of T1CE GLSZM GLNN feature, which indicating the high variability of signal intensity on T1CE image. This radiomic profiling accounts for the gross morphology of Cluster 1 tumors, which exhibited intermingled necrosis within the tumor. Interestingly, genomic signatures associated with lysosomal activity and autophagy were enriched in Cluster 1 GBMs. A lysosome is an important recycling organelle associated with autophagy, and also functions as a relay hub for signaling pathways driven by the mechanistic target of rapamycin complex 1 (mTORC1) ,41. AlthThe radiographic presentation of Cluster 2 (the “rim-enhancing necrotic” subtype) is the prototype of GBMs, with rim-enhancing spherical tumors with a necrotic core. This morphology is well explained by the highlighted features in Cluster 2 (T2 autocorrelation and flat shape feature). Necrosis is the hallmark of GBMs and is known to induce inflammation in the surrounding tissue . In accoCluster 3 (the “cystic” subtype) comprised a minor subset of GBM patients in the training cohort. We could not identify the patients assigned to this cluster in the validation cohort. These tumors grossly exhibited cystic morphology with thin rim-enhancement. The favorable clinical outcome in cystic GBMs has been previously described ,48,49, aPreviously, Itakura et al. identified three distinct phenotypical subtypes of GBM patients using T1CE MR images . These sThese reproducible phenotypical subtypes signify the potential of imaging biomarkers. Importantly, they reflect distinct molecular underpinnings and their distinction may imply the different therapeutic vulnerability. For example, treatment strategy targeting autophagy would be more effective against Cluster 1 tumors, whereas a hostile tumor microenvironment would be a more idealistic target for Cluster 2 tumors considering their predominant inflammatory milieu.Autophagy induction has been implicated in the response to TMZ for malignant gliomas . AutophaTumor microenvironment (TME) is a key target for next-generation therapeutics for gliomas. Despite flourishment of immunotherapies, GBMs remain immune-desert tumors and thus, modulating the immuno-suppressive TME is the priority to overcome their innate treatment resistance against immunotherapy . The intImaging biomarkers are more versatile than molecular biomarkers considering several key challenges against GBMs; they are non-invasive and free from sampling bias, thus reflecting the characteristics of the whole tumor. However, they also have some inherent weakness; data reproducibility and objectivity are the main concerns accompanied by radiographic assessment. Radiomics enabled us to transform the descriptive neuroimaging features into the format of quantitative and objective data, thus contributing the evolution of image-driven data science. Another challenge is their translational ability in terms of biological activity. Many have attempted to annotate the imaging phenotype with understandable molecular correlates; however, no robust association has yet been elucidated. Radiomics is high-dimensional and thus, identifying specific molecular correlates per individual feature would be spurious. Classification analysis is a more applicable approach to explore the molecular rationale behind the radiomics, as shown in present study. This study has several limitations; we retrospectively screened patients and thus, selection bias was inevitable due to the inherent nature of the study design. The number of patients was limited, especially for the validation cohort, where we could not identify Cluster 3. The most significant drawback in this study was the lack of experimental validation. The causal relationship between the highlighted genomic signatures and their corresponding radiomics subtype could be validated through well-designed experiments. We can confirm the therapeutic implication of these image-defined subtypes following experimental validation. As a research field, radiomics is still developing, and thus, further studies focusing on the above limitation are warranted.In conclusion, by assessing the properties of the entire tumor, radiomics may have the potential to provide accurate predictions of patient prognoses. This is a major advantage and may overcome the limitation of molecular biomarkers that draw information from a partial region in the tumor, which is a significant drawback regarding the molecular heterogeneity of GBMs. In the present study, we captured the potential genotype–phenotype association of GBM tumors in terms of radiomics, thereby presenting phenotypic subtypes of GBMs with distinct genomic traits. We applied multi-parametric MR images and multi-habitat ROIs to represent the characteristics of real tumors with sufficient supplements. Radiomics subtypes could serve as imaging biomarkers for prognosis prediction as well as therapeutic strategies. Importantly, these subtypes, much improved in detailed description, are reproducible across studies, which signifies the robustness of phenotypic surrogates for GBM tumors. By integrated radio-genomics approach, we confirmed the potential of radiomics as imaging biomarker for GBMs regarding clinical and genomic significance. The present study has demonstrated the potential of radiomics, an objective and quantitative measure of radiographic phenotype, as an imaging biomarker for the prognosis of GBM patients. Despite previous efforts, biological correlates of imaging phenotype remain elusive. By radiomics, we identified three phenotypic subtypes of GBM tumors, and these subtypes demonstrated distinct prognostic implications as well as genomic characteristics consistent with the prior literature. We showed the molecular correlates of the imaging derived phenotypic subtypes with clinically comprehensible annotations. This approach unraveled the molecular underpinning behind the imaging derived phenotypes, thus supporting the potential of radiomics as an imaging surrogate for GBM tumors.


