Due to their exceptional properties, extracellular vesicles (EVs) receive special attention as next generation biotherapeutics and vehicles for drug delivery. However, despite having many advantages over cell-based therapies, EVs usually exert lower therapeutic efficacy. This results from a number of hurdles that are faced by the EV-based approaches. Administered EVs could be rapidly cleared by the mononuclear phagocytes as well as can randomly distribute within various tissues, making tissue penetration and cell targeting insufficient. However, recent research findings imply that these limitations could be overcome with the use of antigen-specific antibodies and light chains. Major histocompatibility complex (MHC) class II-expressing EVs have been shown to form aggregates after co-incubation with antigen-specific antibodies, which greatly enhanced their biological efficacy. On the other hand, EVs could be coated with antibody light chains of chosen specificity to direct them towards desired target cell population. Both findings open up a promising perspective to achieve the highest efficacy of the EV-based approaches. Herein we discuss the opportunities for enhancing extracellular vesicle’s biological activity by using specific antibodies and light chains in the context of the challenges faced by such therapeutic approach. Extracellular vesicles (EVs) encompass all classes of lipid-membrane vesicles that differ in the formation pathway but are then released by virtually all cells to their surrounding microenvironment . As a neRecent studies and clinical trials uncovered a number of hurdles that are faced by the cell-based therapies. The widely described obstacles affecting the efficacy of stem cell therapies result mainly from a lack of standardized treatment procedures, low percentage of cells that reach the desired tissue/organ, the poor survival of engrafted cells that often rapidly undergo apoptosis in targeted tissues, and finally from the diversity of yielded cell populations between individual donors . In addiAbovementioned obstacles and concerns prompted researchers to search for safer alternatives that could efficiently replace the living cells for therapeutic purposes. Consequently, EVs released by these cells become considered main promising candidates for cell-free therapies .Along these lines, unlike releasing cell, EVs do not contain all components of cellular machinery required for proper functioning of the living cell, but still they can substitute for its activity. The latter is likely possible due to the contained cargo, which enables EVs to perform a specific function. This assumption is supported by the fact that the cargo is sorted into EVs, exosomes especially, in a very sophisticated manner, and represents a unique content of proteins, RNAs, and lipids, usually differing from the parental cell . Thus, sIt is worth noting that, although EV-based strategies also do not have standardized protocols yet, other concerns and hurdles are either not relevant or could be easily avoided, since EVs are much more manipulable than their parental cells. The latter EV feature together with still expanding knowledge about their biology and functions opened up a new research area attempting both to improve the properties of cell-derived EVs and to design and manufacture their artificial counterparts  in orderin vivo, biocompatibility, ability to cross physiological barriers as well as from their rapid uptake into tissues  cell-derived EVs are coated with B1a cell-secreted antibody LCs . Detaileresponse , 2020  would direct them towards PD-L1-expressing tumor cells, while anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies would support regulatory T cell targeting . Hence, On the other hand, almost 20 years ago it was observed that cross-linking of major histocompatibility complex (MHC) class II-expressing EVs with latex beads greatly augments their biological effectiveness . Similarin vitro with transmission electron microscopy and nanoparticle tracking analysis after standardizing and optimizing the protocols of obtaining the aggregates. Whereas the methods to track the stability and bioactivity of EV’s aggregates in in vivo conditions have yet to be developed. Moreover, since aggregation was firstly proved for MHC class II-expressing EVs, the possible MHC-restriction of their activity has to be elucidated with regard to the possibilities to use EVs from allogeneic donors. On the other hand, one can speculate that autologous dendritic cell-derived EVs loaded in vitro with dedicated peptides in a direct or indirect manner (Additionally, some other aspects of this phenomenon require further investigation. Technically, EV’s aggregation could be controlled t manner  could bet manner .Last 2 decades have resulted in tremendous advances in the knowledge of the exceptional functions of EVs, which made them promising candidates for various therapeutic applications. Numerous current research findings have provided the basis for attempting to address the challenges faced by such therapies. Herein discussed possibilities to enhance EV’s biological activity and specifically direct them towards desired cells with the use of antibodies and LCs constitute crucial steps forward to achieve the highest efficacy of EV-based therapeutics.


Diffuse pulmonary meningotheliomatosis (DPM) is reported as a diffuse parenchymal lung disease characterized by disseminating small asymptomatic nodules. These lesions are often detected incidentally as microscopic findings in lung specimens or autopsies examined by a pathologist. We report a case of a 60-year-old male asymptomatic patient presenting with multiple bilateral pulmonary nodules on high-resolution computed tomography and diagnosed by videothoracoscopic surgery. Differential diagnosis of patients presenting with diffuse indeterminate nodules is very important. Definitive diagnosis of DPM requires histopathology and most often videothoracoscopic lung biopsy. Diffuse pulmonary meningotheliomatosis (DPM) is a rare disease characterized by the presence of diffuse benign small nodules of uncertain clinical significance. These nodules consist of epithelioid cells, which have an origin in the mesenchymal meningothelial cell line. These lesions are often detected incidentally as microscopic findings in lung specimens or autopsies examined by pathologist. We report a case of a patient with this rare finding in videothoracoscopic lung biopsy.carotid artery disease with stroke, arterial hypertension, nephrolithiasis, and urolithiasis. He did not have any known malignancy. The physical examination of the patient was within normal limits. Current high-resolution computed tomography (HRCT) of the thorax at the time of admittance displayed diffuse, multiple, 1-5 mm nodular lesions in the parenchyma of both lungs, with a slight predominance in the upper lobes , chronic ischemic heart disease, and pulmonary thromboembolism , 6.Nodules are detected on high-resolution computed tomography as tiny nodules (2-5 mm in diameter) or ground-glass opacity accidentally. DPM can mimic the appearance of a wide spectrum of multiple lung nodule diseases on a chest-computed tomography scan. There are mainly three categories of disease: nonneoplastic, metastatic neoplasms from a known or an unknown primary malignancy, and rare primary lung neoplasms . More coThe differential diagnosis of patients presenting with diffuse nodules is very important; the multidisciplinary team approach is needed. Routine diagnostic work-up is a matter of course, and definitive diagnosis of DPM always requires histopathology. DPM have been diagnosed also by using transbronchial biopsy, but mostly by videothoracoscopic lung biopsy. It is a safe modality with a high rate of diagnostic success. Positron emission tomography imaging in the diagnosis of multiple lung nodules have a role in the evaluation of indeterminate lung nodules larger than 8 mm in patients with low-to-moderate probability of malignancy . SmallerDPM consists of epithelioid cell nests or whorls in a zellballen-like arrangement centered on small veins , 4. HistIn therapeutical management of DPM, conservative treatment with observation and follow-up chest computed tomography imaging is usually favored .This rare eventuality of DPM should be considered in patients with nonspecific diffuse bilateral pulmonary nodules on high-resolution computed tomography. The differential diagnosis of these patients is very important. DPM can mimic several serious lung malignancies, infection, and interstitial lung diseases. Definitive diagnosis of DPM requires histopathology and most often videothoracoscopic lung biopsy.


To develop a new service model that engages and improves the provision of palliative care to PWUS.Although people who use substances (PWUS) continue to die prematurely compared to the general population, they are now more likely to die from chronic diseases rather than from drug-related deaths. Challenges to providing palliative care to PWUS include delayed care-seeking behaviours, complex drug interactions and lack of healthcare provider experience.An informal factorial analysis elucidated population needs through: a review of local databases to estimate the prevalence of palliative need, a thematic review into the deaths of patients in specialist drug services and, a survey of health practitioners’ knowledge and attitudes. These informed the service development phase which involves three key components: 1. A systems approach to increasing patient identification, incorporating key multi-disciplinary stakeholders across hospital- and community-based care 2. Targeted training of healthcare providers and 3. Medicines management for symptom palliation amidst concurrent substance use (including substitution treatments).The palliative needs of PWUS are under-identified: the local substance service was not partaking in the palliative referral pathway. Only 7% of a local hospice's annual caseload was recognised as having substance use problems. The care pathway was described as fragmented. Although >80% of surveyed palliative care practitioners had experienced caring for PWUS, confidence and knowledge around managing withdrawal, pain and opioid substitution therapies was poor.A new pathway is designed to identify PWUS and in their last year of life at key treatment points e.g., accident and emergency, ward-based care. The pathway will then streamline referrals to relevant specialist services depending on complexity of palliative/dependency need. Teaching resources and prescribing guidelines have been developed in collaboration with secondary care pain specialists.