Coral reef ecosystems are among the first to fundamentally change in structure due to climate change, which leads to questioning of whether decades of knowledge regarding reef management is still applicable. Here we assess ecological responses to no-take marine reserves over two decades, spanning a major climate-driven coral bleaching event. Pre-bleaching reserve responses were consistent with a large literature, with higher coral cover, more species of fish, and greater fish biomass, particularly of upper trophic levels. However, in the 16 years following coral mortality, reserve effects were absent for the reef benthos, and greatly diminished for fish species richness. Positive fish biomass effects persisted, but the groups of fish benefiting from marine reserves profoundly changed, with low trophic level herbivores dominating the responses. These findings highlight that while marine reserves still have important roles on coral reefs in the face of climate change, the species and functional groups they benefit will be substantially altered. It is unclear whether rapid climate change will alter the effectiveness of marine reserves. Here Graham et al. use a 20-year time-series from the Seychelles to show that marine reserves may not prevent climate-driven shifts in community composition, and that ecological responses to reserves are substantially altered. Following these climate-driven disturbances, some reefs have undergone regime shifts to alternate dominant taxa such as seaweeds, while others have become characterised by stress tolerant and rapidly recovering coral species5. Similarly, coral reef fishes respond to benthic changes in differing ways, dictated by ecological and life history traits6. Collectively, these non-random species responses are leading to novel ecosystem configurations, and a recognition that coral reef communities are changing substantially in the Anthropocene and are unlikely to return to the stable compositions experienced throughout the Pleistocene7. A fundamental question, which we currently have little understanding of, is how effective current conservation and management approaches will be in these novel ecosystem contexts7.Climate change is altering the composition and structure of coral reef ecosystems8. Evidence from the past 30 years of research on no-take marine reserves, identifies a range of beneficial ecological responses10. Benthic cover of live coral is often higher inside reserves, particularly in the absence of major disturbance12. However, the greatest responses are for reef fish, which are protected from fishing. Species richness of the fish community is typically greater inside marine reserves9, building up over relatively short timescales13. The largest differences in the fish community are detected for fish biomass, which based on a global meta-analysis can be >400% greater in reserves compared to fished areas9, with this biomass enhancement driven primarily by higher trophic level carnivorous fishes15. Whether these expectations of marine reserves, and the knowledge we have built up about them, is now changing is a key question, especially as marine reserves in tropical oceans are expected to experience sea surface temperatures outside historical ranges by mid-century16.No-take marine reserves are one of the most pervasive and successful conservation and management tools used in coral reef ecosystems, where many species are sedentary or territorial, and thus respond well to spatial management17, and longer-term divergent trajectories where some reefs underwent regime shifts to macroalgal dominance while others recovered to hard coral dominance5. We assess the changing role of no-take marine reserves in Seychelles from before to 16 years after this major climatic-disturbance event. Our data span 21 reefs across the entire range of the populated inner Seychelles Islands, including nine reefs within no-take marine reserves  Does the coral reef benthic response to marine reserves change following major coral bleaching?; (2) How does major coral bleaching influence the role of marine reserves in enhancing fish species richness?; and (3) Are reef fish biomass and trophic structure maintained in marine reserves following coral bleaching?Here we use a unique opportunity to understand the role of marine reserves under changing environmental conditions on coral reefs. Seychelles was severely impacted by the 1998 global coral bleaching event, when >90% loss of live coral caused major