Science, Mascharak et al.1 demonstrated that inhibiting Engrailed-1 activation in wound fibroblasts promotes scarless wound repair that phenocopies wound repair by regeneration seen in lower taxon groups and rarely in mammals.In a recent paper published in Many living species of chordates, including vertebrates, respond to injury by regenerating a perfect tissue replica. Mammals and humans on the other hand, with some rare exceptions, replaced true regeneration with scar tissue build-up in wounds. Scars form a quick seal that closes open wounds, and severely restrict tissue biomechanics, function and physiological performance. The mechanistic choice of wounded mammalian tissues to respond by either scarring or regenerating holds tremendous therapeutic potential for preserving and restoring the functions of injured tissues, but remains a biological puzzle.Engrailed-1 (En1). En1 is a homeobox transcription factor, which was thought to be expressed only at the early stages of embryonic development. En1 lineage-positive fibroblasts (EPFs) refers to the progeny of fibroblastic ancestors that had expressed En1 during early embryonic development. Adult EPFs have been demonstrated to be the fibroblastic lineage responsible for connective tissue build up in multiple fibrotic skin models, including excisional wound models and in melanoma tumour stroma formation. Whereas En1 lineage-negative fibroblasts (ENFs) are a separate lineage of skin fibroblasts that does not share a history of En1 expression, and in wounds refrain from contributing to fibrotic responses.2 EPFs and ENFs thus show lineage-specific functions as two specialized adult cell types.The skin serves a prototypical model to explore the underlying mechanisms of scarring and regeneration, because both scarring and regeneration of skin are seen across developmental time, diverse anatomic locations, and injury depths. The current existing cellular resolution of fibroblastic stromal cells in the skin indicates that functionally diverse embryonic lineages of fibroblasts co-inhabit the skin, and to a large degree dictate the skin’s diverging scarring and regeneration responses. In the backskin, two functionally distinct fibroblastic lineages were identified, based on the expression history of 1 have challenged this notion, and called lineage-restriction into question, by proposing that fully committed ENF fibroblasts can respond to tissue challenges, such as injury, by acquiring new fates and converting to EPFs. This phenotypic adaptability is termed plasticity. The authors show that in adult wounds En1 can be reactivated postnatally in ENFs via mechanotransduction signalling. The converted new EPFs are called postnatal EPFs (pEPFs), differentiating them from embryonic EPFs (eEPFs). The authors argue that turning off En1 blocks the ENF-to-pEPF conversion and is sufficient for skin regeneration with regrowth of skin apendages.1Mascharak et al.En1Cre-ERT;Ai6 and En1Cre;R26mTmG) coupled with splinted full-thickness excisional wound models, the authors precisely traced En1-lineage during wound healing processes, and revealed that ~40% of wound fibroblasts were pEPFs. Next, the authors utilized a tension loaded incisional wound model and tissue culture assays to show that mechanical tension significantly increased ENF-to-pEPFs conversion, and was correlated with elevated number of cells expressing Yes-associated protein (YAP) in nuclei, the key effector of the mechanotransduction pathway. The authors further showed that pharmacological inhibition of YAP by verteporfin, or diphtheria toxin-mediated targeted ablation of pEPFs in En1Cre-ERT;Ai6;R26iDTR wounds culminates in skin regeneration 30 days after wounding. To rule out the off-target effect of verteporfin, further investigation was carried in a pEPF-specific YAP knockout models . Almost complete abolishment of ENF-to-pEPF conversion and the same regenerative phenotype was observed.Using tamoxifen-inducible Cre mouse lines , holds tremendous potential for scarless wound repair. It may further provide a novel therapeutic tactic to ameliorate fibrotic responses and induce regeneration, by modulating biomechanical properties of the wound microenvironment or manipulating mechanotransduction in wound fibroblasts  of fibroblasts across all dermal layers.3 Embryonic EPFs should therefore outnumber pEPFs by 4 to 1, even if all adult ENFs convert to pEPFs. Furthermore, EPFs have been shown to be enriched in wounds by collective cell migration that is mediated by intercellular adhesion molecule N-cadherin upregulated specifically on EPFs.4 Therefore, it remains surprising that prevention of ENF-to-pEPF conversion is sufficient to induce regeneration, despite eEPFs being present in wounds and sharing the same profibrotic functions as pEPFs.1Despite providing evidence that biomechanical forces influence the pattern of regulation of En1 need to be reactivated in eEPFs for their scarring functions and what is the role of EN1 in this phenotypic conversion?Mascharak et al. show that ∼40% wound fibroblasts are pEPFs, a percentage that is not reachable via ENF conversion alone. This raises further questions as to whether ENFs include a subset of unipotent fibroblast progenitors that can differentiate into ENFs and EPFs? Is the conversion of wound ENFs to EPFs a differentiation process accompanied with proliferation or with N-Cadherin reactivation? Can this differentiation process be reversed after taking place? Does En1Cre;R26mTmG) resulted in substantial reduction of EPFs, including eEPFs that were ∼60% in wounds. Does inhibition of YAP deplete eEPFs or does it induce a conversion back to ENFs? A transition that would not be noticeable in a Cre-mTmG system, because once GFP starts expression upon Cre recombinase, tdTomato transgene is deleted, shutting down tdTomato protein expression. Importantly, the authors did not address the question of what is the exact functional consequences of En1 and of YAP in ENFs. High nuclear YAP was found in ENFs, but knockout of YAP specifically in pEPFs with En1Cre-ERT;YAPfl/fl transgenic mice leads to regeneration. Is YAP a critical upstream regulator of ENF-to-pEPF conversion, or of EPFs? How does mechanical tension differentially affect ENFs and EPFs to induce formations of two alternative connective tissue structures as in scarring and regeneration also remains an important question clinically requiring further investigation.Third, the treatment of YAP inhibitor verteporfin in wounds of non-inducible Cre lines (En1 transcription activation turns on the matrix remodelling functions in wound fibroblasts remains unknown. The exact downstream target genes of EN1 and the exact function of EN1 in controlling fibrogenicity remains undetermined. The answer to this question may provide additional insights on whether a silencing strategy against EN1 in wound fibroblasts can be used as an alternative approach for skin regeneration.Fourth, how does 5 The fascia fibroblasts have a similar ENF-EPF composition as in reticular dermis, but reside in a distinct viscoelastic jelly-like ECM environment with pre-made matrix fibres. Both excisional and incisional wound models used in the study of Mascharak et al. are full-thickness wounds, which certainly involve subcutaneous fascia. In fact, many YAP+ cells are present in deep wound layers, indicating their fascia origin. Considering that fascia is the tissue specialized in sensing mechanical stress/load, it is plausible that in addition to the reticular dermal fibroblasts, fascia fibroblasts are involved in scarring with the newly discovered mechanical sensing and ENF-EPF conversion mechanisms.A recent study demonstrates that the subcutaneous fascia is critical for the early repair of deep wounds and the subsequent scarring event.Last but not least, for imperative translational purposes, an important question remains about whether equivalent cells of ENFs and EPFs exist in human skin. The precise expression patterns of En1 on various human fibroblast subtypes are not well defined and need to be elucidated.With these questions and perspectives in mind, further investigation building on the basis of the work from Mascharak et al. and others would lead to achieve the ultimate goal of a scarless wound healing.


External beam radiotherapy is indicated in approximately 50-60% of human cancer patients. The prescribed dose of ionizing radiation that can be delivered to a tumor is determined by the sensitivity of the normal surrounding tissues. Despite dose intensification provided by highly conformal radiotherapy, durable locoregional tumor control remains a clinical barrier for recalcitrant tumor histologies, and contributes to cancer morbidity and mortality. Development of target-based radiosensitization strategies that selectively sensitizes tumor tissue to ionizing radiation is expected to improve radiotherapy efficacy. While exploration of radiosensitization strategies has vastly expanded with technological advances permitting the precise and conformal delivery of radiation, maximal clinical benefit derived from radiotherapy will require complementary discoveries that exploit molecularly-based vulnerabilities of tumor cells, as well as the assessment of investigational radiotherapy strategies in animal models that faithfully recapitulate radiobiologic responses of human cancers. To address these requirements, the purpose of this review is to underscore current and emerging concepts of molecularly targeted radiosensitizing strategies and highlight the utility of companion animal models for improving the predictive value of radiotherapy investigations. Radiotherapy is instrumental in treating many cancer types and can be used in curative intent treatments alone or in combination with surgery, chemotherapy, immunotherapy, and hormone therapy. In the setting of advanced cancer, radiotherapy can be used to stabilize and provide analgesia to patients, such as in the management of skeletal metastasis. Overall, it is estimated that external beam radiotherapy is indicated in approximately 50-60% of human cancer patients , 2. Owinin vivo modeling systems, and emphasize key qualities of preclinical animal models that would be expected to increase the predictive value of investigations involving radiotherapy.With intent to improve radiotherapy responses, considerable focus has been to identify mechanistic and specific molecular targets to sensitize tumor tissue to radiotherapy. The clinical success of targeted radiotherapy is dependent on a thorough understanding of molecularly-driven radiobiologic responses, detailed preclinical investigation and appropriate clinical trial design. Despite recognizing these necessary benchmarks, many targeted radiotherapies end in disappointment, failing to provide favorable outcomes in clinical trials. In general, the failures observed in oncology clinical trials suggests the need to improve molecular target evaluation and standard preclinical modeling systems. This review will highlight several targets for radiosensitization, the necessity of appropriate )  . This clInhibiting the function of molecules operative in the DDR, such as ataxia telangiectasia and Rad3-related (ATR), ataxia telangiectasia mutated (ATM), and DNA-dependent protein kinase (DNA-PK), have successfully sensitized cancer cells to ionizing radiation –12. BlocMore sophisticated and selective targeting of tumor cells can be achieved by performing strategic molecular profiling, highlighting specific targets of the DDR and DNA repair pathways to enhance the radiosensitivity of tumor tissues. Impaired cell cycle regulation is a common feature of cancer and most often exemplified by mutated or altered regulatory status of p53, which hampers the tumor cells’ ability to respond to DNA damage. In cells with functional p53, DNA damage results in G1/S checkpoint activation and arrest; allowing time for DNA damage to be faithfully repaired prior to DNA synthesis, and preventing accumulation of DNA damage and its mutagenic consequences. In p53 deficient tumor cells, the G1 checkpoint is not activated, and chromosome replication is initiated despite the existence of DNA damage. With the cell cycle continuing unabated, inhibition of SSB repair (such as PARP inhibition) would ultimately result in augmented DSBs in tumor cells as the replication fork meets the sites of SSBs . AdditioDrawing upon analogous strategies used to identify druggable targets that mitigate the development of chemotherapy resistance, the targeted disruption of molecular radioresistance mechanisms can serve as a synergistic avenue for improving radiotherapy efficacy. Enhanced DNA repair mechanisms may allow tumor cells to resist injury by IR. Overexpression of factors involved in NHEJ –23 and HCells possess signaling pathways that govern cell survival, growth, and proliferation. Signals may be received at the cell surface, or may arise intracellularly, and the response follows a cascade of events in the cytoplasm and nucleus of the cell. Signaling pathways are often dysregulated in cancer cells and these aberrant pathways have been investigated for targeted therapy and the development of personalized medicine. Key signaling pathways harboring potential targetable options to improve radiotherapy response include the PI3K-AKT, MAPK, and NFĸB pathways. A common theme among these pathways is their dysregulated activation favors apoptosis resistance in irradiated cells –34. InteThe PI3K-AKT pathway is traditionally known as a cell survival pathway, though it also plays a role in cell growth and proliferation. In this pathway, Ras phosphorylates and activates PI3K leading to the formation of PIP3 on the inner leaflet of the plasma membrane. AKT recognizes PIP3 and ultimately becomes phosphorylated, activating its kinase function. This pathway can be dysregulated by multiple different mechanisms, and tumor cells with activating mutations in Ras are radioresistant as a consequence of PI3K-AKT pathway activation , 36. AltTranslating these foundational radiobiologic investigations to clinical practice, PI3K inhibition in a human breast cancer study resulted in HR deficiency by downregulating BRCA expression . This leIn addition to the activation of the PI3K-AKT by EGFR, ionizing radiation can cause EGFR, without activating its kinase domain, to be imported into the nucleus where it can directly bind and activate DNA-PK leading to DSB repair by NHEJ , 43. IntIn the MAPK pathway, ligand binding to a receptor tyrosine kinase (RTK) leads to a series of phosphorylation events from Ras → Raf → Mek → Erk1/2, which then results in the activation and production of transcription factors (inclusive of Fos and Jun) that stimulate cell growth and proliferation. There is conflicting evidence about the significance of the MAPK pathway in modulating radiotherapy response, with some evidence suggesting no role of the MAPK pathway , whereasThe nuclear factor-kappa B (NFκB) signaling pathway plays critical roles in inflammation, immune function, and has implications in the development and progression of cancer . (NFκB) While the mechanisms remain to be fully elucidated, other signaling pathways commonly dysregulated in cancers may be targets of radiosensitization strategies. (TGFβ) exerts pleiotropic and complex actions in cancer cells; however, there is evidence that (TGFβ) inhibition can impair DDR, possibly through reduction of ATM kinase activity . Pathwayin vitro experiments and highly manipulatable murine models. However, over the last few decades, a broader horizonal view for improving radiotherapeutic outcomes has been realized based upon the understanding that tumors are complex tissues containing multiple cell types that participate in heterotypic interactions. These scientific revelations now recognize the importance of the tumor microenvironment (TME) in cancer treatment strategies. Research targeting tumor cells directly has traditionally served as the cornerstone strategy for improving radiotherapy response. This approach may be in part attributed to the ease of performing The most extensive investigation of the TME revolves around tumor vasculature and hypoxia. Intra-tumoral hypoxia dynamically evolves during tumor growth and the distribution of hypoxia is determined by the distance to the nearest perfused capillary, the interstitium composition, and oxygen consumption by cells. These factors contribute to diffusion-limiting, or chronic hypoxia. Another form of hypoxia in tumors, referred to as perfusion-limiting or acute hypoxia, occurs as a consequence of having haphazard and abnormal structure of vasculature. Hypoxia occurs frequently in solid tumors and hypoxic cells within tumors are up to three-fold more radioresistant , 61. TheIn response to hypoxia, cells stabilize hypoxia inducible factor (HIF)-1α through decreased activity of prolyl-4-hydroxylases (PDH), which regulates HIF-1α’s half-life. PDH functions to hydroxylate proline residues on HIF-1α in normoxic conditions, allowing it to be recognized by the tumor suppressor protein Von Hippel-Lindau (VHL). VHL ubiquitinates the hydroxylated form of HIF-1α, marking it for proteosomal degradation and preventing its action. The stabilized HIF-1α under hypoxic conditions binds HIF-1α and the heterodimer acts as a transcription factor, binding to hypoxia responsive elements (HRE) and increases the transcription of target genes . It is wOne of the many different target genes upregulated by HIF-1α is vascular endothelial growth factor (VEGF). VEGF is a ligand for a RTK (VEGFR) on the surface of cells. In the case of endothelial cells, VEGF signaling promotes survival and proliferation leading to angiogenesis. Tumors must build a vascular network to provide nutrients and oxygen to permit growth. Given the dependence of oxygen for radiation-induced ROS generation, it seems counterintuitive that inhibiting the VEGF signaling pathway would result in an improved antitumor effect when combined with radiotherapy. In fact, evidence suggests that inhibiting angiogenesis in preclinical models results in an increase in the oxygenation of tumors –74. MechThis notion has led to the concept of normalizing tumor vasculature to improve combination therapies , which rInterestingly, HIF-1α activity increases following radiotherapy as a result of tumor reoxygenation and the formation of reactive oxygen species. The resulting increase in HIF-1 target gene expression has been shown to promote endothelial cell radioresistance, and HIF-1α inhibition enhanced radiotherapy response due to enhanced vascular destruction . This suRepresenting an interesting and underdeveloped field is the influence of radiotherapy-induced tissue stiffness on tumor cell biology. Cells are continuously exposed to physical forces and they respond by modifying their own behavior and remodel the surrounding environment . Such “oin situ vaccine strategy  and be presented to effector T cells. In a form of cell death referred to as immunogenic cell death, irradiated tumor cells display and liberate molecules  to enhance this APC activation. The key immune activating molecules liberated or displayed by tumor cells during radiation-induced immunogenic cell death include calreticulin, high mobility group box protein 1 (HMGB1), and ATP –92. The strategy .Perhaps the best studied and most exciting combinations involving immunotherapy and radiotherapy are checkpoint blockade strategies. Checkpoint inhibitors currently in use are immunomodulatory antibodies, and are so named because they block normal negative regulators of T cell immunity, consequently removing their brake system . The tar+ cytotoxic T lymphocytes (CTLs)  . Studiess (CTLs) –103. Whis (CTLs) . Thus, ps (CTLs) .The contrasting immunostimulatory and immunosuppressive effects of radiotherapy are related to the co-existence of pro-tumor and anti-tumor immune cells within the TME. CTLs, M1 macrophages, and natural killer (NK) cells are considered to be the most effective anti-tumor cells within the TME; whereas pro-tumor immune cells include T regulatory cells (Tregs) and M2 macrophages . Multiplhttps://ccr.cancer.gov/comparative-oncology-program) and there is an increasing awareness of the value of spontaneous tumor models in drug development (https://ccr.cancer.gov/comparative-oncology-program/consortium) is made up of 22 comparative oncology centers, many of which are equipped with state-of-the-art radiotherapy technology. With such a collaborative network in place, companion animal models