short term impacts on benthic structure and fish diversityWe find that prior to coral bleaching, the ecological effects of the marine reserves were consistent with expectations, with greater coral cover, higher species richness, and enhanced fish biomass driven in part by higher trophic level fishes. However, following coral bleaching, there was no difference in coral or macroalgal cover between fished reefs and reefs in marine reserves. Species richness effects of marine reserves were diminished, and declined through time for reefs where coral cover recovered. Greater fish biomass persisted following coral bleaching, but the feeding groups of fish benefiting substantially changed. Upper trophic level piscivores and to a lesser extend mixed-diet carnivores showed little or much reduced reserve effects, with the enhanced biomass in marine reserves driven predominantly by lower trophic level herbivores. Our findings highlight that while marine reserves still drive ecological responses on climate-impacted coral reefs, the groups of species that benefit are substantially different from expectations.19 and the Caribbean20. The reefs in marine reserves also had a higher density of species  than fished reefs, again a well-established response of fish communities to protection13. Coral cover was on average 15% greater in marine reserves, while macroalgal cover and the structural complexity of reefs did not differ between fished and protected reefs. Greater coral cover in protected reefs prior to coral bleaching may be due to placement of the reserves in areas of high cover for tourism opportunities, protection from the impacts of destructive fishing gears, or faster recovery rates from disturbances21.In 1994, prior to the major coral bleaching event of 1998, Seychelles’ marine reserves were performing similarly to many others globally. Fish biomass was on average 75% greater in marine reserves than fished areas Fig. . This gr22. These post-bleaching trajectories are apparent on both fished and protected reefs, with no difference in cover of coral or macroalgae between marine reserves and fished reefs in the years post-bleaching  than on protected reefs , which diminished the reserve effect   Fig. . The inc 2.3, 10.) than on−1 ; protected = −0.2% year−1 ). In contrast, the net reserve benefit on recovering reefs gradually declined to no difference by 2014, which was driven by slightly faster accumulation of species on fished reefs  than protected reefs  to match protected richness levels by 2014 , benefitted the most from protection  . Gains in herbivorous reef fish following coral mortality have been documented before22, and here we demonstrate the role marine reserves play in greatly enhancing these fish groups in novel post-disturbance coral reef configurations2.To investigate the relative role of marine reserves in enhancing carnivorous versus herbivorous processes, we grouped species into those that feed on fish or invertebrates, and those that feed on algal resources. Pre-bleaching, the marine reserve effect was similar for both groups, with biomass 62% greater for herbivores and 77% for carnivores, and both groups also reached similar biomass levels in protected areas (carnivore = 259 kg ha7]) Fig. . While m−1 19, 347; he7]) Fig. . Specifi35, or because the processes of coral recruitment and growth that drive benthic recovery36 are independent of protection from fishing. There is uncertainty regarding what drove higher coral cover in marine reserves pre-bleaching, and reserve placement in higher coral cover locations could have been a factor. However, for one of the marine reserves, which represented three of our studied reef sites, protection was driven by bird conservation on the island. High coral cover habitat was therefore not a factor in reserve selection here, although, even at these sites, coral cover pre-bleaching was ~15% greater than the average on fished reefs.Marine reserves have been a mainstay of the coral reef conservation toolbox for several decades. Here we demonstrate that climate change impacts are fundamentally altering the composition of coral reefs, and the benefits marine reserves can offer. Any influence of protection on the reef benthos was absent post-bleaching, possibly swamped by the overwhelming impact of climatic disturbance13, the diversity of fish on many coral reefs is increasingly governed by disturbance-mediated changes to habitat37. Perhaps most significantly, while the important role of marine reserves in enhancing fish biomass continued post-bleaching, the groups of fish benefiting from the protection drastically changed. A major shift from carnivorous to herbivorous reef fish benefitting from marine reserve protection implies shifts in trophodynamics and ecosystem services. Herbivores deliver key ecosystem functions29, and are important components of fisheries in many geographies. Indeed, herbivorous fish have sustained reef-associated trap fisheries landings in Seychelles following the 1998 coral bleaching event27. The role of marine reserves in exporting herbivorous fish to fisheries through adult spillover or larval export is likely to be substantial, and could play a key role in continued food security as coral reefs degrade through climatic impacts38.The role of marine reserves in enhancing fish biodiversity, captured here simply as species richness, appears to be greatly diminished. While marine reserves can increase species richness in heavily fished seascapes39, where coral reefs are likely to remain most ‘functional’40, where recovery from climate disturbance is most likely5, or where reef conditions are outperforming expectations given local drivers such as proximity to markets41. Of note, emerging benthic configurations such as those dominated by macroalgae may still provide important diversity and fishery benefits42 and, as we show here, also respond positively to marine reserve management. These results may be particularly relevant to Caribbean coral reefs, where shifts from coral to macroalgae have been quite widespread43.The success of new marine reserves will likely benefit from consideration of individual reef exposure and response to climate change and other increasing pressures characterising the Anthropocene. For example, marine reserve placement may be most effective in locations where the rate of warming and threat of frequent bleaching is lowest28. As the scale and frequency of threats to coral reefs increase, so too must the practices and institutions that aim to sustain them45. While marine reserves will be part of this solution, urgent reductions in global greenhouse gas emissions and mitigation of other underlying social and institutional pressures on reefs will be critical46. Unexpected ecological compositions on coral reefs are also changing the ecosystem services relied upon by millions of people across the tropics47. To be successful and equitable in such novel environments, reef conservation and management must strive for continued provision of ecosystem services, livelihoods and food security48. Our findings indicate that ecological responses to other common management approaches on coral reefs and other climate-sensitive ecosystems may also need to be carefully re-evaluated.The changing ecological responses to marine reserves that we document are not likely to be short-lived, given the time between severe coral bleaching events is shortening, and coral reefs are increasingly existing in boom-bust live coral cycles or shifting to alternative habitat-forming taxa such as macroalgae49. Nine sites were located within four no-take marine reserves, which were established between 1968 and 1979; Cousin Special Reserve, Ste Anne Marine Park, Baie Ternay Marine Park, and Curieuse Marine National Parks. Enforcement of these marine reserves is high, and has been consistent since their establishment; two of the reserves have resident rangers on the island they encompass, while the other two are patrolled daily by the Seychelles National Parks Authority. The remaining 12 sites were located in fished areas. Fishing fleet size has remained stable, or increased for one gear and island, through the time period of this study27. The major changes to the reefs have been associated with benthic changes precipitated by the 1998 bleaching event5.Using a 20-year data set (1994–2014), spanning a major climate-induced bleaching event in 1998, we assess the long-term ecosystem dynamics of 21 reef sites across the inner islands of Seychelles. Seychelles’ reefs were among the most severely affected globally by the 1998 coral bleaching event, in which a strong El Niño coincided with a strong positive phase of the Indian Ocean Dipole2 of reef habitat each survey year. Fish abundance and individual body length (to the nearest cm) of diurnally active non-cryptic species (134 species from 16 families) were estimated in each point count area using underwater visual census (UVC). Larger, mobile species were recorded before smaller, more site-attached species to minimize the likelihood of double counting individuals that left and re-entered the survey area. Survey time was not held constant, and varied according to the abundance and diversity of fish in each replicate. The accuracy of fish body length estimations were assessed at the start of each sampling day in all sample years. The length of random sections of PVC pipe were visually estimated prior to the start of data collection, and compared to the actual length of the pipe. Length estimates were consistently within 4% of actual lengths50. We converted data on fish counts to biomass with published length–weight relationships52, and fish were placed into trophic groups based on feeding preferences32.Sites were surveyed using identical methods in 1994, before the mass coral bleaching of 1998, and in 2005, 2008, 2011 and 2014, following the bleaching event. Eight to sixteen replicate 7 m radius point counts were surveyed along the reef slope on each reef, covering up to 0.5 km of reef front and 2500 m53. The structural complexity of the reef was visually estimated on a 6-point scale15. This structural complexity measure captures landscape complexity, including that provided by live corals, the underlying reef matrix, and other geological features, and has been shown to correlate well to other measures of complexity, such as reef height and the linear versus contour chain method53.Within each point count area, the percent