are uniquely positioned to advance drug-radiotherapy combination development. While strong translational value can be gained through purposeful inclusion of companion animal models in radiotherapy-drug development, studies in companion pets should not be viewed as a substitute for traditional preclinical investigations. Instead, companion animal models are expected to improve the predictive value of preclinical investigations.Comparative oncology involves the study of naturally occurring cancers in nonhuman species that function as a complementary model for advancing human cancer research –114. Comelopment . The ComAdditional key qualities provided by companion animal models revolve around the relative ease of sample collection. Companion animals, especially the dog, are frequently used in pharmacokinetic and toxicologic studies during drug development. This information can be used to streamline the use of the tested species in efficacy and proof-of-concept investigations. For the rational selection of targeted molecular therapies, rapid data collection and interpretation of individual tumors is imperative. The COTC previously established molecular characterization techniques for dog tumors . The stuThis review highlights several potential molecular targets and pathways for the rational development of radiosensitizing strategies. Importantly, given the conserved DNA damage responses and tumor histologies shared between human beings and companion animals, unique opportunity is afforded by the purposeful inclusion of pet dogs and cats with naturally occurring cancers for validating investigational radiotherapeutic interventions. In addition to shared tumor histologies, the faithful recapitulation and evolution of key tumor microenvironmental factors inclusive of vasculature, immunocytes, and stroma, can be powerfully leveraged for more accurate preclinical modeling of novel radiotherapeutic protocols. Companion animals have already been used to advance the understanding of tumor radiobiology and treatment. With this proven and existing foundation, companion animals with naturally occurring cancers will likely improve the predictive value of target based radiosensitization strategies and accelerate the translation of innovative radiotherapeutics regimens in human cancer patients.MB and TF envisioned contents of review article. MB wrote original draft and developed all figures. MB and TF revised manuscript and figures for final submission. All authors contributed to the article and approved the submitted version.This review article was supported by Morris Animal Foundation award D19CA-064.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although the survival rate of preterm infants has improved over the years, growth failure and associated impaired neurodevelopmental outcome remains a significant morbidity. Optimal nutrition plays an important role in achieving adequate postnatal growth. Accurate growth monitoring of preterm infants is critical in guiding nutritional protocols. Currently, there is no consensus regarding which growth assessment tool is suitable for monitoring postnatal growth of preterm infants to foster optimal neurodevelopmental outcomes while avoiding future consequences of aggressive nutritional approaches including increased risk for cardiovascular disease and metabolic syndrome. A retrospective single center cohort study was conducted to compare the performance of two growth-assessment tools, Fenton and Intergrowth-21st (IG-21st) in the classification of size at birth, identification of impaired growth and predicting neurodevelopment. A total of 340 infants with mean gestational age of 30 weeks were included. Proportion of agreement between the two tools for identification of small for gestational age (SGA) was high 0.94  however, agreement for classification of postnatal growth failure at discharge was moderate 0.6 . Growth failure at discharge was less prevalent using IG-21st. There was significant association between weight-based growth failure and poor neurodevelopmental outcomes at 12 and 24 months of age. Although the survival rate of the greater than 15 million preterm infants born annually has improved, they have higher morbidity with 20–45% having suboptimal neurodevelopment compared to infants born at term, further challenging clinicians in optimizing care to improve long-term outcomes ,4. Nutri0/7 to 326/7 weeks of gestation admitted to the neonatal intensive care unit at the Children’s Hospital of Richmond (CHoR)who survived to discharge between the years of 2014 and 2018. Infants with major congenital anomalies and chromosomal abnormalities including all aneuploidies, congenital brain malformations, and congenital defects that are require prolonged parenteral nutrition and are associated with poor growth were excluded. Data on infant demographics, anthropometry measurements, short-term neonatal outcomes and neurodevelopment assessment scores were obtained from medical records. Short-term neonatal outcomes included bronchopulmonary dysplasia , necrotizing enterocolitis (Bell stage ≥ II), sepsis (positive blood culture), retinopathy of prematurity (retinopathy requiring treatment), any intraventricular hemorrhage and periventricular leukomalacia. Growth parameters such as weight, length, and head circumference were measured at regular intervals by NICU nurses per unit protocol. Weight, length, and head circumference centiles and Z-scores were determined using bulk calculators for Fenton (2013) and IG-21st growth chart, which are publicly available at Fenton Growth Chart|University of Calgary ), and INTERGROWTH-21st (ox.ac.uk (accessed on 9 April 2021)) respectively. While using IG-21st, we utilized newborn size for preterm infants’ chart to assess size at birth and the postnatal growth of preterm infants’ charts to assess postnatal growth. Neurodevelopment was assessed using Bayley Scales of Infant Development 3rd edition (BSID-III) at 12 and 24 months by trained and certified examiners. The primary outcome of the study was poor neurodevelopmental outcome defined as composite score < 85 in any of the development subsets tested either at 12 or 24 months. The study protocol was approved by the Virginia Commonwealth University Institutional Review Board.This is a retrospective cohort study of preterm infants 24t-tests were used to compare Z-scores between the two charts. Unadjusted Logistic regression models were constructed to quantify the differences in poor neurodevelopment outcome between infants with and without growth failure in weight as classified by each growth chart. Additionally, an adjusted model was performed after controlling for SGA that is known to affect neurodevelopment [p-values are presented. For each growth reference, the diagnostic performance and discriminatory ability was calculated via area under the receiver operating characteristic curves (AUROC) and compared between the two tools.Descriptive statistics is provided for demographic data and baseline characteristics. Median and IQR were used to summarize all continuous variables, while frequencies and percentages summarized all categorical variables. In addition, the preterm infant’s growth z-score in weight, length, and head circumference at birth was summarized by both Fenton and IG-21st. The Z-score at discharge, and the change in z-score between birth and discharge for all growth parameters was also summarized by each chart. Cohen’s Kappa  was used to assess the agreement between Fenton and IG-21st, and paired elopment . Unadjus0/7 and 326/7 weeks of gestation during the study period of January 2014–December 2018, 42 (9.8%) were excluded for congenital or chromosomal abnormalities and 46 (11.9%) died while in hospital. A total of 340 infants who survived to discharge were included and analyzed  with an average birth weight of 1310 g  and 55% male infants. Demographics and prevalence of common neonatal morbidities are shown in Follow-up for standardized neurodevelopmental testing was 37% and 22% at 12 and 24 months, respectively. There was no difference in identifying SGA infants at birth using Fenton versus IG-21st; however, the prevalence of growth failure for both weight and length were higher by Fenton than IG-21st charts . The prop = 0.094). It is notable that the variability with this association is considerably larger than all other associations of interest as evident by the very wide confidence interval. There appears to be insufficient evidence of any other growth failures (in both tools) being associated with any of the other outcomes of interest. While the AUROC for this near significant predictor is larger than all other predictors (AUROC = 0.608) it is still low implying poor predictive capability of adverse neurodevelopmental outcome in language.The unadjusted logistic regressions  between p = 0.049). The odds of a poor neurodevelopmental outcome in language in infants with weight-based growth failure per IG-21st are nearly four times greater  than infants who do not have such growth failure per IG-21st after adjusting for SGA. In terms of predictive capability, it appears that adjusting for SGA slightly improved AUROC (by about 0.05) in both tools for the language neurodevelopmental outcome, while having negligible or no improvement in AUROC for the cognition and motor outcomes. Due to the small event size from small sample size, the variability in the association between the outcomes and SGA in both tools is quite large, leading to very wide confidence intervals. The small event size did not allow for the adjustment of other predictors known to affect neurodevelopmental outcomes such as IVH, BPD, NEC, IVH, and PVL.However, as shown by Growth charts based on cross-sectional size-at-birth measurements by gestational age should be used cautiously to monitor postnatal growth as intrauterine and extrauterine growth is not comparable owing to the different biological processes, environmental and nutritional approaches that preterm infants are subjected to after birth . In thisSeveral definitions have been used to define postnatal growth failure with weight percentiles at discharge below a previously set cut-off value the 10th percentile being the most common one. However, this definition can potentially misclassify infants born SGA who exhibit desirable growth patterns but may be discharged at <10th percentile. Furthermore, other studies have suggested the use of standard deviation scores (Z-score) for longitudinal assessment of growth as it describes growth regardless of centiles at birth and discharge. Based on available literature, we defined growth failure at discharge by a Z-score drop >1 from birth to discharge in weight, linear, or head growth ,21,22. ISince the development of this growth reference tool, few studies have compared its performance against the fetal-based growth charts commonly used in classifying growth abnormalities, and they fall short of recommending its clinical adoption due to lack of long-term outcome studies on large population samples of preterm infants. This study highlights a potential difference in predictive ability of fetal-based charts versus postnatal growth charts based on healthy preterm longitudinal data. A future study on a large multicenter population-based data to further explore the functional implications of use of specific growth charts in assessing long term outcome risk is warranted.In this single center retrospective study, we have demonstrated that postnatal weight and length-based growth failure was less prevalent among preterm infants using IG-21st compared to Fenton charts. The difference in growth is persistent throughout the postnatal period. Weight-based growth failure defined by IG-21st may be more strongly associated with poor neurodevelopmental outcomes than Fenton. Given this significant difference in growth pattern assessment using these two growth charts, we believe future large observational studies are warranted to further delineate the predictive ability of these growth charts in identifying postnatal growth failure that is independently associated with long-term morbidity. This will allow for discerning the relevant functional impact of each tool and support or refute their clinical use.


DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks. DNA replication is therefore a fundamental process necessary for cell division, organism development, tissue homeostasis, and cell renewal. Genome duplication occurs during S-phase, and the associated DNA synthesis is overall highly accurate. Nonetheless, many endogenous and exogenous factors can challenge the process of DNA replication, a phenomenon that is referred to as replication stress. Replication stress can be defined as any event that alters the rate of DNA replication. This includes the deceleration of replication fork progression, a well-recognized feature of replication stress, as consequence of a myriad of fork obstacles . In. In77]. proteins . Defectsproteins ,80. Inteproteins ,82,83. Bproteins .Beyond the function of SUMOylation in regulating DNA repair and replication factors’ activity and cellular localization, SUMO metabolism is critical to the mobility of DNA lesions and their anchorage to NPC.S. pombe), known as the Nup84 complex in budding yeast [The double-layered nuclear membrane is penetrated by large macromolecular structures called NPCs that have an estimated mass of ~50 MDa in yeast and 112 MDa in vertebrates . Cryo-elng yeast ,87,88.nup84Δ or nup133Δ) lead to sensitivity to genotoxic drugs and replication stress [NUP84 was reported to cause a delay in replication fork progression in the presence of DNA damage [NUP133 in budding yeast and humans) leads to sensitivity to replication stress but not to DSBs or UV-induced DNA damage, and Nup132 promotes DNA replication recovery upon transient fork stalling [Beyond the canonical function of NPCs in the selective import/export of proteins and RNAs, those large structures contribute to the regulation of gene expression, 3D organization of the genomes, DNA repair processes, and maintenance of genome integrity ,90,91. Sn stress ,92. AddiA damage . In fissDrosophila, heterochromatic DSBs relocate to the nuclear periphery in an Nse2- and PIAS-dependent manner [Evidence gathered over the past two decades from numerous studies support the concept that NPCs act as docking sites for different types of DNA lesions. However, the exact NPC components involved in anchoring DNA lesions are unknown. The anchorage of DNA lesions is dependent on SUMO metabolism and both monoSUMOylation and SUMO chains’ formation, indicating that SUMO constitutes the key signal for NPC anchorage . A curret manner . In thisIn budding yeast, SUMOylation plays a key role in the nucleolar dynamics by ensuring the compartmentalization of HR activities. Indeed, replication-born DSBs within rDNA sequences shift from the nucleolus to anchor to NPCs and maintain repeat integrity . RecentlA recent study on budding yeast highlighted SUMO-independent alternative mechanisms by which NPCs regulate HR activity . ReplicaBoth chromatin context and repeated sequences affect the scenario by which DSBs are repaired; SUMOylation events appear necessary to move DSBs away from their compartments and prevent RAD51 loading until DSBs are relocated in a “safer” environment to complete DNA repair. Both similar and distinct scenarios have emerged for the repair of arrested forks.Akin to SUMO’s role as a nuclear positioning signal for DSBs, SUMO-dependent relocation of replication stress sites was recently discovered. In budding yeast, tri-nucleotide repeats, such as CAG, have a tendency to form secondary DNA structures prone to stall replication forks . Such stRTS1-RFB, which mediates a DNA-bound protein block to replisomes, were recently shown to relocate and anchor to NPCs in S-phase  also stimulated the efficiency of RDR. NPC anchorage is then necessary to clear off SUMO conjugates by the proteasome and the SENP protease Ulp1, two activities enriched at the nuclear periphery. In the absence of Nup132, Ulp1 is delocalized from NPC and less expressed . In thisOverall, these finding reveal that the switch between monoSUMOylation and the SUMO chain formation at arrested replisomes likely constitutes a quality-control step that dictates the choice of replication fork repair pathways in the nuclear space. Moreover, the SUMO metabolism differentially influences the fate of arrested replisomes according to sequences’ context; at repeated sequences, SUMOylation restrains Rad51 loading until the stalled forks anchor to NPCs, whereas at unique sequences, SUMOylation is necessary to maintain fork integrity until SUMO chains trigger NPC anchorage to allow an efficient fork restart. This suggests that additional features such as chromatin landscape influences SUMOylation features at sites of replication stress.SUMOylation connects replication stress sites to NPCs that act as molecular hubs to regulate HR activity. Different scenarios have emerged according to the type of fork obstacles and their surrounding sequences environment and organisms. The mechanisms triggering the relocation of forks arrested at repeated sequences, such as at expanded CAG, are presumably distinct from those involved in the relocation of protein-mediated fork stalling. It is evident that cells have evolved pathways to restrict the access of Rad51 at repeated sequences to limit deleterious HR events and preserve a constant size of repeats. Such pathways may limit fork-restart efficiency when the forks arrest at unique sequences. However, all the scenarios reveal that a spatially segregated SUMO metabolism is critical to ensure genome integrity at replication stress sites. Many questions remain to be addressed: How is the division of labor organized between distinct E3 SUMO ligases in yeast and human nuclei upon replication stress? What are the mechanisms engaged at the NPC or nuclear periphery that ensure an efficient and error-free fork restart? How are these NPC-related mechanisms coordinated with the global DDR response? How do chromatin organization and potential histone marks influence SUMO metabolism at sites of replication stress? Finally, most DSBs do not relocalize to the nuclear periphery or NPCs, raising questions about how the molecular and structural determinants make replication stress sites prone to relocation and NPC anchorage. Deciphering the repertoire of SUMOylated factors at replication forks upon various replication-blocking agents will certainly provide additional layers to answer these questions.