cover of live hard coral, soft coral, macroalgae, sand, rubble, and rock was quantified visually and using 10 m line intercept transects5. While data were also collected in 2017, a number of sites could not be visited, including three of the four regime-shifted marine reserve reefs. We have therefore included data in our analyses up to 2014 to (1) ensure our study includes reef sites which were consistently monitored in the post-bleaching period, and (2) avoid skewing analyses towards recovering reef characteristics. However, while recognizing the smaller sample size in 2017, the data indicate that the post-1998 bleaching trends do continue to 2017 . Thus, our data indicate that the altered role of the marine reserves were established following the first major bleaching disturbance of 1998, and may persist through subsequent climate disturbances.A major global bleaching event in 2016 severely impacted Seychelles’ reefs5. Among fished sites, seven were classified as recovering and five as regime-shifting. Among protected sites, five were classified as recovering and four as regime-shifting.A regime-shifting reef was defined as one where post-disturbance macroalgal cover becomes greater than coral cover and trajectories through time indicate that cover of macroalgae remains high or is increasing. Recovering reefs, conversely, are defined as those reefs where post-disturbance coral cover becomes greater than macroalgal cover and remains greater or increases through time−1), and species richness of coral reef fishes. For each variable, we measured (1) the average difference between fished and protected reefs and (2) the reserve response ratio (protected/fished). We used a drop-one jackknife approach to estimate uncertainty in reserve effects. Specifically, we estimated the difference between fished and protected reefs after excluding one site, and repeated the process by excluding each site in turn . The minimum and maximum jackknife estimates represent uncertainty in reserve effect estimates introduced by individual reefs with exceptionally high or low values for fish biomass or species richness. For benthic changes, we estimated the mean benthic cover  and structural complexity in each year, separately for fished and protected reefs in recovering coral and regime-shifted habitats.We examined marine reserve effects on community biomass, functional group biomass , survey year , and fishing management (protected or fished). We accounted for temporal correlations within reefs by fitting separate survey year slopes and intercepts for each reef r, and fitted interacting fixed effects between survey year*habitat, and survey year*reserve status to estimate linear trends in each habitat regime for fished and protected areas. Priors were weakly informative ; σ = Cauchy). Predictions were visualized by drawing 1000 samples from the posterior distribution across each survey year, for each fishing and habitat combination, and extracting the posterior mean and 95% certainty intervals. Model fits were assessed by drawing posterior predictions for individual reefs . Posterior predictions of biomass and richness absolute difference values were rescaled to represent the percent recovery from pre-bleaching levels .We modelled post-bleaching recovery trajectories in fish biomass and species richness at the scale of individual reefs. For each reef, we converted fish biomass and species richness into the absolute difference between pre- and post-bleaching values . Using these response data for y was the carnivore or herbivore biomass (kg ha−1) estimated in each survey i, and management is fished or protected effect estimated for each functional group FG (carnivore or herbivore), with reef-level intercept terms (r). For post-bleaching reserve effects, we fitted the same model structure as (2) for Gamma-distributed y (3), but including hierarchical fixed effects of habitat (recovering or regime-shifted), survey year, management for each FG:We also modelled reserve effects in promoting carnivore and herbivore biomass before and after bleaching. Using reef-level estimates of herbivore and carnivore biomass, where herbivores included all grazing, scraping, excavating and browsing species and carnivores included all mixed-diet feeders and piscivores, we assessed pre-bleaching reserve effects by fitting the linear model:These additional fixed covariates enabled us to estimate carnivore and herbivore biomass in different habitat regimes while accounting for temporal effects of survey year. Using these two models, we visualized reserve effects on carnivore and herbivore biomass by drawing from the posterior distribution of biomass under fished and protected conditions, for pre-bleaching (4), post-bleaching recovering coral (5), and post-bleaching regime-shifted reefs (5), and for each individual reef .54. Model convergence was assessed using 55  sampling of 7000 iterations (warmup of 1500) across three chains, using the No-U-Turn-Sampler implemented in Stanentclass1pt{minimaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal hematopoietic disorders, which is characterized by cytopenias in the peripheral blood and bone marrow dysplasia due to ineffective hematopoiesis. Patients with