Microsatellite instability (MSI) is a molecular fingerprint for defects in the mismatch repair system (dMMR) and is associated with higher risks of cancers. MSI/dMMR tumours are characterized by the accumulation of mutations throughout the genome, and particularly in microsatellite (MS) DNA repeat sequences. MSI stands as a major biomarker for familial cancer risk assessment, cancer prognosis, and therapeutic choices. Standard-of-care classification of MSI/dMMR tumours is most frequently achieved using immunohistochemistry or PCR-based assay directed against a set of five MS regions. However, novel molecular methods based on tumour tissue or plasma samples have been developed and could enter in the future trends of MSI testing. Here, we provide insights into these emerging approaches and discuss their advantages and limitations.Microsatellite instability (MSI) is a molecular scar resulting from a defective mismatch repair system (dMMR) and associated with various malignancies. MSI tumours are characterized by the accumulation of mutations throughout the genome and particularly clustered in highly repetitive microsatellite (MS) regions. MSI/dMMR status is routinely assessed in solid tumours for the initial screening of Lynch syndrome, the evaluation of cancer prognosis, and treatment decision-making. Currently, pentaplex PCR-based methods and MMR immunohistochemistry on tumour tissue samples are the standard diagnostic methods for MSI/dMMR. Other tissue methods such as next-generation sequencing or real-time PCR-based systems have emerged and represent viable alternatives to standard MSI testing in specific settings. The evolution of the standard molecular techniques has offered the opportunity to extend MSI determination to liquid biopsy based on the analysis of cell-free DNA (cfDNA) in plasma. This review aims at synthetizing the standard and emerging techniques used on tumour tissue samples for MSI/dMMR determination. We also provide insights into the MSI molecular techniques compatible with liquid biopsy and the potential clinical consequences for patients with solid cancers. The mismatch repair (MMR) machinery is an evolutionarily conserved system responsible for the preservation of DNA homeostasis in cells . The MMRV600E mutation  . In. In® MSI methods ,76,77,78 methods . The IdyPCR (ddPCR)–based assay  using the same five MS markers than those previously described in the Promega® Analysis system    78]. Th. ThPCR ) in colorectal cancer samples [Next-Generation Sequencing (NGS) currently represents a widely used technology that facilitates personalized cancer therapy through the research of numerous actionable alterations in a single assay. Recently, NGS has been adapted for the focused purpose of MSI testing. Some groups proposed NGS panels that integrate the specific detection of MSI ,82,83. A samples .The analysis of long mononucleotide repeats (>15 bp in length) has long been preferred for MSI determination considering their high level instability in tumours . HoweverThe MSI status could also be inferred using NGS data from whole-genome, whole-exome, gene targeted, or RNA sequencing that were not originally developed for MSI diagnosis . During The advantage of NGS methods over PCR-based assays is that they provide the MSI status at the same time as other relevant cancer-related alterations. This one-step approach is of particular interest for cancer types with low MSI frequency for which MSI-specific testing is not systematically performed. The widespread deployment of NGS notably highlighted the fact that the MSI status is much more prevalent than previously thought . MoreoveTumour biopsy represents the reference source of material for MSI determination; however, its clinical use has several limitations. First, tissue material is sometimes inaccessible due to tumour location and results from an invasive procedure associated with potential surgical complications . Second,Liquid biopsy (LB), which refers to the analysis of circulating tumour DNA (ctDNA) shed into the body fluids  by tumour cells, has emerged last few years as a promising surrogate for tumour biopsy. LB is presented as a minimally-invasive and easily-repeatable tool, which overcomes the issue of spatial and temporal heterogeneity and allows the longitudinal monitoring of the disease through iterative sampling . LB has Considering the current trend toward tumour molecular diagnostics based on ctDNA analysis and the multiple stakes of MSI on cancer management, there is a growing need to develop novel approaches for MSI diagnosis on blood samples (bMSI). One of the major technical challenges for bMSI determination is that it requires highly sensitive methods due to the highly fragmented nature of ctDNA and the small fraction of ctDNA among total cfDNA in the body fluids (as low as 0.01% in early stages of cancers) . In the In cfDNA, altered microsatellite sequences can be missed due to their high contamination with germline DNA molecules and the presence of stutter bands at homopolymer regions when CE or HRM is employed for end-point detection. The enrichment of unstable microsatellites was proposed as an option prior to PCR-based amplification to improve the detection of low-frequency mutant alleles. The NaMe-PrO  approach consists of overlapping oligonucleotide probes and double-strand-specific nucleases, which specifically eliminate long unaltered homopolymer regions while sparing those harbouring indels. NaMe-PrO combined with CE or HRM attained a limit of detection of 0.01% mutant microsatellite allele frequencies ,113. HowIn order to efficiently capture low fraction cfDNA present in plasma samples, Yu et al. developed an inter-Alu-PCR-NGS approach that combines a fast and easy-to perform PCR assay with a NGS-based broad molecular profiling . Alu seqSilveira et al. evaluated the analytical performance of the previously described 3-marker  drop-off ddPCR approach on blood samples from patients with advanced or metastatic CRC and endometrial cancers . Using t® CDx  and FoundationOne® Liquid CDx  are commercial FDA-approved blood-based companion diagnostics that have been adapted for specific bMSI determination. The Guardant360® CDx, FoundationOne® Liquid CDx, OncoLBx and Georgiadis approaches all employ hybrid-capture enrichment of target regions and rely on molecular barcoding to help filter false positive events arising due to technical PCR errors. They also integrate in silico error correction approaches in order to reduce the background noise and accurately recover true insertion-deletion events in MS regions. Due to the fragmented nature of ctDNA, Willis et al. showed that some MS loci traditionally used for tissue-based MSI testing are not suitable for ctDNA-based NGS approaches due to low coverage or background noise among these regions [® panel, they selected limited but highly informative MS loci based on their coverage and noise profiles in order to improve molecular capture and mapping efficiency. Along with bMSI status, Georgiadis and FoundationOne® Liquid CDx methods offer the possibility to determine blood based tumour mutation burden (bTMB), a complementary biomarker that help inform ICI treatment. For all panels described, a minimum of 5–30 ng cfNDA input was required. To note, some groups implemented specific bMSI computational algorithms for NGS data in the context of liquid biopsy analysis, enabling the detection of very low fraction of ctDNA (0.05–0.5%)  .A good overall agreement has been observed between conventional MSI tissue-based testing and newly developed ctDNA-based approaches ,117,118.Determination of MSI status in cancers is of particular clinical importance considering its diagnostic, prognostic, and therapeutic significance. IHC and pentaplex PCR account for MSI/dMMR standard reference methods on tumour tissue specimens. However, other approaches  have emerged and progressively entered into clinical practice. Given the multiple methods currently available, the approach to be used should be chosen considering the cancer type, the preanalytical conditions, the lab resources and technical expertise, and the availability of paired normal tissues.In the last few years, liquid biopsy led to a major paradigm shift in oncology, providing a viable surrogate to tissue biopsy for molecular investigations. As other ctDNA markers, bMSI can be detected in body fluids and contributes to predict treatment efficacy and follow disease evolution over time. The use of bMSI-based strategies has been initially confounded by lack of sensitivity; however, recent technological advances in the field showed potential in reducing background noises and enhancing detection efficiency. Further translational studies are needed to confirm the clinical utility of bMSI-based approaches and delineate their potentials applications in routine practice.


Recently, the STOP Persistent Atrial Fibrillation trial demonstrated cryoballoon (CB) ablation to be an effective and safe tool as an initial ablation approach in patients with drug-resistant PSAF.2 However, a perceived drawback of utilizing balloon catheter technologies in patients with PSAF has traditionally been the perception of lack of ability to perform linear ablation lesion sets or address reentrant atrial arrhythmias adequately without using radiofrequency ablation (RFA) catheters.Achieving sustained freedom from atrial arrhythmias following catheter ablation can be an elusive endpoint, especially in individuals with long-standing persistent atrial fibrillation (AF) (PSAF) for whom multiple mechanisms may be operant.1 describe the successful use of an extensive integrated left atrial (LA) ablation approach using only a third-generation CB catheter in a patient with persistent AF who developed LA atrial flutter. In addition, the case report also reinforces the feasibility of LA appendage (LAA) electrical isolation (LAAEI) followed by implantation of an LAA occlusion device concurrently in the same procedure to avoid hazards of stasis/potential thrombosis after LAAEI in an individual for whom long-term oral anticoagulation may not be the preferred option.In this issue of the journal, Aryana et al.3 Furthermore, even if patients remain free from AF recurrence post-ablation, those with diseased substrate still often have a higher proclivity for developing reentrant atrial arrhythmias, which must be addressed with additional linear lesion sets. Thus, an argument could be made that upfront extensive ablation approaches may be beneficial in some cases to debulk and potentially address problematic substrate and limit the “real estate” available for the perpetuation of reentrant arrhythmias.Central to the rationale for the approach utilized in this case was the recognition that pulmonary vein (PV) isolation (PVI) alone is often insufficient in patients with longstanding PSAF. It has been shown that the substrate for the initiation and maintenance of AF involves anatomical sites outside the PVs, such as the posterior LA wall, LAA, superior vena cava, and bundles of the LAA–left superior PV (LSPV) ridge.4 the acute procedural success rate of PVI + posterior wall isolation (PWI) was 94.1% . Among patients with PSAF, 12-month freedom from atrial arrhythmias following a single procedure was 61.9% , which was similar to the effectiveness of PVI only. Notably, two atrioesophageal fistulas occurred , both cases in the RF–PWI group. A subsequent randomized controlled trial (RCT) by Lee et al.5 also found that a PWI strategy did not lead to a significant difference in freedom from AF  at 16 months. However, an RCT by Aryana et al.6 comparing PVI alone to PVI + PWI in patients with longstanding PSAF demonstrated a significant reduction in recurrence of AF at one year  using CB ablation with an adjunctive RFA approach. A large multicenter study—Left Atrial Posterior Wall and Posterior Vein Isolation Using Cryoballoon for Treatment of Persistent Atrial Fibrillation (PIVoTAL-IDE)—is currently underway to further evaluate the efficacy and safety of this approach.7The posterior wall of the LA has been implicated in the initiation and maintenance of PSAF. In a meta-analysis of 17 studies by Thiyagarajah et al.1 was LAAEI using an ostial CB ablation approach. Previously Di Biase et al.8 examined 987 patients  and observed that, in 27% of patients referred for a redo ablation, the LAA appeared to be the source of drivers for AF. When LAAEI was performed, the rate of AF recurrence was 15%, compared to 74% in patients who did not receive ablation and 68% in patients undergoing only focal ablation (P < .001). These findings were further supported in the Left Atrial Appendage Isolation in Patients with Longstanding Persistent Atrial Fibrillation Undergoing Catheter Ablation (BELIEF) trial.9 However, some concerns have been raised about the risk of post-procedural LAA thrombus, which was detected in 23.2% of patients who underwent LAAEI versus 2.2% in the control arm (P < .001) in a prospective study despite appropriate anticoagulation.10 A subsequent systematic review and meta-analysis including 2,336 patients in 9 studies by Romero et al.11 found no significant difference in the rates of cerebral thromboembolism with LAAEI versus standard LA ablation .9 Nonetheless, in the case presented by Aryana et al.,1 an LAA occlusion device was implanted in the same procedure to mitigate the risk of post-procedural thromboembolism.Another strategy employed in this case report by Aryana et al.12 several prospective studies examining combined CB PVI and LAAO seem to suggest a low incidence of developing significant peri-device leaks post-procedure.14 Thus far, only a single study15 (n = 22) has prospectively studied the LAAEI approach versus RF PVI only and found higher freedom from AF in the LAAEI group  and an LAAEI time of 33.5 ± 27.7 minutes. In the cohort, 5% of patients developed peri-device leaks greater than 5 mm on 45-day transesophageal echocardiography imaging. Whether the CB technique can be utilized as a more efficient approach for LAAEI with perhaps a lower risk of peri-device leak after device implant may be an important area of future study.Although concurrent RF PVI and LAAO implantation has been associated with the incidence of increased or new significant (>5 mm) peri-device leak on post-procedural imaging,1 demonstrates the feasibility and importance of developing innovative techniques for single-modality ablation, especially as recent trends in catheter technology development have focused more so on improving acute procedural endpoints mainly for PVI. As increasing financial constraints limit the practicality of mixing energy sources in the same procedure, development of single-modality techniques for extensive LA ablation may become of increasing importance. Patients with longstanding PSAF have traditionally fared poorly with ablation strategies. Looking forward, larger clinical studies will be needed to validate the generalizability of the approach used in this case study given the higher operator learning curve expected to master techniques in this report.The case presented by Aryana et al.16 Also, it may be more difficult to judge proper placement of CB lesions when attempting linear ablation compared to point-by-point RFA where catheter contact and location can be accurately assessed in real time due to integration with 3-dimensional mapping systems. In this instance, the authors address this limitation by utilizing intracardiac echocardiography (ICE) visualization before cryoenergy applications. Finally, heat sink effects on energy transfer, which affect other thermal energy sources similarly, exist during cryoenergy delivery and may attenuate the depth of tissue injury in thicker areas adjacent to the coronary sinus or LAA–LSPV ridge  in a way that could affect the durability of LAA isolation or ability to address certain circuits of perimetral flutter .17Intriguing is that utilization of a CB approach for the creation of linear lesion sets may have the potential to significantly shorten the procedure time needed for debulking due to a wider electrical effect on tissue than that with point-by-point RFA. At the same time, it may be more difficult to discern regions with sustained reversible injury versus areas destined for irreversible necrosis following cryoenergy applications as durable lesion formation is most likely in areas in direct contact with the balloon surface .In summary, the case study presented by Aryana et al. is an elegant and convincing initial case demonstration that, at experienced centers, the CB catheter is a potentially capable multifaceted tool when extensive LA ablation is required in patients with PSAF. This case report also emphasizes the importance of consideration of LAA placement to mitigate the risk of thromboembolism after LAAEI.MD1 and Henry D. Huang, MD, FHRS (henry_d_huang@rush.edu)1Jamario R. Skeete, 1Rush University Medical Center, Chicago, IL, USAThe authors report no conflicts of interest for the published content.1.AryanaALafortunePDi BiaseLNataleACryoballoon ablation of the pulmonary veins, the posterior wall, the left atrial appendage, and the mitral isthmus guided by intracardiac echocardiography and three-dimensional mappingJ Innov Cardiac Rhythm Manage20211314865487010.19102/icrm.2022.1301052.IwasakiYKNishidaKKatoTNattelSAtrial fibrillation pathophysiology: implications for managementCirculation20111242022642274[CrossRef][PubMed]220831483.SuWWReddyVYBhasinKCryoballoon ablation of pulmonary veins for persistent atrial fibrillation: results from the multicenter STOP persistent AF trialHeart Rhythm2020171118411847[CrossRef][PubMed]325901514.ZhaoYDi BiaseLTrivediCImportance of non-pulmonary vein triggers ablation to achieve long-term freedom from paroxysmal atrial fibrillation in patients with low ejection fractionHeart Rhythm2016131141149[CrossRef][PubMed]263047135.ThiyagarajahAKadhimKLauDHFeasibility, safety, and efficacy of posterior wall isolation during atrial fibrillation ablationCirc Arrhythm Electrophysiol2019128e007005[CrossRef][PubMed]314018536.LeeJMShimJParkJThe electrical isolation of the left atrial posterior wall in catheter ablation of persistent atrial fibrillationJACC Clin Electrophysiol201951112531261[CrossRef][PubMed]317534297.AryanaAAllenSLPujaraDKConcomitant pulmonary vein and posterior wall isolation using cryoballoon with adjunct radiofrequency in persistent atrial fibrillationJACC Clin Electrophysiol202172187196[CrossRef][PubMed]336023998.AryanaAPujaraDKAllenSLLeft atrial posterior wall isolation in conjunction with pulmonary vein isolation using cryoballoon for treatment of persistent atrial fibrillation (PIVoTAL): study rationale and designJ Interv Card Electrophysiol2021621187198[CrossRef][PubMed]330096459.Di BiaseLBurkhardtJDMohantyPLeft atrial appendage: an underrecognized trigger site of atrial fibrillationCirculation20101222109118[CrossRef][PubMed]2060612010.Di BiaseLBurkhardtJDMohantyPLeft atrial appendage isolation in patients with longstanding persistent AF undergoing catheter ablation: BELIEF trialJ Am Coll Cardiol2016681819291940[CrossRef][PubMed]2778884711.HeegerCHRilligAGeislerDLeft atrial appendage isolation in patients not responding to pulmonary vein isolationCirculation20191395712715[CrossRef][PubMed]3068941612.RomeroJGabrMPatelKEfficacy and safety of left atrial appendage electrical isolation during catheter ablation of atrial fibrillation: an updated meta-analysisEuropace2021232226237[CrossRef][PubMed]3332497813.PhillipsKPWalkerDTHumphriesJA® left atrial appendage occlusion procedures: five-year experienceCombined catheter ablation for atrial fibrillation and WatchmanJ Arrhythm2016322119126[CrossRef][PubMed]2709219314.FassiniGContiSMoltrasioMConcomitant cryoballoon ablation and percutaneous closure of left atrial appendage in patients with atrial fibrillationEuropace2016181117051710[CrossRef][PubMed]2740262315.RenZZhangJWangSTwo-year outcome from combining cryoballoon ablation and left atrial appendage closure: CLACBAC studyFront Cardiovasc Med20217610537[CrossRef][PubMed]3350599416.PanikkerSJarmanJWEVirmaniRLeft atrial appendage electrical isolation and concomitant device occlusion to treat persistent atrial fibrillationCirc Arrhythm Electrophysiol201697e003710[CrossRef][PubMed]2740660217.TakamiMMisiriJLehmannHISpatial and time-course thermodynamics during pulmonary vein isolation using the second-generation cryoballoon in a canine in vivo modelCirc Arrhythm Electrophysiol201581186192[CrossRef][PubMed]2553252918.JonesDLGuiraudonGMSkanesACGuiraudonCMAnatomical pitfalls during encircling cryoablation of the left atrium for atrial fibrillation therapy in the pigJ Interv Card Electrophysiol2008213187193[CrossRef][PubMed]183244591 report a case presentation of a patient with persistent AF treated by combining PVI, PWI, LAA isolation, mitral isthmus ablation by CB ablation, and eventually LAA mechanical closure with an endocardial device. This is a clear example of how feasible and safe CB (and thus cryoenergy) is, even outside of the PVs, as a more aggressive approach for restoring and maintaining regular sinus rhythm in a patient with advanced atrial arrhythmia.Aryana et al.3 Furthermore, atrial myocytes in LPW have a higher incidence of delayed afterdepolarizations and larger late sodium