MDS have an increased risk of transformation to acute myeloid leukemia (AML). Although the molecular basis of MDS is heterogeneous, several studies demonstrated the significant contribution of the dysregulated immune system in accelerating MDS progression. The immunosuppressive tumor microenvironment is shown to induce tolerance of MDS blasts, which may result in a further accumulation of genetic aberrations and lead to the disease progression. Increasing evidence shows an expansion of myeloid-derived suppressor cells (MDSCs), a population of inflammation-associated immature cells, in patients with MDS. Interestingly, the increased MDSC populations are shown to be correlated with a risk of disease progression in MDS. In addition, MDS is highly prevalent in aged individuals with non-hematology co-morbidities who are fragile for chemotherapy. Increasing research effort is devoting to identify novel agents to specific targeting of the MDSC population for MDS treatment. Myelodysplastic syndrome (MDS) is a group of clonal hematologic disorders characterized by the abnormal and ineffective hematopoiesis with an increased risk of acute myeloid leukemia (AML) transformation . PatientAs a standard prognostic tool, in MDS patients, International Prognostic Scoring System (IPSS) is the clinical and pathological assessment of morphology, periphery cytopenias, and karyotype , 8, whicClinically, the management for MDS is often complex due to the age, disease stage, and co-morbidities of individual patients. The treatment options for MDS patients range from supportive care to aggressive treatment, such as chemotherapy and hematopoietic stem cell transplantation , 14. MDSWhile the molecular basis of MDS is heterogeneous, increasing evidence revealed the significant contribution of the dysregulated immune system in accelerating MDS progression . The immIn the tumor microenvironment, the primary immunosuppressive cell types include activated immature myeloid cells, Tregs, and regulatory B cells. Upon activation, these immunosuppressive cells reduce the T cell proliferation and type II interferon secretion, inhibit antigen presentation, and inhibit natural killer cell function . MDSCs sBelow as illustrated in Fig. −), HLA-DR− [Phenotypic studies on human MDSCs revealed that MDSCs lack the traditional surface markers of mature immune cells, including lineage negative  and can form a complex that can signal through the cell membrane , 39. Twocontrols .− HLA-DRlow CD11b+ CD33+ MDSCs and the frequency of Lin− HLA-DRlowCD11b+CD33+CD27L+ MDSCs is much higher in the BM than that in the peripheral blood of MDS patients [CD27, a costimulatory receptor of the TNF superfamily, is constitutively expressed on lymphocytes and hematopoietic stem/progenitor cells . We founpatients .+ cell population on immune-suppressive MDSC [CD38 is a glycoprotein with ectoenzymatic functions, which is expressed on different cells, including lymphocytes and myeloid cell populations. We have preliminary data showing that MDS patient has a higher frequency of CD38ive MDSC .The fact that MDSCs function as a key cellular component of the BM microenvironment of MDS patients points to a potential role of MDSCs in the initiation and progression of MDS. It is conceivable to hypothesize that inhibition of the MDSC population may benefit to MDS patients. In fact, several studies have revealed the potential of depleting MDSCs or inhibiting MDSC activity in the treatment of MDS patients.+ cell elimination [Gleason and colleagues developed a single-chain variable fragment (scFv) recombinant reagent, so-called bispecific BiKE, which targets CD16 as well as the myeloid differentiation antigen CD33 (CD16xCD33) and facilitates CD33mination . They fumination . This stmination , blast, mination .+ MDS blasts, leukemic stem cells, and MDSCs thus offers a new therapeutic strategy for AML patients [Anti-CD123 is a humanized IgG1 monoclonal antibody that specifically targets the α-subunit of the IL-3 receptor (CD123) to effectively neutralize IL-3 signaling . This thpatients .+ immunosuppressive cell populations, including MDSCs, Tregs, and Bregs [Daratumumab is a human IgG1ĸ monoclonal antibody (mAb) that binds with high affinity to a unique epitope on CD38, a transmembrane glycoprotein. It is a targeted immunotherapy directed towards tumor cells expressing high levels of CD38, such as plasma cells from patients with multiple myeloma. Daratumumab is shown to eliminate CD38nd Bregs . Due to Treatment of patients with MDS has improved in recent years, but remains challenging. Drugs targeting different pathways or different cell populations might show a better overall response in MDS to minimize side effects. Synergistic effect by targeting tumor cells and immunosuppressive populations shows a promising result in improving bone marrow hematopoiesis. Combinatory application of ezatiostat hydrochloride and epoetin alpha in MDS patients at lower risk may be able to reduce the red blood cell transfusion dependence. Combination therapy using hypomethylating agents or Revlimid together with an antigen-specific therapeutic antibody would increase the response rates and improve patient quality of life with minimal adverse side effects.