currents in addition to larger intracellular Ca transients and sarcoplasmic reticulum Ca contents. This can promote a greater propensity to arrhythmogenic behavior and lead to distinctive electrophysiological properties that may contribute to the pathophysiology of AF.4There are reasons why PWI of the LA (LPWI) needs to be included when ablation of persistent AF is planned. First, LPW shares the embryological origin of the PVs, which implies that the two structures are intertwined, potentially favoring creation of different and complex circuits. In this respect, it is worth mentioning that, as the LA develops, the PVs represent an outgrowth tissue of the LPW. This explains why the embryologic origin of the LPW and PVs can provide the anatomic basis for the development of atrial arrhythmias and, in particular, of AF.6 Moreover, spontaneous trigger activity and so-called rotors have been previously reported in persistent AF patients.8 Therefore, since secondary triggers and more complex atrial circuitries take place in the LPW, it appears reasonable that their elimination may be a crucial adjuvant strategy for providing a better clinical outcome. Aryana et al.1 elegantly describe how to safely maneuver the CB in the LA so as to properly cover the entire LPW. As the ablative techniques and the technology in the field evolve, how to perform isolation of the LPW remains a very debatable, controversial issue. Currently, the majority of operators use point-by-point radiofrequency current for LPWI by performing a roof line and a floor line . In addition, mitral isthmus ablation can be added. Several issues need to be addressed when linear lesions in the LA posterior wall are taken into account. There is an inherent technical difficulty in achieving a successful electrical LPW isolation by a set of linear radiofrequency lesions due to the complex anatomical architecture of the atrial musculature. Again, even if conduction block along the lines is achieved during the procedure, one cannot rule out the occurrence of gaps over time and, thus, dormant conduction may take place during the follow-up. Aryana et al.,1 by adopting a CB technique, have demonstrated the feasibility of creating an effective homogenization of the LPW. It looks like the specific design of CB facilitates a more homogenous tissue ablation.The LPW in patients with persistent AF is an ideal anatomic location for significant atrial remodeling, including fibrosis and lymphomononuclear infiltration.9 showing the feasibility, effectiveness, and safety of such an approach. Electrical LAA isolation is surely to be acknowledged as an adjunctive maneuver than can improve the clinical outcome of persistent AF ablation. What appears very promising is how fast is LAA isolation promoted by CB can occur as compared to the long time required when RF energy is applied.Moreover, what makes this clinical case unique is the additional LAA isolation and, then, the mitral isthmus ablation for the occurring mitral-dependent atrial flutter following LPWI. Since the LAA can harbor secondary, non-PV triggers responsible for recurrent and self-sustained AF, the choice to electrically isolate the LAA appears reasonable, especially when the mechanical closure of this anatomical structure is planned . Concomitant CB ablation and LAA closure have already been described by our group,The authors of the present case are known as masters of the CB technique and, thus, mitral isthmus line creation can also be safely and effectively performed. Moreover, this clinical case highlights the role of ICE in guiding the 3-dimensional reconstruction of the LA and also showing the position of CB when it is maneuvered toward the several anatomic ablation sites in the LPW and the entrance of the LAA. Also, the ICE probe, when moved transeptally to the LA, can offer excellent views of the LAA and, thus, guide the implant of an occluder.In summary, the overall information that we collect from this clinical report notifies us that the LPW plays a pivotal role in the maintenance of AF and should be included in the ablative approach of patients with persistent AF. Probably, in order to obtain an effective and durable LPWI, the scheme of lesions should be properly established so as to reduce the likelihood of electrical reconnection of the posterior wall. The result achieved by Aryana et al. also reinforces the concept that a more aggressive approach needs to be carried out in patients with more advanced AF, adding LAA isolation followed by mechanical closure with an endocardial occluder.Even with the inherent limitations of a single clinical case, we should applaud Aryana et al. for drawing our attention to the pivotal role of the LPW in the initiation and maintenance of AF and therefore underlying the value of walking out of the PVs toward the posterior wall. Achieving proven isolation of LPW is essential along with the abolition of sources of non-PV triggers (such as the LAA) in order to promote additional benefits over PVI alone in the treatment of persistent AF.MD, PhD, FESC, FHRS (claudio.tondo@cardiologicomonzino.it)1Claudio Tondo, 1Centro Cardiologico Monzino, Heart Rhythm Center, Department of Clinical Electrophysiology and Cardiac Pacing, Department of Biochemical, Surgical and Dentist Sciences, University of Milan, Milan, ItalyThe author reports no conflicts of interest for the published content.


Vertebral artery ostial stenosis is implicated in one-fifth of all posterior circulation cerebrovascular accidents. However, uniform treatment guidelines and data on the different treatment modalities are still lacking. Endovascular stenting is an emerging therapy for symptomatic vertebral artery ostial stenosis when medical management fails. This manuscript will examine the safety and efficacy of endovascular revascularization with drug-eluting stents in a series of ten consecutive patients that had failed medical management. We also report the rate of complications and in-stent restenosis, as well as the rate of recurrent cerebrovascular accidents. Vertebral artery ostial stenosis is common amongst patients with cardiovascular risk factors . VertebrAlthough poorly studied, optimal medical management of symptomatic vertebral artery stenosis is the current treatment of choice . HoweverEndovascular stenting with drug-eluting stents (DES) is the mainstay for coronary interventions as rates of restenosis and neo-intimal hyperplasia are significantly lower . The diaPatient selectionThis is a retrospective study of all patients who underwent endovascular stenting of the vertebral artery origin with Resolute Onyx stent between the years 2018 and 2021. A total of 10 patients were included in this study. Included patients had either confirmation of stroke isolated to the posterior circulation with magnetic resonance imaging (MRI) or transient ischemic attack (TIA) symptoms localizing to the posterior circulation as deemed by a stroke neurologist. All patients had a computed tomography angiography (CTA) or magnetic resonance angiography (MRA) demonstrating 50% or more stenosis of the dominant vertebral artery or bilateral vertebral artery stenosis greater than 50%  for one year. Acutely symptomatic patients were loaded with Plavix 600 mg and Aspirin 325 mg prior to the procedure. Heparin was given at the start of the procedure at a dose of 100 mg/kg to maintain an activated coagulation time > 250 seconds. The symptomatic vertebral artery was accessed via either the ipsilateral femoral or radial artery. A 6-french 80 cm Cook Shuttle  was utilized with either approach. The shuttle was parked proximal to the origin of the vertebral artery within the subclavian artery for femoral arterial access and distal to the vertebral artery within the subclavian artery when the radial approach was undertaken Figure , 2b. AngAll patients were admitted to the neuro-critical care unit for 24 hours post-procedure for close observation.Assessment of clinical and radiographical outcomesAll patients were seen in follow-up at six months and 12 months post-stenting. National Institute of Health stroke scale (NIHSS) and modified Rankin scale (mRS) were performed during each visit. Every patient underwent serial radiographic imaging, with either CTA or MRA, at six months and one year to evaluate stent patency. The imaging studies were independently reviewed by neuroradiology. Information regarding procedural morbidity, mortality, successful revascularization, recurrent stroke or TIA, and stent patency at follow-up was also collected.Ten patients with symptomatic vertebral artery ostial stenosis were included in the study. Baseline characteristics of included patients are summarized in Table The median mRS score at presentation and at six-month follow-up was one, going down to zero at 12-month follow-up (Table Atherosclerotic vertebral artery ostial stenosis carries a significant risk of recurrent stroke . MedicalThese studies underscore the safety of vertebral artery ostial stenting peri- and post-procedurally. As with endovascular coronary artery revascularization, treatment with angioplasty alone or with the use of bare-metal stents carries a higher risk of in-stent restenosis. With the introduction of DES, stent patency has significantly improved as DES has anti-proliferative effects, reducing neo-intimal hyperplasia.The previous case series confirmed the safety and low rate of in-stent restenosis using several different DES for vertebral artery ostial stenosis -18. The This study has several limitations. Namely, the study was a single-arm study that was performed at a single center. There was no control group. Moreover, the sample size was small. Finally, patients were consistently followed for one year only, thus the possibility of delayed in-stent stenosis or recurrent stroke was not evaluated.Vertebral artery stenosis accounts for a significant proportion of all ischemic strokes. However, the management and treatment of symptomatic vertebral artery stenosis are poorly studied. Current treatment guidelines are based on a low level of evidence. In our study, all patients underwent endovascular stenting with an Resolute Onyx stent and were followed for one year after the intervention. None of our patients experienced adverse outcomes. All stents remained patent after one year, with no patient having a recurrent stroke or TIA. Our current study builds on previously published data and adds to the body of evidence that attests to the safety and efficacy of stenting in cases of symptomatic vertebral artery ostial stenosis. Large, multicenter studies are needed to corroborate and prove the external validity of our findings.